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Sample records for adhesion modification protein

  1. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli.

    Science.gov (United States)

    Choi, Yoo Seong; Yang, Yun Jung; Yang, Byeongseon; Cha, Hyung Joon

    2012-10-24

    In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in practical applications and

  2. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  3. Photorhabdus adhesion modification protein (Pam binds extracellular polysaccharide and alters bacterial attachment

    Directory of Open Access Journals (Sweden)

    Joyce Susan A

    2010-05-01

    Full Text Available Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C and human (37°C temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect

  4. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    LENUS (Irish Health Repository)

    Jones, Robert T

    2010-05-12

    Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

  5. Cohesion and Adhesion with Proteins

    Science.gov (United States)

    Charles R. Frihart

    2016-01-01

    With increasing interest in bio-based adhesives, research on proteins has expanded because historically they have been used by both nature and humans as adhesives. A wide variety of proteins have been used as wood adhesives. Ancient Egyptians most likely used collagens tobond veneer to wood furniture, then came casein (milk), blood, fish scales, and soy adhesives, with...

  6. Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

    Science.gov (United States)

    Monniot, Céline; Boisramé, Anita; Da Costa, Grégory; Chauvel, Muriel; Sautour, Marc; Bougnoux, Marie-Elisabeth; Bellon-Fontaine, Marie-Noëlle; Dalle, Frédéric; d'Enfert, Christophe; Richard, Mathias L

    2013-01-01

    Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

  7. Soy protein adhesives

    Science.gov (United States)

    Charles R. Frihart

    2010-01-01

    In the quest to manufacture and use building materials that are more environmentally friendly, soy adhesives can be an important component. Trees fix and store carbon dioxide in the atmosphere. After the trees are harvested, machinery converts the wood into strands, which are then bonded together with adhesives to form strandboard, used in constructing long-lasting...

  8. Adhesives from modified soy protein

    Science.gov (United States)

    Sun, Susan [Manhattan, KS; Wang, Donghai [Manhattan, KS; Zhong, Zhikai [Manhattan, KS; Yang, Guang [Shanghai, CN

    2008-08-26

    The present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  9. Post-translational modification-regulated leukocyte adhesion and migration.

    Science.gov (United States)

    Loh, Jia Tong; Su, I-Hsin

    2016-06-14

    Leukocytes undergo frequent phenotypic changes and rapidly infiltrate peripheral and lymphoid tissues in order to carry out immune responses. The recruitment of circulating leukocytes into inflamed tissues depends on integrin-mediated tethering and rolling of these cells on the vascular endothelium, followed by transmigration into the tissues. This dynamic process of migration requires the coordination of large numbers of cytosolic and transmembrane proteins whose functional activities are typically regulated by post-translational modifications (PTMs). Our recent studies have shown that the lysine methyltransferase, Ezh2, critically regulates integrin signalling and governs the adhesion dynamics of leukocytes via direct methylation of talin, a key molecule that controls these processes by linking integrins to the actin cytoskeleton. In this review, we will discuss the various modes of leukocyte migration and examine how PTMs of cytoskeletal/adhesion associated proteins play fundamental roles in the dynamic regulation of leukocyte migration. Furthermore, we will discuss molecular details of the adhesion dynamics controlled by Ezh2-mediated talin methylation and the potential implications of this novel regulatory mechanism for leukocyte migration, immune responses, and pathogenic processes, such as allergic contact dermatitis and tumorigenesis.

  10. Chapter 16: Soy Proteins as Wood Adhesives

    Science.gov (United States)

    Charles R. Frihart; Christopher G. Hunt; Michael J. Birkeland

    2014-01-01

    Protein adhesives allowed the development of bonded wood products such as plywood and glulam in the early 20th century. Petrochemical-based adhesives replaced proteins in most wood bonding applications because of lower cost, improved production efficiencies, and enhanced durability. However, several technological and environmental factors have led to a resurgence of...

  11. Protein adsorption, platelet adhesion, and bacterial adhesion to polyethylene-glycol-textured polyurethane biomaterial surfaces.

    Science.gov (United States)

    Xu, Li-Chong; Siedlecki, Christopher A

    2017-04-01

    Traditional strategies for surface modification to enhance the biocompatibility of biomaterials often focus on a single route utilizing either chemical or physical approaches. This study combines the chemical and physical treatments as applied to poly(urethane urea) (PUU) biomaterials to enhance biocompatibility at the interface for inhibiting platelet-related thrombosis or bacterial adhesion-induced microbial infections. PUU films were first textured with submicron patterns by a soft lithography two-stage replication process, and then were grafted with polyethylene glycol (PEG). A series of biological response experiments including protein adsorption, platelet adhesion/activation, and bacterial adhesion/biofilm formation showed that PEG-grafted submicron textured biomaterial surfaces were resistant to protein adsorption, and greatly increased the efficiency in reducing both platelet adhesion/activation and bacterial adhesion/biofilm formation due to the additive effects of physical topography and grafted PEG. Results suggest that a combination of chemical modification and surface texturing will be more efficient in preventing biomaterial-associated thrombosis and infection of biomaterials. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 668-678, 2017. © 2015 Wiley Periodicals, Inc.

  12. Soy protein modification: A review

    Directory of Open Access Journals (Sweden)

    Barać Miroljub B.

    2004-01-01

    Full Text Available Soy protein products such as flour, concentrates and isolates are used in food formulation because of their functionality, nutritional value and low cost. To obtain their optimal nutritive and functional properties as well as desirable flavor different treatments are used. Soybean proteins can be modified by physical, chemical and enzymatic treatments. Different thermal treatments are most commonly used, while the most appropriate way of modifying soy proteins from the standpoint of safety is their limited proteolysis. These treatments cause physical and chemical changes that affect their functional properties. This review discusses three principal methods used for modification of soy protein products, their effects on dominant soy protein properties and some biologically active compounds.

  13. Plasma surface modification of rigid contact lenses decreases bacterial adhesion.

    Science.gov (United States)

    Wang, Yingming; Qian, Xuefeng; Zhang, Xiaofeng; Xia, Wei; Zhong, Lei; Sun, Zhengtai; Xia, Jing

    2013-11-01

    Contact lens safety is an important topic in clinical studies. Corneal infections usually occur because of the use of bacteria-carrying contact lenses. The current study investigated the impact of plasma surface modification on bacterial adherence to rigid contact lenses made of fluorosilicone acrylate materials. Boston XO and XO2 contact lenses were modified using plasma technology (XO-P and XO2-P groups). Untreated lenses were used as controls. Plasma-treated and control lenses were incubated in solutions containing Staphylococcus aureus or Pseudomonas aeruginosa. MTT colorimetry, colony-forming unit counting method, and scanning electron microscopy were used to measure bacterial adhesion. MTT colorimetry measurements showed that the optical density (OD) values of XO-P and XO2-P were significantly lower than those of XO and XO2, respectively, after incubation with S. aureus (P plasma technology in contact lens surface modification.

  14. Adhesion protein protocols [Methods in molecular biology, v. 96

    National Research Council Canada - National Science Library

    Dejana, Elisabetta; Corada, Monica

    1999-01-01

    "An international corps of expert investigators describe their optimized techniques for both the identification of new cell adhesion proteins and for the characterization of novel adhesive structures...

  15. Wood adhesives containing proteins and carbohydrates

    Science.gov (United States)

    In recent years there has been resurgent interest in using biopolymers as sustainable and environmentally friendly ingredients in wood adhesive formulations. Among them, proteins and carbohydrates are the most commonly used. In this chapter, an overview is given of protein-based and carbohydrate-...

  16. Effects of some chemical surface modifications on resin zirconia adhesion.

    Science.gov (United States)

    Liu, Dan; Tsoi, James Kit-Hon; Matinlinna, Jukka Pekka; Wong, Hai Ming

    2015-06-01

    To evaluate the effects of various chemical surface modifications on adhesion between zirconia and resin adhesive. Pre-sintered zirconia discs were sectioned from commercial cylindrical blocks and polished with abrasive papers under running tap water. All the discs were randomly divided into five study groups according to the methods of surface treatment, including: the control group (fully sintered, without any modification), group S (fully sintered and sandblasted with silica coated alumina particles), group HN (fully sintered and etched with a blend of mineral acid solution at 100 °C for 25 min), group HF (fully sintered and etched with 48% hydrofluoric acid solution at 100 °C for 25 min), and group Si (coated with silica particles and then fully sintered). The mean value of surface roughness was evaluated before further treatment. Resin stubs (3.6mm in diameter and 3mm in height) were adhered and light cured on each zirconia disc after the application of a silane coupling agent. In each group, all the samples were further divided into three subgroups with each n=12, one for the measurement of initial adhesion strength (shear bond) value and the other two were tested after thermal cycling for 10,000 and 20,000 cycles, respectively. The results were analyzed with two-way ANOVA and Turkey HSD (pzirconia surface crystallinity. The morphological appearance of zirconia surface after surface treatment was observed with SEM. The control group showed the lowest initial shear bond strength (SBS) value (16.8 ± 2.4 MPa) and did not survive the aging treatments. All the investigated surface treatments improved resin zirconia bond strength significantly, the group S displaying the highest initial value of 25.1 ± 2.7 MPa. However, the highest resistance to the aging effects of thermal cycling was found in group Si. It was further shown in the XRD examination that only the grit-blasting caused the crystalline transformation from tetragonal phase to monoclinic phase (T

  17. The role of the focal adhesion protein PINCH1 for the radiosensitivity of adhesion and suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Veit Sandfort

    Full Text Available Focal adhesion (FA signaling mediated by adhesion to extracellular matrix and growth factor receptors contributes to the regulation of the cellular stress response to external stimuli. Critical to focal adhesion assembly and signaling is the adapter protein PINCH1. To evaluate whether the prosurvival function of PINCH1 in radiation cell survival depends on cell adhesion, we examined PINCH1(fl/fl and PINCH1(-/- mouse embryonic fibroblasts and human cancer cell lines. Here, we found that the enhanced cellular radiosensitivity mediated by PINCH1 depletion observed under adhesion conditions is conserved when cells are irradiated under suspension conditions. This unsuspected finding could not be explained by the observed modification of adhesion and growth factor associated signaling involving FAK, Paxillin, p130(CAS, Src, AKT, GSK3β and ERK1/2 under suspension and serum withdrawal relative to adhesion conditions with serum. Our data suggest that the adapter protein PINCH1 critically participates in the regulation of the cellular radiosensitivity of normal and malignant cells similarly under adhesion and suspension conditions.

  18. SPARC fusion protein induces cellular adhesive signaling.

    Directory of Open Access Journals (Sweden)

    Lamei Cheng

    Full Text Available Secreted protein, acidic and rich in cysteine (SPARC has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ∼44 kDa under reducing conditions, and ∼38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.

  19. Wood adhesive properties of cottonseed protein with denaturant additives

    Science.gov (United States)

    Most commercial wood adhesive use either formaldehyde-based resins or polyurethanes, both of which include potentially toxic chemicals in their formulations. As a result, proteins are being considered as greener and more sustainable wood adhesives. While most of the protein adhesive studies focus ...

  20. Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion.

    Science.gov (United States)

    Sallee, Jennifer L; Wittchen, Erika S; Burridge, Keith

    2006-06-16

    Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.

  1. A bioinspired elastin-based protein for a cytocompatible underwater adhesive.

    Science.gov (United States)

    Brennan, M Jane; Kilbride, Bridget F; Wilker, Jonathan J; Liu, Julie C

    2017-04-01

    The development of adhesives that can be applied and create strong bonds underwater is a significant challenge for materials engineering. When the adhesive is intended for biomedical applications, further criteria, such as biocompatibility, must be met. Current biomedical adhesive technologies do not meet these needs. In response, we designed a bioinspired protein system that shows promise to achieve biocompatible underwater adhesion coupled with environmentally responsive behavior that is "smart" - that is, it can be tuned to suit a specific application. The material, ELY 16 , is constructed from an elastin-like polypeptide (ELP) that can be produced in high yields from Escherichia coli and can coacervate in response to environmental factors such as temperature, pH, and salinity. To confer wet adhesion, we utilized design principles from marine organisms such as mussels and sandcastle worms. When expressed, ELY 16 is rich in tyrosine. Upon modification with the tyrosinase enzyme to form mELY 16 , the tyrosine residues are converted to 3,4-dihydroxyphenylalanine (DOPA). Both ELY 16 and mELY 16 exhibit cytocompatibility and significant dry adhesion strength (>2 MPa). Modification with DOPA increases protein adsorption to glass and provides moderate adhesion strength (∼240 kPa) in a highly humid environment. Furthermore, this ELP exhibits a tunable phase transition behavior that can be formulated to coacervate in physiological conditions and provides a convenient mechanism for application underwater. Finally, mELY 16 possesses significantly higher adhesion strength in dry, humid, and underwater environments compared with a commercially available fibrin sealant. To our knowledge, mELY 16 provides the strongest bonds of any rationally designed protein when used completely underwater, and its high yields make it more viable for commercial application compared to natural adhesive proteins. In conclusion, this ELP shows great potential to be a new "smart" underwater

  2. Surface Modification of Titanium and Polyimide Sheet for Adhesive Bonding

    NARCIS (Netherlands)

    Akram, M.

    2015-01-01

    Major industrial sectors like automotive, aerospace and others are increasingly using polymer composites in their structural parts. Polyimide sheet and adhesives, are high performance polymers. They are widely used in various engineering applications due to their excellent thermal, mechanical and

  3. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation......A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...

  4. Spatial control of cell adhesion and patterning through mussel-inspired surface modification by polydopamine.

    Science.gov (United States)

    Ku, Sook Hee; Lee, Joon Seok; Park, Chan Beum

    2010-10-05

    The spatial control and patterning of mammalian cells were achieved by using the universal adhesive property of mussel-inspired polydopamine (PDA). The self-polymerization of dopamine, a small molecule inspired by the DOPA motif of mussel foot proteins, resulted in the formation of a PDA adlayer when aqueous dopamine solution was continuously injected into poly(dimethylsiloxane) microchannels. We found that various cells (fibrosarcoma HT1080, mouse preosteoblast MC3T3-E1, and mouse fibroblast NIH-3T3) predominantly adhered to PDA-modified regions, maintaining their normal morphologies. The cells aligned in the direction of striped PDA patterns, and this tendency was not limited by the type of cell line. Because PDA modification does not require complex chemical reactions and is applicable to any type of material, it enables cell patterning in a simple and versatile manner as opposed to conventional methods based on the immobilization of adhesive proteins. The PDA-based method of cell patterning should be useful in many biomaterial research areas such as the fabrication of tissue engineering scaffolds, cell-based devices for drug screening, and the fundamental study of cell-material interactions.

  5. Milk Protein Polymer and Its Application in Environmentally Safe Adhesives

    Directory of Open Access Journals (Sweden)

    Mingruo Guo

    2016-08-01

    Full Text Available Milk proteins (caseins and whey proteins are important protein sources for human nutrition; in addition, they possess important natural polymers. These protein molecules can be modified by physical, chemical, and/or enzymatic means. Casein is one of the oldest natural polymers, used for adhesives, dating back to thousands years ago. Research on milk-protein-based adhesives is still ongoing. This article deals with the chemistry and structure of milk protein polymers, and examples of uses in environmentally-safe adhesives. These are promising routes in the exploration of the broad application of milk proteins.

  6. Oxidative stress causes plasma protein modification.

    Science.gov (United States)

    Tetik, Sermin; Kiliç, Arzu; Aksoy, Halil; Rizaner, Nahit; Ahmad, Sarfraz; Yardimci, Turay

    2015-01-01

    We investigated the effect of oxidative systems on plasma proteins using Chloramine-T, a source of free radicals. Plasma specimens from 10 healthy volunteers were treated with 40 mmol/L Chloramine-T (1:1 v/v). Total protein and plasma carbonyl levels were evaluated spectrophotometrically. Identification of plasma proteins modifications was performed by SDS-PAGE, protein and lipid electrophoresis. Protein fragmentation was evaluated by HPLC. Total protein levels of oxidised plasmas were significantly lower (4.08 ± 0.12 g/dL) than control (7.86 ± 0.03 g/dL) (P < 0.01). Plasma carbonyl levels were higher (1.94 ± 0.38 nmol/mg protein) in oxidised plasma than that of control (0.03 ± 0.01 nmol/mg protein) (P < 0.01). Plasma oxidation had no significant effect on the levels of proteins and lipids. Protein fragmentations were detected in oxidised groups compared to those of the control. We conclude that protein modifications have direct effect on the protein functions, which are related to stress agent, its treatment period(s), and the methodology used for evaluating such experimental results.

  7. Urea modified cottonseed protein adhesive for wood composite products

    Science.gov (United States)

    Cottonseed protein has the potential to be used as renewable and environmentally friendly adhesives in wood products industry. However, the industry application was limited by its low mechanical properties, low water resistance and viscosity. In this work, urea modified cottonseed protein adhesive w...

  8. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins.

    Science.gov (United States)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N; Patil, Navinkumar J; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-28

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  9. 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

  10. Laser processing of natural mussel adhesive protein thin films

    Energy Technology Data Exchange (ETDEWEB)

    Doraiswamy, A. [Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7575 (United States); Narayan, R.J. [Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7575 (United States)]. E-mail: roger_narayan@unc.edu; Cristescu, R. [Plasma and Radiation Physics, National Institute for Lasers, Bucharest-Magurele (Romania); Mihailescu, I.N. [Plasma and Radiation Physics, National Institute for Lasers, Bucharest-Magurele (Romania); Chrisey, D.B. [United States Naval Research Laboratory, Washington, DC (United States)

    2007-04-15

    A novel laser processing technique is presented for depositing mussel adhesive protein thin films. Synthetic adhesives (e.g., acrylics, cyanoacrylates, epoxies, phenolics, polyurethanes, and silicones) have largely displaced natural adhesives in the automotive, aerospace, biomedical, electronic, and marine equipment industries over the past century. However, rising concerns over the environmental and health effects of solvents, monomers, and additives used in synthetic adhesives have led the adhesives community to seek natural alternatives. Marine mussel adhesive protein is a formaldehyde-free natural adhesive that demonstrates excellent adhesion to several classes of materials, including pure metals, metal oxides, polymers, and glasses. We have demonstrated the deposition of Mytilus edulis foot protein-1 thin films using matrix assisted pulsed laser evaporation (MAPLE). The Fourier transform infrared spectrum data suggest that the matrix assisted pulsed laser evaporation process does not cause significant damage to the chemical structure of M. edulis foot protein-1. In addition, matrix assisted pulsed laser evaporation appears to provide a better control over film thickness and film roughness than conventional solvent-based thin film processing techniques. MAPLE-deposited mussel adhesive protein thin films have numerous potential electronic, medical, and marine applications.

  11. Surface chemical modification of poly(dimethylsiloxane) for the enhanced adhesion and proliferation of mesenchymal stem cells.

    Science.gov (United States)

    Kuddannaya, Shreyas; Chuah, Yon Jin; Lee, Min Hui Adeline; Menon, Nishanth V; Kang, Yuejun; Zhang, Yilei

    2013-10-09

    The surface chemistry of materials has an interactive influence on cell behavior. The optimal adhesion of mammalian cells is critical in determining the cell viability and proliferation on substrate surfaces. Because of the inherent high hydrophobicity of a poly(dimethylsiloxane) (PDMS) surface, cell culture on these surfaces is unfavorable, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. Here, (3-aminopropyl)triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry was employed to immobilize either fibronectin (FN) or collagen type 1 (C1) on PDMS. The efficiency of these surfaces to support the adhesion and viability of mesenchymal stem cells (MSCs) was analyzed. The hydrophobicity of the native PDMS decreased significantly with the mentioned surface functionalization. The adhesion of MSCs was mostly favorable on chemically modified PDMS surfaces with APTES + GA + protein. Additionally, the spreading area of MSCs was significantly higher on APTES + GA + C1 surfaces than on other unmodified/modified PDMS surfaces with C1 adsorption. However, there were no significant differences in the MSC spreading area on the unmodified/modified PDMS surfaces with FN adsorption. Furthermore, there was a significant increase in cell proliferation on the PDMS surface with APTES + GA + protein functionalization as compared to the PDMS surface with protein adsorption only. Therefore, the covalent surface chemical modification of PDMS with APTES + GA + protein could offer a more biocompatible platform for the enhanced adhesion and proliferation of MSCs. Similar strategies can be applied for other substrates and cell lines by appropriate combinations of self-assembly monolayers (SAMs) and extracellular matrix proteins.

  12. The Influence of Biochemical Modification on the Properties of Adhesive Compounds

    Directory of Open Access Journals (Sweden)

    Anna Rudawska

    2016-12-01

    Full Text Available The main objective of this study was to determine the effect of biochemical modification of epoxy adhesive compounds on the mechanical properties of a cured adhesive exposed to various climatic factors. The epoxy adhesive was modified by lyophilized fungal metabolites and prepared by three methods. Additionally, the adhesive compound specimens were seasoned for two months at a temperature of 50 °C and 50% humidity in a climate test chamber, Espec SH 661. The tensile strength tests of the adhesive compounds were performed using a Zwick/Roell Z150 testing machine in compliance with the DIN EN ISO 527-1 standard. The examination of the adhesive specimens was performed using two microscopes: a LEO 912AB transmission electron microscope equipped with Quantax 200 for EDS X-ray spectroscopy and a Zeiss 510 META confocal microscope coupled to an AxioVert 200M. The experiments involved the use of a CT Skyscan 1172 tomograph. The results revealed that some mechanical properties of the modified adhesives were significantly affected by both the method of preparation of the adhesive compound and the content of the modifying agent. In addition, it was found that seasoning of the modified adhesives does not lead to a decrease in some of their mechanical properties.

  13. Improvement of the Adhesion Between TiO2 Nanofilm and Glass Substrate by Roughness Modifications

    Science.gov (United States)

    Cedillo-Gonzalez, Erika Iveth; Montorsi, Monia; Mugoni, Consuelo; Montorsi, Mauro; Siligardi, Cristina

    An increase of the adhesion between soda-lime glass substrate and TiO2 nanofilm was achieved by roughness modifications, treating the glass surface with acid or basic solutions. The study was organised through a D-optimal experimental design. The roughness (measured by AFM) and the weight loss were statistically analysed using MODDE 9.0 software. Subsequently, the correlation between the surface roughness and the adhesion (measured by scratch test) of the films was studied. The statistic analysis of the results indicates how the chemical treatments modify the roughness of the glasses and it was found that smooth surfaces enhance the adhesion of the films.

  14. Protein covalent modification by biologically active quinones

    Directory of Open Access Journals (Sweden)

    MIROSLAV J. GASIC

    2004-11-01

    Full Text Available The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4 upon modification toward lower values (from pI 5.0 to 5.3 indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

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

    Science.gov (United States)

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

    2016-11-01

    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 (-NH2) 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 zwitterion modified PP surface.

  16. Optimized Baxter model of protein solutions : Electrostatics versus adhesion

    NARCIS (Netherlands)

    Prinsen, P.; Odijk, T.

    2004-01-01

    A theory is set up of spherical proteins interacting by screened electrostatics and constant adhesion, in which the effective adhesion parameter is optimized by a variational principle for the free energy. An analytical approach to the second virial coefficient is first outlined by balancing the

  17. Urinary proteins with post-translational modifications.

    Science.gov (United States)

    Liu, Liu; Liu, Xuejiao

    2015-01-01

    Research on the human urine proteome may lay the foundation for the discovery of relevant disease biomarkers. Posttranslational modifications (PTMs) have important effects on the functions of protein biomarkers. Identifying PTMs without enrichment adds no extra steps to conventional identification procedures for urine proteomics. The only difference is that this method requires software that can conduct unrestrictive identifications of PTMs. These PTMs include methylation, dehydration, oxidation, hydroxylation, phosphorylation, or dihydroxylation. These data are useful reference for PTM biomarker discovery in the future.

  18. Surface modification of an epoxy resin with polyamines and polydopamine: Adhesion toward electroless deposited copper

    Energy Technology Data Exchange (ETDEWEB)

    Schaubroeck, David, E-mail: David.Schaubroeck@elis.ugent.be [Center for Microsystems Technology (CMST), IMEC and Ghent University, Technologiepark 914A, B-9052 Ghent (Belgium); Mader, Lothar [Center for Microsystems Technology (CMST), IMEC and Ghent University, Technologiepark 914A, B-9052 Ghent (Belgium); Dubruel, Peter [Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281 S4 bis, B-9000 Ghent (Belgium); Vanfleteren, Jan [Center for Microsystems Technology (CMST), IMEC and Ghent University, Technologiepark 914A, B-9052 Ghent (Belgium)

    2015-10-30

    Highlights: • Surface modifications of epoxy resins with polydopamine and grafted polyamines can significantly increase the adhesion toward electroless deposited copper. • A clear characterization of the copper/epoxy interphase is provided by SEM analyses of cross sections. • Tailored conditions such as etching time (roughness) and electroless deposition temperature are needed to increase the adhesion of the modified surfaces. - Abstract: In this paper the influence of the epoxy roughness, surface modifications and ELD (electroless copper deposition) temperatures on the adhesive strength of the copper is studied. Good adhesion at low roughness values is targeted due to their applicability in high density electronic circuits. Roughened epoxy surfaces are modified with adsorbed polyamines, polydopamine and polyamines grafted to polydopamine. Next the, adhesive strength of ELD copper is determined with peel strength measurements and the interphases are examined with SEM (scanning electron microscopy). Polydopamine and polyamines grafted to polydopamine can lead to increased adhesive strength at lower roughness values compared to the non-modified samples at specific plating temperatures.

  19. High-adhesion Cu patterns fabricated by nanosecond laser modification and electroless copper plating

    Science.gov (United States)

    Lv, Ming; Liu, Jianguo; Zeng, Xiaoyan; Du, Qifeng; Ai, Jun

    2015-10-01

    Adhesion strength is a crucial factor for the performance and reliability of metallic patterns on insulator substrates. In this study, we present an efficient technique for selective metallization of alumina ceramic with high adhesion strength by using nanosecond laser modification and electroless copper plating. Specifically, a 355 nm Nd:YVO4 ultraviolet (UV) laser was employed not only to decompose palladium chloride film locally for catalyzing the electroless reaction, but also to modify the ceramic surface directly using its high fluence. An orthogonal experiment was undertaken to study the effects of processing parameters including laser fluence, scanning speed and scanning line interval on adhesion strength. The adhesion strength was measured by pulling a metallic wire soldered into the copper coating perpendicular to the substrate using a pull tester. The results have shown that a strong adhesion between the copper coating and the alumina ceramic, higher than the tensile strength of tin-lead solder was obtained. Surface and interface characteristics were investigated to understand that, whose results have shown that the high-aspect-ratio microstructures formed by the laser modification is the major reason for the improvement of adhesion.

  20. Post-translational modifications of exosomal proteins.

    Science.gov (United States)

    Moreno-Gonzalo, Olga; Villarroya-Beltri, Carolina; Sánchez-Madrid, Francisco

    2014-01-01

    Exosomes mediate intercellular communication and participate in many cell processes such as cancer progression, immune activation or evasion, and the spread of infection. Exosomes are small vesicles secreted to the extracellular environment through the release of intraluminal vesicles contained in multivesicular bodies (MVBs) upon the fusion of these MVBs with the plasma membrane. The composition of exosomes is not random, suggesting that the incorporation of cargo into them is a regulated process. However, the mechanisms that control the sorting of protein cargo into exosomes are currently elusive. Here, we review the post-translational modifications detected in exosomal proteins, and discuss their possible role in their specific sorting into exosomes.

  1. Absolute quantitation of protein posttranslational modification isoform.

    Science.gov (United States)

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

  2. Improved adhesion performances of aramid fibers with vinyl epoxy via supercritical carbon dioxide modification

    Science.gov (United States)

    Qin, M. L.; Kong, H. J.; Yu, M. H.; Teng, C. Q.

    2017-06-01

    In this paper, aramid fibers were treated under supercritical carbon dioxide (SCCO2) with isocyanate terminated liquid nitrile rubber to improve the adhesion performances of vinyl epoxy composites. The interfacial shear strength (IFSS) of vinyl epoxy composites was investigated by micro-bond test. The results indicate that the surface modification of aramid fibers in SCCO2 was an efficient method to increase the adhesion performances between fibers and vinyl epoxy. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were adopted to investigate the surface structure and composition of aramid fibers. The flexural strength and interlaminar shear strength (ILSS) of treated aramid fibers/vinyl epoxy composites was improved by 18.1% and 28.9% compared with untreated aramid fibers, respectively. Furthermore, the fractured surfaces of the composites were observed by SEM, which showed that the interfacial adhesion of composites has been remarkably changed.

  3. Covalent and stable CuAAC modification of silicon surfaces for control of cell adhesion

    DEFF Research Database (Denmark)

    Vutti, Surendra; Buch-Månson, Nina; Schoffelen, Sanne

    2015-01-01

    Stable primary functionalization of metal surfaces plays a significant role in reliable secondary attachment of complex functional molecules used for the interfacing of metal objects and nanomaterials with biological systems. In principle, this can be achieved through chemical reactions either...... in the vapor or liquid phase. In this work, we compared these two methods for oxidized silicon surfaces and thoroughly characterized the functionalization steps by tagging and fluorescence imaging. We demonstrate that the vapor-phase functionalization only provided transient surface modification that was lost......-transfer reaction. Subsequently, D-amino acid adhesion peptides could be immobilized on the surface by use of Cu(I)-catalyzed click chemistry. This enabled the study of cell adhesion to the metal surface. In contrast to unmodified surfaces, the peptide-modified surfaces were able to maintain cell adhesion during...

  4. Effect of Atmospheric Pressure Plasma Modification on Polyimide and Adhesive Joining with Titanium

    Science.gov (United States)

    Akram, M.; Jansen, K. M. B.; Ernst, L. J.; Bhowmik, S.; Ajeesh, G.; Ahmed, S.; Chakraborty, D.

    2015-10-01

    This investigation highlights the effect of surface modification on polyimide by atmospheric pressure plasma treatment with different exposure time. Surface modification of polymer by plasma treatment essentially creates physical and chemical changes such as cross-linking and formation of free radicals. It also forms oxygen functionalization in the form of polar groups on polymer surface, hence improving the wetting and adhesion properties. It is observed that surface energy of the polymer increases with increasing exposure time of atmospheric pressure plasma. However, prolonged exposure time of plasma results in deterioration of the surface layer of polyimide resulting in degradation and embrittlement. Scanning electron microscopy and atomic force microscopy analysis reveal that there is a considerable morphological change on the polymer surface due to atmospheric pressure plasma treatment. X-ray photo electron spectroscopy analysis reveals that the oxygen functionalities of polymer surface increases significantly when polyimide is exposed to atmospheric pressure plasma. Untreated and atmospheric pressure plasma-treated polyimide sheet are adhesive bonded by employing polyimide adhesive as well as with titanium substrate. Due to surface modification of polyimide, it is observed that there is a significant increase in lap shear tensile strength, and therefore, this technology is highly acceptable for aviation and space applications.

  5. Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid).

    Science.gov (United States)

    Cai, Ning; Gong, Yingxue; Chian, Kerm Sin; Chan, Vincent; Liao, Kin

    2008-03-01

    Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10(-7) J m(-2)) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

  6. Chemical Modification of Soy Flour Protein and its Properties

    Science.gov (United States)

    Yuzhi Xu; Chunpeng Wang; Fuxiang Chu; Charles R. Frihart; Linda F. Lorenz; Nicole M. Stark

    2012-01-01

    This work is to examine ways to chemically modify soy proteins flours and analyze the results and determine the adhesive performance. Reaction with acetic anhydride converts amine and hydroxyl groups to amides and esters, respectively that are less polar and can make the adhesive more water resistant.The succinic anhydride reacts with these same groups but the products...

  7. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one...... physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...

  8. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    Directory of Open Access Journals (Sweden)

    Catríona M. Dowling

    2015-07-01

    Full Text Available The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

  9. Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling

    Energy Technology Data Exchange (ETDEWEB)

    Dowling, Catríona M., E-mail: Catriona.Dowling@ul.ie; Kiely, Patrick A., E-mail: Catriona.Dowling@ul.ie [Department of Life Sciences, Materials and Surface Science Institute and Stokes Institute, University of Limerick, Limerick 78666 (Ireland); Health Research Institute (HRI), University of Limerick, Limerick 78666 (Ireland)

    2015-07-15

    The signaling outputs of Receptor Tyrosine Kinases, G-protein coupled receptors and integrins converge to mediate key cell process such as cell adhesion, cell migration, cell invasion and cell proliferation. Once activated by their ligands, these cell surface proteins recruit and direct a diverse range of proteins to disseminate the appropriate response downstream of the specific environmental cues. One of the key groups of proteins required to regulate these activities is the family of serine/threonine intracellular kinases called Protein Kinase Cs. The activity and subcellular location of PKCs are mediated by a series of tightly regulated events and is dependent on several posttranslational modifications and the availability of second messengers. Protein Kinase Cs exhibit both pro- and anti-tumorigenic effects making them an interesting target for anti-cancer treatment.

  10. Dancing to Another Tune—Adhesive Moonlighting Proteins in Bacteria

    Directory of Open Access Journals (Sweden)

    Veera Kainulainen

    2014-03-01

    Full Text Available Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions.

  11. Post-Translational Modifications and RNA-Binding Proteins.

    Science.gov (United States)

    Lovci, Michael T; Bengtson, Mario H; Massirer, Katlin B

    RNA-binding proteins affect cellular metabolic programs through development and in response to cellular stimuli. Though much work has been done to elucidate the roles of a handful of RNA-binding proteins and their effect on RNA metabolism, the progress of studies to understand the effects of post-translational modifications of this class of proteins is far from complete. This chapter summarizes the work that has been done to identify the consequence of post-translational modifications to some RNA-binding proteins. The effects of these modifications have been shown to increase the panoply of functions that a given RNA-binding protein can assume. We will survey the experimental methods that are used to identify the presence of several protein modifications and methods that attempt to discern the consequence of these modifications.

  12. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  13. Fats and function: protein lipid modifications in plant cell signalling.

    Science.gov (United States)

    Turnbull, Dionne; Hemsley, Piers A

    2017-12-01

    The post-translational lipid modifications N-myristoylation, prenylation and S-acylation are traditionally associated with increasing protein membrane affinity and localisation. However this is an over-simplification, with evidence now implicating these modifications in a variety of roles such as membrane microdomain partitioning, protein trafficking, protein complex assembly and polarity maintenance. Evidence for a regulatory role is also emerging, with changes or manipulation of lipid modifications offering a means of directly controlling various aspects of protein function. Proteomics advances have revealed an enrichment of signalling proteins in the lipid-modified proteome, potentially indicating an important role for these modifications in responding to stimuli. This review highlights some of the key themes and possible functions of lipid modification during signalling processes in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Dual Coordination of Post Translational Modifications in Human Protein Networks

    Science.gov (United States)

    Woodsmith, Jonathan; Kamburov, Atanas; Stelzl, Ulrich

    2013-01-01

    Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling. PMID:23505349

  15. Diagonal chromatography to study plant protein modifications

    NARCIS (Netherlands)

    Walton, Alan; Tsiatsiani, Liana; Jacques, Silke; Stes, Elisabeth; Messens, Joris; Van Breusegem, Frank; Goormachtig, Sofie; Gevaert, Kris

    An interesting asset of diagonal chromatography, which we have introduced for contemporary proteome research, is its high versatility concerning proteomic applications. Indeed, the peptide modification or sorting step that is required between consecutive peptide separations can easily be altered and

  16. Prokaryotic Ubiquitin-Like Protein Modification

    OpenAIRE

    Maupin-Furlow, Julie A.

    2014-01-01

    Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier protein...

  17. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    Science.gov (United States)

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants.

  18. Manipulating the adhesion of electroless nickel-phosphorus film on silicon wafers by silane compound modification and rapid thermal annealing

    OpenAIRE

    Hsu, Chin-Wei; Wang, Wei-Yen; Wang, Kuan-Ting; Chen, Hou-An; Wei, Tzu-Chien

    2017-01-01

    In this study, the effect of 3-2-(2-aminoethylamino) ethylamino propyl trimethoxysilane (ETAS) modification and post rapid thermal annealing (RTA) treatment on the adhesion of electroless plated nickel-phosphorus (ELP Ni-P) film on polyvinyl alcohol-capped palladium nanoclusters (PVA-Pd) catalyzed silicon wafers is systematically investigated. Characterized by pull-off adhesion, atomic force microscopy, X-ray spectroscopy and water contact angle, a time-dependent, three-staged ETAS grafting m...

  19. Adsorption of intrinsically disordered barnacle adhesive proteins on silica surface

    Science.gov (United States)

    Wang, Xiaoqiang; Wang, Chao; Xu, Baomei; Wei, Junting; Xiao, Yang; Huang, Fang

    2018-01-01

    The adsorption of recombinant barnacle proteins Bacp19k and Mrcp19k on hydrophilic silica surface was characterized by spectroscopic ellipsometry in artificial seawater (pH = 8.2). They are homologous adhesive proteins destined for underwater adhesion but bear opposite net charges in seawater. As assessed with their primary and secondary structures, both proteins are intrinsically disordered and thus distinct from globular proteins that have dominated research in the field. Different from Mrcp19k, higher initial rate and adsorbed amount were obtained via curve fitting for Bacp19k in kinetic studies, due to favorable charge interactions with silica surface. The good fitting with the same dynamic model also indicates the formation of monolayer coverage in both cases. The two adsorption isotherms of Bacp19k and Mrcp19k are different in the initial change and maximum adsorption level, indicating different protein-surface affinities and charge interactions. Each isotherm fits the Langmuir model well, which is commonly used to describe monolayer adsorption, thus consistent with the predication from kinetic fitting. To further examine the effect of electrostatic interaction on the adsorption, the isotherm of the 1:1 mixture of Bacp19k and Mrcp19k was also constructed, which showed a higher correlation fit for Jovanovic than for Langmuir model. The presence of electrostatic attraction between Bacp19k and Mrcp19k deviated from one of the required conditions for Langmuir behavior, which may also result in the highest coadsorption level but slowest initial change among the three isotherms. The surface state of the adhesive proteins and the change with adsorption time were also examined by atomic force microscopy. The results thus obtained are in good agreement with the corresponding ellipsometric measurement.

  20. Adhesions

    Science.gov (United States)

    Adhesions are bands of scar-like tissue. Normally, internal tissues and organs have slippery surfaces so they can shift easily as the body moves. Adhesions cause tissues and organs to stick together. They ...

  1. Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio myogenesis

    Directory of Open Access Journals (Sweden)

    M.L. Costa

    2003-08-01

    Full Text Available The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

  2. Regulation of autophagy by protein post-translational modification

    National Research Council Canada - National Science Library

    Wani, Willayat Yousuf; Boyer-Guittaut, Michaël; Dodson, Matthew; Chatham, John; Darley-Usmar, Victor; Zhang, Jianhua

    2015-01-01

    ... status, and has an important role in quality control of macromolecules and organelles. As with other major cellular pathways, autophagy proteins are subjected to regulatory post-translational modification...

  3. Influence of silane surface modification of veneer on interfacial adhesion of wood-plastic plywood

    Science.gov (United States)

    Fang, Lu; Chang, Liang; Guo, Wen-jing; Chen, Yongping; Wang, Zheng

    2014-01-01

    In this study, wood-plastic plywood was fabricated with high density polyethylene (HDPE) film and poplar veneer by hot-pressing. To improve the interfacial adhesion between the wood veneer and HDPE film, silane A-171 (vinyltrimethoxysilane) was used to treat the surface of poplar veneer by spraying. The effects of silane agent on the veneer surface properties as well as the physical-mechanical performance of wood-plastic plywood were evaluated. The adsorption of several prehydrolyzed alkoxysilanes onto the veneer surface and the existence of a covalent bonding between the wood veneer and silane agent were confirmed using FTIR, XPS and contact angle. Silane surface treatment resulted in enhancement of shear strength and water resistance. When one layer HDPE film was used as adhesive, it caused 293.2% increase in shear strength, 34.6% and 40.8% reduction in water absorption and thickness swelling, respectively. In addition, the wood failure also increased from 5% to 100% due to the silane modification. Dynamic mechanical analysis (DMA) results showed that treated plywood have higher storage modulus, lower tan δ peak value and lagged temperature for tan δ peak value with respect to untreated plywood. Experimental results of interfacial morphology by SEM further revealed better interaction between silane A-171 treated veneer and HDPE film.

  4. Structure and Modification of Electrode Materials for Protein Electrochemistry.

    Science.gov (United States)

    Jeuken, Lars J C

    The interactions between proteins and electrode surfaces are of fundamental importance in bioelectrochemistry, including photobioelectrochemistry. In order to optimise the interaction between electrode and redox protein, either the electrode or the protein can be engineered, with the former being the most adopted approach. This tutorial review provides a basic description of the most commonly used electrode materials in bioelectrochemistry and discusses approaches to modify these surfaces. Carbon, gold and transparent electrodes (e.g. indium tin oxide) are covered, while approaches to form meso- and macroporous structured electrodes are also described. Electrode modifications include the chemical modification with (self-assembled) monolayers and the use of conducting polymers in which the protein is imbedded. The proteins themselves can either be in solution, electrostatically adsorbed on the surface or covalently bound to the electrode. Drawbacks and benefits of each material and its modifications are discussed. Where examples exist of applications in photobioelectrochemistry, these are highlighted.

  5. High efficient and continuous surface modification of carbon fibers with improved tensile strength and interfacial adhesion

    Science.gov (United States)

    Sun, Jingfeng; Zhao, Feng; Yao, Yue; Jin, Zhen; Liu, Xu; Huang, Yudong

    2017-08-01

    Most of the surface modification technologies for carbon fibers, no matter in laboratory scale or for commercial manufacture, are accompanied by a simultaneous decrease in tensile strength. In this paper, a feasible and high efficient strategy for carbon fiber treatment which could obviously improve both tensile strength and interfacial adhesion was proposed. Continuously moving carbon fibers were treated with atmospheric helium plasma for 1 min, followed by a 5 min pyrolytic carbon deposition using ethanol as precursor at 800 °C. The effects of the new approach were characterized by SEM, AFM, nanoindentation, XPS, Raman, wettability analysis, single fiber tensile strength testing and single fiber pull-out testing. After modification, pyrolytic carbon coating was deposited on the fiber surface uniformly, and the roughness and surface energy increased significantly. The single fiber tensile testing results indicate that the resulting fiber strength increased 15.7%, rising from 3.13 to 3.62 GPa. Meanwhile, the interfacial shear strength of its epoxy composites increased from 65.3 to 83.5 MPa. The comparative studies of carbon fibers modified with commercial anodic oxidation and sizing were also carried out. The results demonstrate that the new method can be utilized in the carbon fiber manufacture process and is more efficient than the traditional approaches.

  6. Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes

    DEFF Research Database (Denmark)

    Lyles, J M; Linnemann, D; Bock, E

    1984-01-01

    Posttranslational modifications and intracellular transport of the D2-cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A...

  7. Small heat shock proteins in cellular adhesion and migration

    Science.gov (United States)

    Montagna, Georgina N.; Matuschewski, Kai; Buscaglia, Carlos A.

    2012-01-01

    Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells. PMID:22568951

  8. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one...... that has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...

  9. Regulation of dynamin family proteins by post-translational modifications.

    Science.gov (United States)

    Kar, Usha P; Dey, Himani; Rahaman, Abdur

    2017-06-01

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.

  10. Short-term adhesion and long-term biofouling testing of polydopamine and poly(ethylene glycol) surface modifications of membranes and feed spacers for biofouling control

    KAUST Repository

    Miller, Daniel J.

    2012-08-01

    Ultrafiltration, nanofiltration membranes and feed spacers were hydrophilized with polydopamine and polydopamine- g-poly(ethylene glycol) surface coatings. The fouling propensity of modified and unmodified membranes was evaluated by short-term batch protein and bacterial adhesion tests. The fouling propensity of modified and unmodified membranes and spacers was evaluated by continuous biofouling experiments in a membrane fouling simulator. The goals of the study were: 1) to determine the effectiveness of polydopamine and polydopamine- g-poly(ethylene glycol) membrane coatings for biofouling control and 2) to compare techniques commonly used in assessment of membrane biofouling propensity with biofouling experiments under practical conditions. Short-term adhesion tests were carried out under static, no-flow conditions for 1 h using bovine serum albumin, a common model globular protein, and Pseudomonas aeruginosa, a common model Gram-negative bacterium. Biofouling tests were performed in a membrane fouling simulator (MFS) for several days under flow conditions similar to those encountered in industrial modules with the autochthonous drinking water population and acetate dosage as organic substrate. Polydopamine- and polydopamine- g-poly(ethylene glycol)-modified membranes showed significantly reduced adhesion of bovine serum albumin and P. aeruginosa in the short-term adhesion tests, but no reduction of biofouling was observed during longer biofouling experiments with modified membranes and spacers. These results demonstrate that short-term batch adhesion experiments using model proteins or bacteria under static conditions are not indicative of biofouling, while continuous biofouling experiments showed that membrane surface modification by polydopamine and polydopamine- g-poly(ethylene glycol) is not effective for biofouling control. © 2012 Elsevier Ltd.

  11. Short-term adhesion and long-term biofouling testing of polydopamine and poly(ethylene glycol) surface modifications of membranes and feed spacers for biofouling control.

    Science.gov (United States)

    Miller, Daniel J; Araújo, Paula A; Correia, Patricia B; Ramsey, Matthew M; Kruithof, Joop C; van Loosdrecht, Mark C M; Freeman, Benny D; Paul, Donald R; Whiteley, Marvin; Vrouwenvelder, Johannes S

    2012-08-01

    Ultrafiltration, nanofiltration membranes and feed spacers were hydrophilized with polydopamine and polydopamine-g-poly(ethylene glycol) surface coatings. The fouling propensity of modified and unmodified membranes was evaluated by short-term batch protein and bacterial adhesion tests. The fouling propensity of modified and unmodified membranes and spacers was evaluated by continuous biofouling experiments in a membrane fouling simulator. The goals of the study were: 1) to determine the effectiveness of polydopamine and polydopamine-g-poly(ethylene glycol) membrane coatings for biofouling control and 2) to compare techniques commonly used in assessment of membrane biofouling propensity with biofouling experiments under practical conditions. Short-term adhesion tests were carried out under static, no-flow conditions for 1 h using bovine serum albumin, a common model globular protein, and Pseudomonas aeruginosa, a common model Gram-negative bacterium. Biofouling tests were performed in a membrane fouling simulator (MFS) for several days under flow conditions similar to those encountered in industrial modules with the autochthonous drinking water population and acetate dosage as organic substrate. Polydopamine- and polydopamine-g-poly(ethylene glycol)-modified membranes showed significantly reduced adhesion of bovine serum albumin and P. aeruginosa in the short-term adhesion tests, but no reduction of biofouling was observed during longer biofouling experiments with modified membranes and spacers. These results demonstrate that short-term batch adhesion experiments using model proteins or bacteria under static conditions are not indicative of biofouling, while continuous biofouling experiments showed that membrane surface modification by polydopamine and polydopamine-g-poly(ethylene glycol) is not effective for biofouling control. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. The modification of proteins by 3-hydroxykynurenine.

    Science.gov (United States)

    Stutchbury, G M; Truscott, R J

    1993-08-01

    The autoxidation and reactivity towards proteins of 3-hydroxykynurenine, a tryptophan metabolite found in the human lens, has been studied. At neutral pH, 3-hydroxykynurenine was readily oxidized using molecular oxygen with the formation of several coloured products. The autoxidation of both 3-hydroxykynurenine and the related aminophenol, 3-hydroxyanthranilic acid, was inhibited by the inclusion of sulphydryl compounds such as glutathione or cysteine. Covalent adducts involving the thiols were not observed with either aminophenol. 3-Hydroxykynurenine was found to react with proteins, including lens proteins, to produce brown-coloured polypeptides characterized by an indistinct long wavelength absorption. This protein tanning was inhibited by glutathione. Despite the presence of an amino group in the side chain of 3-hydroxykynurenine, this tanning of proteins was found to involve amino groups including those of lysine residues, as has been found for 3-hydroxyanthranilic acid. Although both aminophenols tanned polylysine, only 3-hydroxykynurenine induced precipitation of the polyamino acid. 3-Hydroxykynurenine tanned all of the purified crystallins but induced precipitation only in the case of alpha A-crystallin. The implications of these findings for senile cataract are discussed.

  13. Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

    Science.gov (United States)

    Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

    2016-01-01

    Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

  14. Solid-binding Proteins for Modification of Inorganic Substrates

    Science.gov (United States)

    Coyle, Brandon Laurence

    Robust and simple strategies to directly functionalize graphene- and diamond-based nanostructures with proteins are of considerable interest for biologically driven manufacturing, biosensing and bioimaging. In this work, we identify a new set of carbon binding peptides that vary in overall hydrophobicity and charge, and engineer two of these sequences (Car9 and Car15) within the framework of various proteins to exploit their binding ability. In addition, we conducted a detailed analysis of the mechanisms that underpin the interaction of the fusion proteins with carbon and silicon surfaces. Through these insights, we were able to develop proteins suitable for dispersing graphene flakes and carbon nanotubes in aqueous solutions, while retaining protein activity. Additionally, our investigation into the mechanisms of adhesion for our carbon binding peptides inspired a cheap, disposable protein purification system that is more than 10x cheaper than commonly used His-tag protein purification. Our results emphasize the importance of understanding both bulk and molecular recognition events when exploiting the adhesive properties of solid-binding peptides and proteins in technological applications.

  15. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

  16. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Aanei, Carmen Mariana, E-mail: caanei@yahoo.com [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Eloae, Florin Zugun [Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Flandrin-Gresta, Pascale [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Tavernier, Emmanuelle [Service Hematologie Clinique, Institut de Cancerologie de la Loire, 42270, Saint-Priest-en-Jarez (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Carasevici, Eugen [Department of Immunology, Gr. T. Popa University of Medicine and Pharmacy, 700115, Iasi (Romania); Guyotat, Denis [Service Hematologie Clinique, Institut de Cancerologie de la Loire, 42270, Saint-Priest-en-Jarez (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France); Campos, Lydia [Laboratoire Hematologie, CHU de Saint-Etienne, 42055, Saint-Etienne (France); CNRS UMR 5239, Universite de Lyon, 42023, Saint-Etienne (France)

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  17. Olefin metathesis for site-selective protein modification.

    Science.gov (United States)

    Lin, Yuya A; Chalker, Justin M; Davis, Benjamin G

    2009-04-17

    For a reaction to be generally useful for protein modification, it must be site-selective and efficient under conditions compatible with proteins: aqueous media, low to ambient temperature, and at or near neutral pH. To engineer a reaction that satisfies these conditions is not a simple task. Olefin metathesis is one of most useful reactions for carbon-carbon bond formation, but does it fit these requirements? This minireview is an account of the development of olefin metathesis for protein modification. Highlighted below are examples of olefin metathesis in peptidic systems and in aqueous media that laid the groundwork for successful metathesis on protein substrates. Also discussed are the opportunities in protein engineering for the genetic introduction of amino acids suitable for metathesis and the related challenges in chemistry and biology.

  18. Femtosecond laser induced surface modification for prevention of bacterial adhesion on 45S5 bioactive glass

    Science.gov (United States)

    Shaikh, Shazia; Singh, Deepti; Subramanian, Mahesh; Kedia, Sunita; Singh, Anil Kumar; Singh, Kulwant; Gupta, Nidhi; Sinha, Sucharita

    2018-02-01

    Bacterial attachment and biofilm formation on implant surface has been a major concern in hospital and industrial environment. Prevention of bacterial infections of implant surface through surface treatment could be a potential solution and hence this has become a key area of research. In the present study, the antibacterial and biocompatible properties of femtosecond laser surface treated 45S5 bioactive glass (BG) have been investigated. Adhesion and sustainability of both gram positive S. aureus and gram negative P.aeruginosa and E. coli nosocomial bacteria on untreated and laser treated BG samples has been explored. An imprint method has been used to visualize the growth of bacteria on the sample surface. We observed complete bacterial rejection potentially reducing risk of biofilm formation on laser treated surface. This was correlated with surface roughness, wettability and change in surface chemical composition of the samples before and after laser treatment. Biocompatibility of the laser treated BG was demonstrated by studying the anchoring and growth of human cervix cell line INT407. Our results demonstrate that, laser surface modification of BG enables enhanced bacterial rejection without affecting its biocompatibility towards growth of human cells on it. These results open a significantly potential approach towards use of laser in successfully imparting desirable characteristics to BG based bio-implants and devices.

  19. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...... in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...

  20. Dissecting signaling and functions of adhesion G protein-coupled receptors

    NARCIS (Netherlands)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide; Engel, Felix B.; Formstone, Caroline; Goffinet, André; Hamann, Jörg; Kittel, Robert J.; Liebscher, Ines; Lin, Hsi-Hsien; Monk, Kelly R.; Petrenko, Alexander; Piao, Xianhua; Prömel, Simone; Schiöth, Helgi B.; Schwartz, Thue W.; Stacey, Martin; Ushkaryov, Yuri A.; Wobus, Manja; Wolfrum, Uwe; Xu, Lei; Langenhan, Tobias

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix

  1. Micropatterning of hydrophilic polyacrylamide brushes to resist cell adhesion but promote protein retention.

    Science.gov (United States)

    Hou, Jianwen; Shi, Qiang; Ye, Wei; Stagnaro, Paola; Yin, Jinghua

    2014-12-11

    Contrary to a prevailing concept on protein adsorption and cell adhesion, novel micropatterned polyacrylamide (PAAm) brushes that can resist cell adhesion but promote protein retention are created through patterning of ATRP initiators and surface-initiated ATRP on a polymer substrate.

  2. Enterococcus faecalis surface proteins determine its adhesion mechanism to bile drain materials

    NARCIS (Netherlands)

    Waar, K; van der Mei, HC; Harmsen, HJM; Degener, JE; Busscher, HJ

    An important step in infections associated with biliary drains is adhesion of micro-organisms to the surface. In this study the role of three surface proteins of Enterococcus faecalis (enterococcal surface protein, aggregation substances 1 and 373) in the adhesion to silicone rubber,

  3. Posttranslational modifications of proteins : tools for functional proteomics [Methods in molecular biology, v. 194

    National Research Council Canada - National Science Library

    Kannicht, Christoph

    2002-01-01

    ... single glycosylation sites in a protein. Additional powerful techniques facilitate the analysis of glycosylphosphatidylinositols, lipid modifications, protein phosphorylation and sulfation, protein methylation and acetylation, a-amidation...

  4. Cell signaling, post-translational protein modifications and NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Theillet, Francois-Xavier [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany); Smet-Nocca, Caroline [Universite Lille Nord de France, CNRS UMR 8576 (France); Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany); Yoon, Mi-Kyung; Kriwacki, Richard W. [St. Jude Children' s Research Hospital, Department of Structural Biology (United States); Landrieu, Isabelle; Lippens, Guy [Universite Lille Nord de France, CNRS UMR 8576 (France); Selenko, Philipp, E-mail: selenko@fmp-berlin.de [In-cell NMR Group, Department of NMR-Supported Structural Biology, Leibniz Institute of Molecular Pharmacology (FMP Berlin) (Germany)

    2012-11-15

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

  5. PLMD: An updated data resource of protein lysine modifications.

    Science.gov (United States)

    Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

    2017-05-20

    Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  6. Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

    KAUST Repository

    Zernickel, Anna

    2014-10-01

    With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the

  7. Chemoenzymatic methods for site-specific protein modification.

    Science.gov (United States)

    Rabuka, David

    2010-12-01

    In the past decade, numerous chemical technologies have been developed to allow the site-specific post-translational modification of proteins. Traditionally covalent chemical protein modification has been accomplished by the attachment of synthetic groups to nucleophilic amino acids on protein surfaces. These chemistries, however, are rarely sufficiently selective to distinguish one residue within a literal sea of chemical functionality. One solution to this problem is to introduce a unique chemical handle into the target protein that is orthogonal to the remainder of the proteome. In practice, this handle can be a novel peptide sequence, which forms a 'tag' that is selectively and irreversibly modified by enzymes. Furthermore, if the enzymes can tolerate substrate analogs, it becomes possible to engineer chemically modified proteins in a site-specific fashion. This review details the significant progress in creating techniques for the chemoenzymatic generation of protein-small molecule constructs and provides examples of novel applications of these methodologies. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. [Structural biology of post-translational modifications of proteins].

    Science.gov (United States)

    Kato, Koichi

    2012-01-01

    A majority of proteins encoded in genomes of limited size are post-translationally diversified by covalent modifications such as glycosylation and ubiquitination. Although recent advances in structural proteomics have enabled high-throughput structure determination of proteins, structural analyses of post-translationally modified proteins remain challenging because of the lack of appropriate determination methods. Therefore, we developed methodologies for characterizing the post-translational modifications of proteins from the structural viewpoint, focusing especially on glycosylation and ubiquitination. For instance, we established a systematic method for structural glycomics to address broader issues, including glycosylation profiling and 3D structure analyses of glycoproteins. Our stable-isotope-assisted NMR techniques in conjunction with X-ray crystallographic approach provide valuable information at the atomic level on conformations, dynamics, and interactions of glycoproteins such as antibody and proteins involved in the ubiquitin-proteasome system. These studies provide the structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans and mechanistic insights into ubiquitination reactions in glycoprotein-fate determination in cells. These approaches will allow new possibilities for structural studies on post-translationally modified proteins of clinical, pathological, and pharmaceutical interests.

  9. Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene

    Energy Technology Data Exchange (ETDEWEB)

    Gabriel, Matthias; Dahm, Manfred; Vahl, Christian-F, E-mail: mgabriel@uni-mainz.de [Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University School of Medicine, Mainz (Germany)

    2011-06-15

    Polytetrafluoroethylene (PTFE), a frequently utilized polymer for the fabrication of synthetic vascular grafts, was surface-modified by means of a wet-chemical process. The inherently non-cell-adhesive polymer does not support cellular attachment, a prerequisite for the endothelialization of luminal surface grafts in small diameter applications. To impart the material with cell-adhesive properties a treatment with sodium-naphthalene provided a basis for the subsequent immobilization of the adhesion promoting RGD-peptide using a hydroxy- and amine-reactive crosslinker. Successful conjugation was shown with cell culture experiments which demonstrated excellent endothelial cell growth on the modified surfaces.

  10. New insights into subcomplex assembly and modifications of centrosomal proteins

    Directory of Open Access Journals (Sweden)

    Habermann Karin

    2012-07-01

    Full Text Available Abstract This review provides a brief overview of the recent work on centrosome proteomics, protein complex identification and functional characterization with an emphasis on the literature of the last three years. Proteomics, genetic screens and comparative genomics studies in different model organisms have almost exhaustively identified the molecular components of the centrosome. However, much knowledge is still missing on the protein-protein interactions, protein modifications and molecular changes the centrosome undergoes throughout the cell cycle and development. The dynamic nature of this large multi-protein complex is reflected in the variety of annotated subcellular locations and biological processes of its proposed components. Some centrosomal proteins and complexes have been studied intensively in different organisms and provided detailed insight into centrosome functions. For example, the molecular, structural and functional characterization of the γ-Tubulin ring complex (γ-TuRC and the the discovery of the Augmin/HAUS complex has advanced our understanding of microtubule (MT capture, nucleation and organization. Surprising findings revealed new functions and localizations of proteins that were previously regarded as bona fide centriolar or centrosome components, e.g. at the kinetochore or in the nuclear pore complex regulating MT plus end capture or mRNA processing. Many centrosome components undergo posttranslational modifications such as phosphorylation, SUMOylation and ubiquitylation that are critical in modulating centrosome function and biology. A wealth of information has recently become available driven by new developments in technologies such as mass spectrometry, light and electron microscopy providing more detailed molecular and structural definition of the centrosome and particular roles of proteins throughout the cell cycle and development.

  11. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths

    Science.gov (United States)

    Thormann, Esben; MizunoPresent Address: Nihon L'Oreal, Research; Innovation Center, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa, Japan., Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M. Soledad; Arias, José Luis; Rutland, Mark W.; PaiPresent Address: CenterFunctional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973., Ranjith Krishna; Bergström, Lennart

    2012-06-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude.The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30536d

  12. Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

    DEFF Research Database (Denmark)

    Tan, Minjia; Peng, Chao; Anderson, Kristin A.

    2014-01-01

    We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods...

  13. Surface Modification of PMMA to Improve Adhesion to Corneal Substitutes in a Synthetic Core-Skirt Keratoprosthesis.

    Science.gov (United States)

    Riau, Andri K; Mondal, Debasish; Yam, Gary H F; Setiawan, Melina; Liedberg, Bo; Venkatraman, Subbu S; Mehta, Jodhbir S

    2015-10-07

    Patients with advanced corneal disease do poorly with conventional corneal transplantation and require a keratoprosthesis (KPro) for visual rehabilitation. The most widely used KPro is constructed using poly(methyl methacrylate) (PMMA) in the central optical core and a donor cornea as skirt material. In many cases, poor adherence between the PMMA and the soft corneal tissue is responsible for device "extrusion" and bacterial infiltration. The interfacial adhesion between the tissue and the PMMA was therefore critical to successful implantation and device longevity. In our approach, we modified the PMMA surface using oxygen plasma (plasma group); plasma followed by calcium phosphate (CaP) coating (p-CaP); dopamine followed by CaP coating (d-CaP); or plasma followed by coating with (3-aminopropyl)triethoxysilane (3-APTES). To create a synthetic KPro model, we constructed and attached 500 μm thick collagen type I hydrogel on the modified PMMA surfaces. Surface modifications produced significantly improved interfacial adhesion strength compared to untreated PMMA (p < 0.001). The p-CaP group yielded the best interfacial adhesion with the hydrogel (177 ± 27 mN/cm(2)) followed by d-CaP (168 ± 31 mN/cm(2)), 3-APTES (145 ± 12 mN/cm(2)), and plasma (119 ± 10 mN/cm(2)). Longer-term stability of the adhesion was achieved by d-CaP, which, after 14 and 28 days of incubation in phosphate buffered saline, yielded 164 ± 25 mN/cm(2) (p = 0.906 compared to adhesion at day 1) and 131 ± 20 mN/cm(2) (p = 0.053), respectively. In contrast, significant reduction of adhesion strength was observed in p-CaP group over time (p < 0.001). All surface coatings were biocompatible to human corneal stromal fibroblasts, except for the 3-APTES group, which showed no live cells at 72 h of culture. In contrast, cells on d-CaP surface showed good anchorage, evidenced by the expression of focal adhesion complex (paxillin and vinculin), and prominent filopodia protrusions. In conclusion, d-CaP can

  14. Potential protein post-translational modification in ERp57: A phenotype marker for male fertility

    OpenAIRE

    Viroj Wiwanitkit

    2010-01-01

    Background : In protein expression, post-translational modification is an important process. It is also an important process in human reproductive science. ERp57 is a molecule that is mentioned for post-translational modification. ERp57 is a component of human sperm acrosome proteins. However, the data on post-translational modifications of ERp57 is limited. Aim: The aim of this work is to assess potential protein post-translational modifications in ERp57 protein. Settings and Design : A desc...

  15. Software eyes for protein post-translational modifications.

    Science.gov (United States)

    Na, Seungjin; Paek, Eunok

    2015-01-01

    Post-translational modifications (PTMs) are critical to almost all aspects of complex processes of the cell. Identification of PTMs is one of the biggest challenges for proteomics, and there have been many computational studies for the analysis of PTMs from tandem mass spectrometry (MS/MS). Most early PTM identification studies have been performed by matching MS/MS data to protein databases, using database search tools, but they are prohibitively slow when a large number of PTMs is given as a search parameter. In this article, we present recent developments to search for more types of PTMs and to speed up the search, and discuss many computational issues and solutions in terms of identifying multiply modified peptides or searching for all possible modifications at once in unrestrictive mode. Apart from the most common type of PTMs involving covalent addition of functional groups to proteins, PTMs such as disulfide linkage require dedicated software for the analysis because they may involve cross-linking between two different parts of proteins. Finally, methods for identification of protein disulfide bonds are presented. © 2014 Wiley Periodicals, Inc.

  16. Post-translational regulation and modifications of flavivirus structural proteins.

    Science.gov (United States)

    Roby, Justin A; Setoh, Yin Xiang; Hall, Roy A; Khromykh, Alexander A

    2015-07-01

    Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.

  17. [Effects of epigallocatechin-3-gallate modification on the bonding stability of an etch-and-rinse adhesive to intraradicular dentin].

    Science.gov (United States)

    Yu, H H; Zhang, L; Yu, F; Zhou, H; Shen, L J; Chen, J H

    2017-02-09

    Objective: To evaluate the effects of epigallocatechin-3-gallate (EGCG) modification on the bonding stability of an etch-and-rinse adhesive to intraradicular dentin, and to find a new strategy to improve the stability of bonding interface. Methods: EGCG was incorporated into Single Bond 2 (SB2) with the concentration of 200 mg/L and 400 mg/L respectively to fabricated experimental adhesives group A and group B, while Single Bond 2 without EGCG was used as control group. Laser scanning confocal microscope (LSCM) and scanning electron microscope (SEM) were used to observe the bacterial biofilm adherent to the surface of the cured adhesive. Micro-Raman spectrum was used to test the degree of conversion (DC) of adhesives. The push-out bond strength of instant testing and aging with thermocycling for 5 000 times were also tested. Results: Group A and group B showed inhibiting effect on the biofilm formation of Enterococcus faecalis and performed better with higher concentration. No significant differences were detected in DC among group A ([69.73±0.68]%), group B ([69.03±1.65]%) and control group ([70.06±1.62]%) (P>0.05), and the immediate push-out bond strength of control group ([10.45±2.00] MPa) was not compromised compared to group A ([10.02±2.03] MPa) and group B ([9.95±3.03] MPa) (P>0.05). After thermocycling for 5 000 times, group A ([7.01±1.39] MPa) and group B ([7.62±1.88] MPa) showed significantly higher push-out bond strength than control group did ([5.08±1.56] MPa) (Padhesives showed anti-bacterial effect and enhanced bonding stability of intraradicular dentin-adhesive interfaces.

  18. Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

    Science.gov (United States)

    von Bilderling, Catalina; Caldarola, Martín; Masip, Martín E.; Bragas, Andrea V.; Pietrasanta, Lía I.

    2017-01-01

    The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.

  19. Dissecting signaling and functions of adhesion G protein-coupled receptors.

    Science.gov (United States)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide; Engel, Felix B; Formstone, Caroline; Goffinet, André; Hamann, Jörg; Kittel, Robert J; Liebscher, Ines; Lin, Hsi-Hsien; Monk, Kelly R; Petrenko, Alexander; Piao, Xianhua; Prömel, Simone; Schiöth, Helgi B; Schwartz, Thue W; Stacey, Martin; Ushkaryov, Yuri A; Wobus, Manja; Wolfrum, Uwe; Xu, Lei; Langenhan, Tobias

    2012-12-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas, such as immunity, neuroscience, and development and tumor biology has been intensified in the recent years, the general biological and pharmacological properties of adhesion-GPCRs are not well known, and they have not yet been used for biomedical purposes. The "6th International Adhesion-GPCR Workshop," held at the Institute of Physiology of the University of Würzburg on September 6-8, 2012, assembled a majority of the investigators currently actively pursuing research on adhesion-GPCRs, including scientists from laboratories in Europe, the United States, and Asia. The meeting featured the nascent mechanistic understanding of the molecular events driving the signal transduction of adhesion-GPCRs, novel models to evaluate their functions, and evidence for their involvement in human disease. © 2012 New York Academy of Sciences.

  20. Thin films of polymer mimics of cross-linking mussel adhesive proteins deposited by matrix assisted pulsed laser evaporation

    Science.gov (United States)

    Cristescu, R.; Mihailescu, I. N.; Stamatin, I.; Doraiswamy, A.; Narayan, R. J.; Westwood, G.; Wilker, J. J.; Stafslien, S.; Chisholm, B.; Chrisey, D. B.

    2009-03-01

    Mussels secrete specialized adhesives known as mussel adhesive proteins, which allow attachment of the organisms to underwater marine environments. Obtaining large quantities of naturally derived mussel adhesive proteins adhesives has proven to date rather problematic, thus, synthetic analogs of mussel adhesive proteins have recently been developed. We report deposition of 1:100 and 1:1000 poly[(3,4-dihydroxystyrene)-co-styrene)] mussel adhesive protein analogs by matrix assisted pulsed laser evaporation (MAPLE) using an ArF* excimer laser source. The deposited films have been evaluated for their antifouling behavior. The MAPLE-deposited synthetic mussel adhesive protein analog thin films are homogenous and adhesive, making the use of these materials in thin film form a viable option.

  1. Evaluation of milk powder quality by protein oxidative modifications.

    Science.gov (United States)

    Scheidegger, Dana; Radici, Paola M; Vergara-Roig, Víctor A; Bosio, Noelia S; Pesce, Silvia F; Pecora, Rolando P; Romano, José C P; Kivatinitz, Silvia C

    2013-06-01

    The objective of the present research was to evaluate commercially available milk powders according to their protein oxidative modifications and antioxidant capacity, and to evaluate if these characteristics are related to physical quality parameters such as dispersibility or stability during storage. Fifteen commercially processed spray-dried milk powders were evaluated: 6 whole milk powders (WMP), 4 skim milk powders (SMP), and 5 infant formula powders (IFP). Protein oxidative status was measured as protein carbonyl (PC) content, dityrosine content, and extent of protein polymerization. The level of PC was slightly lower in SMP than in WMP, whereas IFP had more than twice as much PC as WMP (2.8 ± 0.4, 2.1 ± 0.2, and 6.5 ± 1.3 nmol/mg of protein for WMP, SMP, and IFP, respectively). No differences were detected in dityrosine accumulation. Although all the possible pairs of parameters were tested for correlations, we found that 4 parameters were linked: PC, whey content, protein aggregate level, and dispersibility. After 9 mo of storage at -20°C or room temperature, all milk samples were analyzed to evaluate changes in protein oxidative status (PC, dityrosine, and protein integrity) and related parameters. Compared with the initial condition, PC increased in all tested samples after 9 mo of storage at -20°C or at room temperature. Stored milk powders had increased PC and decreased dispersibility compared with prestorage levels. Our results highlight the importance of protein oxidative status in milk powder and its relationship to other related quality parameters, such as protein integrity and dispersibility. Our findings suggest that the understanding of such relationships could help in developing quality differentiation for different types of milk powders in the product market. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  2. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, Heather G.; Roberto, Francisco F.

    2006-01-17

    The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  3. Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Silverman, Heather G.; Roberto, Francisco F.

    2006-02-07

    The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  4. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    Science.gov (United States)

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013.

  5. Proteins and their modifications in a medieval mummy

    Science.gov (United States)

    Sedláková, Pavla; Pataridis, Statis; Bortolotti, Federica; Gottardo, Rossella

    2016-01-01

    Abstract Proteins and their modifications of the natural mummy of Cangrande della Scala (Prince of Verona, Northern Italy, 1291–1329) were studied. The nano‐LC‐Q‐TOF analysis of samples of rib bone and muscle from the mummy showed the presence of different proteins including Types I, III, IV, V, and XI collagen, hemoglobin (subunits alpha and beta), ferritin, biglycan, vitronectin, prothrombin, and osteocalcin. The structure of Type I and Type III collagen was deeply studied to evaluate the occurrence of modifications in comparison with Type I and Type III collagen coming from tissues of recently died people. This analysis showed high percentage of asparaginyl and glutaminyl deamidation, carbamylation and carboxymethylation of lysine, as well as oxidation and dioxidation of methionine. The most common reaction during the natural mummification process was oxidation—the majority of lysine and proline of collagen Type I was hydroxylated whereas methionine was oxidated (oxidated or dioxidated). To the best of our knowledge, this is the first study which reports the protein profile of a natural mummified human tissue and the first one which describes the carbamylation and carboxymethylation of lysine in mummified tissues. PMID:27543755

  6. MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

    Directory of Open Access Journals (Sweden)

    N. G. Zemlianskykh

    2016-10-01

    Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

  7. Effect of Atmospheric Pressure Plasma Modification on Polyimide and Adhesive Joining with Titanium

    NARCIS (Netherlands)

    Akram, M.; Jansen, K.M.B.; Ernst, L.J.; Bhowmik, S.; Ajeesh, G.; Ahmed, S.; Chakraborty, D.

    2015-01-01

    This investigation highlights the effect of surface modification on polyimide by atmospheric pressure plasma treatment with different exposure time. Surface modification of polymer by plasma treatment essentially creates physical and chemical changes such as cross-linking and formation of free

  8. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    Directory of Open Access Journals (Sweden)

    Chunxiang Yao

    Full Text Available Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2, react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat, an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  9. Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells

    Science.gov (United States)

    do Carmo, Fillipe L. R.; Rabah, Houem; Huang, Song; Gaucher, Floriane; Deplanche, Martine; Dutertre, Stéphanie; Jardin, Julien; Le Loir, Yves; Azevedo, Vasco; Jan, Gwénaël

    2017-01-01

    Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favored by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species. PMID:28642747

  10. Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells.

    Science.gov (United States)

    do Carmo, Fillipe L R; Rabah, Houem; Huang, Song; Gaucher, Floriane; Deplanche, Martine; Dutertre, Stéphanie; Jardin, Julien; Le Loir, Yves; Azevedo, Vasco; Jan, Gwénaël

    2017-01-01

    Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favored by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species.

  11. Relation of proteins, platelets, and gas nuclei in adhesion to a synthetic material.

    Science.gov (United States)

    Ward, C A; Stanga, R D; Zingg, W; Herbert, M A

    1977-07-01

    We report the result of exposing silicone rubber to washed pig platelet suspensions that contained on average 0.018 mg of proteins/ml in solutions. This protein content is sufficiently low to reasonably neglect the protein adhesion to the material. On comparing the measured platelet adhesion from the platelet suspensions with that from blood, we find that when the gas nuclei normally present in the surface roughness of the material are removed the number of adhering platelets is the same. Thus, in the absence of the gas nuclei, the proteins in blood plasma play a negligible role in the platelet adhesion. In contrast, when both the gas nuclei and proteins are present, the maximum platelet adhesion was observed. From this and the above observation, it appears the gas nuclei affect one or more of the proteins, and this brings about an increased platelet adhesion. Finally, the platelet adhesion from the platelet suspensions was reduced after the removal of the gas nuclei. Thus the platelets themselves sense the change in the surface resulting from the removal of the gas nuclei.

  12. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium); Noppe, Gauthier; Horman, Sandrine [Pôle de Recherche Cardiovasculaire, IREC, Université Catholique de Louvain (Belgium); Morel, Nicole, E-mail: nicole.morel@uclouvain.be [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium)

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  13. The SRC-associated protein CUB Domain-Containing Protein-1 regulates adhesion and motility.

    Science.gov (United States)

    Benes, C H; Poulogiannis, G; Cantley, L C; Soltoff, S P

    2012-02-02

    Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.

  14. [Surface modification of RGD peptides onto acellularized porcine aortic valve to promote cell adhesion].

    Science.gov (United States)

    Guo, Li-ming; Zeng, Xiao-fei; Ma, Rui-dong; Shang, Guan-sheng; Hao, Ming; Yi, Ding-hua

    2010-11-01

    To investigate the impact of RGD peptides on cell adhesion to acellularized procine aortic valve. The acellular porcine aorta valve (APAV) was prepared by removing the cells and cellular components from porcine aortic valve using trypsin and hyposmosis TritonX-100. With the help of epoxy chloropropane (EC), the decelluarized valve scaffolds were immobilized with YGRGDSP peptide. MFBs were seeded onto four groups [acellularized value (AV) group, EC group, glutaraldehyde+EC (GE) group and EC+ RGD group or GE+RGD group] of coupled, coated and untreated decelluarized valve scaffolds. Ninhydrin reaction, cell count and fluorescent imaging test were employed to examine the efficiency of cell adhesion. More cells were attached to the decellularized valve scaffolds when the cells were coupled with RGD peptides compared with the others. The adhesive effect was correlated with the concentration of the RGD peptide and the attaching time. With the help of EC, YGRGDSP peptides can be immobilized by covalent bonding. RGD peptides improve cell adhesion to decellularized valve scaffolds.

  15. Epigenetic regulation of tumor endothelial cell anergy : Silencing of intercellular adhesion molecule-1 by histone modifications

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Castermans, Karolien; Vire, Emmanuelle; Dings, Ruud P. M.; Hoebers, Nicole T. H.; Mayos, Kevin H.; Egbrink, Mirjam G. A. Oude; Molema, Grietje; Fuks, Francois; Griffloen, Arjan W.

    2006-01-01

    Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel

  16. Atmospheric pressure plasma surface modification of titanium for high temperature adhesive bonding

    NARCIS (Netherlands)

    Akram, M.; Jansen, K.M.B.; Ernst, L.J.; Bhowmik, S.

    2011-01-01

    In this investigation surface treatment of titanium is carried out by plasma ion implantation under atmospheric pressure plasma in order to increase the adhesive bond strength. Prior to the plasma treatment, titanium surfaces were mechanically treated by sand blasting. It is observed that the

  17. Tyrosine Sulfation as a Protein Post-Translational Modification

    Directory of Open Access Journals (Sweden)

    Yuh-Shyong Yang

    2015-01-01

    Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

  18. Myristoylation: An Important Protein Modification in the Immune Response

    Directory of Open Access Journals (Sweden)

    Daniel Ikenna Udenwobele

    2017-06-01

    Full Text Available Protein N-myristoylation is a cotranslational lipidic modification specific to the alpha-amino group of an N-terminal glycine residue of many eukaryotic and viral proteins. The ubiquitous eukaryotic enzyme, N-myristoyltransferase, catalyzes the myristoylation process. Precisely, attachment of a myristoyl group increases specific protein–protein interactions leading to subcellular localization of myristoylated proteins with its signaling partners. The birth of the field of myristoylation, a little over three decades ago, has led to the understanding of the significance of protein myristoylation in regulating cellular signaling pathways in several biological processes especially in carcinogenesis and more recently immune function. This review discusses myristoylation as a prerequisite step in initiating many immune cell signaling cascades. In particular, we discuss the hitherto unappreciated implication of myristoylation during myelopoiesis, innate immune response, lymphopoiesis for T cells, and the formation of the immunological synapse. Furthermore, we discuss the role of myristoylation in inducing the virological synapse during human immunodeficiency virus infection as well as its clinical implication. This review aims to summarize existing knowledge in the field and to highlight gaps in our understanding of the role of myristoylation in immune function so as to further investigate into the dynamics of myristoylation-dependent immune regulation.

  19. Surface modification of polyester fabrics by atmospheric-pressure air/He plasma for color strength and adhesion enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chunming, E-mail: zcm1229@126.com [College of Textiles and Clothing, Qingdao University, Qingdao 266071 (China); Sunvim Grp Co Ltd, Gaomi 261500 (China); Zhao, Meihua; Wang, Libing; Qu, Lijun [College of Textiles and Clothing, Qingdao University, Qingdao 266071 (China); Men, Yajing [Sunvim Grp Co Ltd, Gaomi 261500 (China)

    2017-04-01

    Highlights: • Air/He plasma gave hydrophilicity on polyester surface and decreased contact angle to 18°. • The roughness of polyester increased and pit-like structures appeared on the surface after plasma treatment. • XPS confirmed the generation of new functional groups on polyester fabric. • The improved pigment color yield and anti-bleeding performance were contributed by the alteration of pigment adhesion. • The air/He plasma was more effective than air plasma at the same treatment time. - Abstract: Surface properties of water-based pigmented inks for ink-jet printed polyester fabrics were modified with atmospheric-pressure air/He plasma to improve the color strength and pigment adhesion of the treated surfaces. The influence of various parameters, including the surface morphology, chemical compositions, surface energy and dynamic contact angles of the control and plasma treated samples was studied. Color strength and edge definition were used to evaluate the ink-jet printing performance of fabrics. The change in pigment adhesion to polyester fibers was analyzed by SEM (scanning electron microscopy). AFM (Atomic force microscope) and XPS (X-ray photoelectron spectroscopy) analyses indicated the increase in surface roughness and the oxygen-containing polar groups(C=O, C−OH and COOH) reinforced the fixation of pigments on the fiber surface. The result from this study suggested that the improved pigment color yield was clearly affected by alteration of pigment adhesion enhanced by plasma surface modification. Polyester fabrics exhibited better surface property and ink-jet printing performance after the air/He mixture plasma treatment comparing with those after air plasma treatment.

  20. PTM-SD: a database of structurally resolved and annotated posttranslational modifications in proteins

    OpenAIRE

    Craveur, Pierrick; Rebehmed, Joseph; de Brevern, Alexandre G.

    2014-01-01

    Posttranslational modifications (PTMs) define covalent and chemical modifications of protein residues. They play important roles in modulating various biological functions. Current PTM databases contain important sequence annotations but do not provide informative 3D structural resource about these modifications. Posttranslational modification structural database (PTM-SD) provides access to structurally solved modified residues, which are experimentally annotated as PTMs. It combines differen...

  1. Adhesive Proteins of Stalked and Acorn Barnacles Display Homology with Low Sequence Similarities

    Science.gov (United States)

    Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

    2014-01-01

    Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa). PMID:25295513

  2. Simple modifications to methimazole that enhance its inhibitory effect on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression by human endothelial cells.

    Science.gov (United States)

    Alapati, Anuja; Deosarkar, Sudhir P; Lanier, Olivia L; Qi, Chunyan; Carlson, Grady E; Burdick, Monica M; Schwartz, Frank L; McCall, Kelly D; Bergmeier, Stephen C; Goetz, Douglas J

    2015-03-15

    The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor-α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves׳ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Hamann, Jörg; Aust, Gabriela; Araç, Demet

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic...... and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative......-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential....

  4. Transience of plasma surface modification as an adhesion promoter for polychlorotrifluorethylene

    CERN Document Server

    Subramanian, S; Love, B J; Romand, M; Charbonnier, M

    2002-01-01

    Poly(chlorotrifluoroethylene) (PCTFE) and other fluoropolymers are increasingly used as inner layer dielectrics. However, these polymers have low surface energies and correspondingly poor adhesive properties. Results are presented on the use of a low-pressure ammonia plasma to enhance the surface bondability of PCTFE. The plasma modified PCTFE film surfaces were characterized by x-ray photoelectron spectroscopy and contact angle measurements. Surface modified films exhibited improved adhesion to electroless copper deposits (180 deg. peel test) compared to coated PCTFE controls and that underwent no plasma exposure. Annealing studies were conducted between 30 and 100 deg. C to examine the stability of the plasma-modified surfaces. For samples annealed below T sub g , contact angle measurements indicated that the plasma-introduced groups remained bound on the surface for four weeks. For specimens annealed above T sub g , the surface functionalities were absorbed within the bulk and surface rearrangement occurre...

  5. Functional Characteristics of Milk Protein Concentrates and Their Modification.

    Science.gov (United States)

    Uluko, Hankie; Liu, Lu; Lv, Jia-Ping; Zhang, Shu-Wen

    2016-05-18

    A major deterrent to the usage of milk protein concentrate (MPC), a high-protein milk product with increasing demand as a food and sports drink ingredient, has been its poor functional characteristics when compared with other milk protein products such as whey protein concentrate and sodium caseinates. This review discusses the recent research on functional properties of MPC, focusing on factors that may contribute to the poor functional characteristics before, during, and after production. Current research, methods employed, and new understanding on the causes of poor solubility of MPC at mild temperatures (about 20°C) has been presented, including loss of solubility during storage as these areas have received unprecedented attention over the past decade, and also affects other useful functional properties of MPC, such as emulsifying properties, gelation, and foaming. Processing methods, which include heat treatment, high-pressure application, microwave heating, ultrasound application, and enzyme and salts modification, have been used or have potential to modify or improve the functional properties of MPCs. Future research on the effects of these processing methods on the functional properties, including effects of enzyme hydrolysis on bitterness and bioactivity, has also been discussed.

  6. Dual ion beam irradiation of polymeric materials for the modification of optical properties with improved adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae-Won, E-mail: pjw@kaeri.re.kr [Korea Atomic Energy Research Institute, 1045 Deadeock-Daero, Yuseong-Gu, Daejeon 305-353 (Korea, Republic of); Lee, Eal H.; Lee, Jae-Sang [Korea Atomic Energy Research Institute, 1045 Deadeock-Daero, Yuseong-Gu, Daejeon 305-353 (Korea, Republic of); Lee, Byung-hoon; Kim, Min-kyu [Shinki Intermobile Co. Ltd., 69-4 Junam-Dong, Yangsan-City, Kyungnam (Korea, Republic of); Lee, Chan-Young [Korea Atomic Energy Research Institute, 1045 Deadeock-Daero, Yuseong-Gu, Daejeon 305-353 (Korea, Republic of); Kim, Hyung-jin [Korea Atomic Energy Research Institute, 1045 Deadeock-Daero, Yuseong-Gu, Daejeon 305-353 (Korea, Republic of); Shinki Intermobile Co. Ltd., 69-4 Junam-Dong, Yangsan-City, Kyungnam (Korea, Republic of); Choi, Byung-Ho [Korea Atomic Energy Research Institute, 1045 Deadeock-Daero, Yuseong-Gu, Daejeon 305-353 (Korea, Republic of)

    2012-06-15

    Metallic (chromium) coating has often been applied on the surface of polymeric components, mainly to improve their appearance with a metallic luster and to protect from degradation under UV and visible light. However, the toxic nature of hexavalent chromium and delamination problems are an increasing concern in the plating industry. A similar metallic luster and the UV-visible light protection can be achieved by treating the surface of polymers by ion beams. However, a degradation by weathering including cracks, loss of glossiness, blistering, and eventual delamination have been problematic for ion beam processed polymers, particularly with a single ion beam irradiation. The main cause of adhesion failure is the abrupt change in material properties at the interface between coating and polymer or ion beam treated surface and the underlying untreated bulk polymer. In this work, therefore, a method is developed that improves adhesion by producing a graded interface by employing a dual ion beam processing. For demonstration purposes in this work, polycarbonate/acrylonitrile butadiene styrene blends were irradiated first with nitrogen ions followed by helium ions, achieving the desired metallic luster with improved adhesion. The experimental findings are explained in light of the stopping range of ions in materials and their interaction mechanisms with polymeric materials.

  7. Alteration and modulation of protein activity by varying post-translational modification

    Science.gov (United States)

    Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

    2015-03-03

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  8. Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

    Science.gov (United States)

    Hauf, Matthias; Richter, Florian; Schneider, Tobias; Faidt, Thomas; Martins, Berta M; Baumann, Tobias; Durkin, Patrick; Dobbek, Holger; Jacobs, Karin; Möglich, Andreas; Budisa, Nediljko

    2017-09-19

    Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Soy Flour Adhesive Strength Compared with That of Purified Soy Proteins*

    Science.gov (United States)

    Linda Lorenz; Michael Birkeland; Chera Daurio; Charles R. Frihart

    2015-01-01

    Except for the substitution of soy flour in phenolic resins (Frihart et al. 2013) and the use of soy flour at high pHs (Lambuth 2003), the literature on soy protein properties for adhesives has mainly focused on soy protein isolate and specific protein fractions (Sun 2005b). The assumption is that proteins are the main portion of soy flour giving bond strength and the...

  10. Post-translational protein modifications in type 1 diabetes

    DEFF Research Database (Denmark)

    Wägner, A M; Cloos, P; Bergholdt, R

    2007-01-01

    that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces......-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans...... expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low...

  11. Assays for Post-translational Modifications of Intermediate Filament Proteins

    Science.gov (United States)

    Omary, M. Bishr

    2016-01-01

    Intermediate filament (IF) proteins are known to be regulated by a number of post-translational modifications (PTMs). Phosphorylation is the best studied IF PTM, whereas ubiquitination, sumoylation, acetylation, glycosylation, ADP-ribosylation, farnesylation and transamidation are less understood in functional terms but are known to regulate specific IFs under various contexts. The number and diversity of IF PTMs is certain to grow along with rapid advances in proteomic technologies. Therefore, the need for a greater understanding of the implications of PTMs to the structure, organization, and function of the IF cytoskeleton has become more apparent with the increased availability of data from global profiling studies of normal and diseased specimens. This chapter will provide information on established methods for the isolation and monitoring of IF PTMs along with the key reagents that are necessary to carry out these experiments. PMID:26795469

  12. Preliminary study on chicken feather protein-based wood adhesives

    Science.gov (United States)

    Zehui Jiang; Daochun Qin; Chung-Yun Hse; Monlin Kuo; Zhaohui Luo; Ge Wang; Yan Yu

    2008-01-01

    The objective of this preliminary study was to partially replace phenol in the synthesis of phenol-formaldehyde resin with feather protein. Feather protein–based resins, which contained one part feather protein and two parts phenol, were formulated under the conditions of two feather protein hydrolysis methods (with and without presence of phenol during...

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

    Science.gov (United States)

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

    2015-01-01

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

  14. Potential protein post-translational modification in ERp57: A phenotype marker for male fertility

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2010-01-01

    Full Text Available Background : In protein expression, post-translational modification is an important process. It is also an important process in human reproductive science. ERp57 is a molecule that is mentioned for post-translational modification. ERp57 is a component of human sperm acrosome proteins. However, the data on post-translational modifications of ERp57 is limited. Aim: The aim of this work is to assess potential protein post-translational modifications in ERp57 protein. Settings and Design : A descriptive computational bioinformatics study. Materials and Methods : In this work, potential protein post-translational modifications in ERp57 protein were assessed via a standard bioinformatics technique. Statistical Analysis Used : Bioinformatics analysis. Results : There are three post-translational modifications within ERp57 from bioinformatics analysis. Conclusion : This new knowledge can be useful for better realization on molecular process of male infertility.

  15. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Gonzalo P Solis

    Full Text Available Analyses of cultured cells and transgenic mice expressing prion protein (PrP deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1 the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2 the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and

  16. Protein modification responds to exercise intensity and antioxidant supplementation.

    Science.gov (United States)

    Lamprecht, Manfred; Oettl, Karl; Schwaberger, Guenther; Hofmann, Peter; Greilberger, Joachim F

    2009-01-01

    To assess the effects of different exercise intensities and antioxidant supplementation on plasma protein modification. Trained men (n = 41) from a homogenous population were randomly assigned to perform cycle ergometer exercise either at 70% or 80% of individual .VO2max. Each intensity group was randomly assigned to receive either juice powder concentrate (JPC 70%, n = 11; JPC 80%, n = 10) or placebo (Plac 70%, n = 10; Plac 80%, n = 10) capsules for 28 wk. Four controlled exercise bouts and blood collections were conducted at baseline and study weeks 4, 16, and 28. Blood samples were drawn before (BE), immediately after (IE), and 30 min (30M) and 30 h (30H) postexercise. These samples were analyzed to estimate concentrations of carbonyl groups on plasma proteins (CP) and the redox state of human serum albumin (HSA). In the Plac group, CP concentrations increased at 80% of .VO2max IE and 30M, returning to preexercise concentrations by 30H (P JPC groups (P JPC group had lower baseline CP levels after 16 and 28 wk and no exercise-induced CP increase. HSA is reversibly shifted to a more oxidized state by recent intense exercise.

  17. Effect of dentin surface modification on the microtensile bond strength of self-adhesive resin cements.

    Science.gov (United States)

    Broyles, Allison C; Pavan, Sabrina; Bedran-Russo, Ana Karina

    2013-01-01

    To explore the potential to modify human dentin surface as a means of improving the microtensile bond strength (μTBS) of resin cement to dentin. Sound human molars were collected, and their occlusal surfaces were ground flat to expose polished dentin. Indirect composite resin cylinders were cemented to the teeth with RelyX Unicem or G-Cem self-adhesive cements following dentin surface treatments: 6.5% grape-seed extract, 5% glutaraldehyde, or 25% polyacrylic acid and control (no pretreatment). After 24 hours, the teeth were sectioned into beams to produce a cross-sectional area of 1.0 mm(2). Specimens of each group (n = 25) were individually mounted on a jig and placed on a tensile testing machine. A tensile force was applied to failure at a 1 mm/min crosshead speed. The use of polyacrylic acid on dentin prior to cementation with RelyX Unicem resulted in a statistically significant increase in μTBS compared to the control group (p= 0.0282). Polyacrylic acid (p= 0.0016) or glutaraldehyde (p= 0.0043) resulted in a statistically significant increase in μTBS of G-Cem to dentin when compared to the control group. Treatment with grape-seed extract did not result in a statistically significant increase in μTBS for either cement (p > 0.05). Priming dentin surfaces prior to the use of self-adhesive resin cements may be a promising means of improving μTBS. In addition, it was concluded that the results of this study are material dependent as well as being dependent of the type of dentin primer. © 2012 by the American College of Prosthodontists.

  18. Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

    Science.gov (United States)

    Kord Forooshani, Pegah

    2016-01-01

    ABSTRACT Marine mussels secret protein‐based adhesives, which enable them to anchor to various surfaces in a saline, intertidal zone. Mussel foot proteins (Mfps) contain a large abundance of a unique, catecholic amino acid, Dopa, in their protein sequences. Catechol offers robust and durable adhesion to various substrate surfaces and contributes to the curing of the adhesive plaques. In this article, we review the unique features and the key functionalities of Mfps, catechol chemistry, and strategies for preparing catechol‐functionalized polymers. Specifically, we reviewed recent findings on the contributions of various features of Mfps on interfacial binding, which include coacervate formation, surface drying properties, control of the oxidation state of catechol, among other features. We also summarized recent developments in designing advanced biomimetic materials including coacervate‐forming adhesives, mechanically improved nano‐ and micro‐composite adhesive hydrogels, as well as smart and self‐healing materials. Finally, we review the applications of catechol‐functionalized materials for the use as biomedical adhesives, therapeutic applications, and antifouling coatings. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 9–33 PMID:27917020

  19. Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

    Science.gov (United States)

    Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

    2017-07-21

    Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

  20. Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications

    DEFF Research Database (Denmark)

    Bouaidat, Salim; Berendsen, C.; Thomsen, P.

    2004-01-01

    conventional cleanroom photolithography and lift-off. Single cell arrays showed sharp contrast in cell adhesion between the untreated glass surface and the ppCrown layer. Similarly, proteins adsorbed selectively to untreated glass but not to ppCrown. The simplicity of the liftoff technique and the sturdiness...... cells ( HeLa) and fluorescence labeled proteins (isothiocyanate-labeled bovine serum albumin, i.e. FITC-BSA). The PEO-like coatings were fabricated by plasma polymerization of 12-crown-4 (ppCrown) with plasma polymerized hexene (ppHexene) as adhesion layer. The coatings were micro patterned using...

  1. Disulfide Bonds: A Key Modification in Bacterial Extracytoplasmic Proteins.

    Science.gov (United States)

    Lee, S F; Davey, L

    2017-12-01

    Disulfide bonds are a common posttranslational modification that contributes to the folding and stability of extracytoplasmic proteins. Almost all organisms, from eukaryotes to prokaryotes, have evolved enzymes to make and break these bonds. Accurate and efficient disulfide bond formation can be vital for protein function; therefore, the enzymes that catalyze disulfide bond formation are involved in multiple biological processes. Recent advances clearly show that oral bacteria also have the ability to from disulfide bonds, and this ability has an effect on a range of dental plaque-related phenotypes. In the gram-positive Streptococcus gordonii, the ability to form disulfide bonds affected autolysis, extracellular DNA release, biofilm formation, genetic competence, and bacteriocin production. In Actinomyces oris, disulfide bond formation is needed for pilus assembly, coaggregation, and biofilm formation. In other gram-positive bacteria, such as Enterococcus faecalis, disulfide bonds are formed in secreted bacteriocins and required for activity. In these oral bacteria, the enzymes that catalyze the disulfide bonds are quite diverse and share little sequence homology, but all contain a CXXC catalytic active site motif and a conserved C-terminal cis-proline, signature features of a thiol-disulfide oxidoreductase. Emerging evidence also indicates that gram-negative oral bacteria, such as Porphyromonas gingivalis and Tannerella forsythia, use disulfide bonds to stabilize their outer membrane porin proteins. Bioinformatic screens reveal that these gram-negative bacteria carry genes coding for thiol-disulfide oxidoreductases in their genomes. In conclusion, disulfide bond formation in oral bacteria is an emerging field, and the ability to form disulfide bonds plays an important role in dental plaque formation and fitness for the bacteria.

  2. Considering Protonation as a Post-translational Modification Regulating Protein Structure and Function

    Science.gov (United States)

    Schönichen, André; Webb, Bradley A.; Jacobson, Matthew P.; Barber, Diane L.

    2014-01-01

    Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes. PMID:23451893

  3. Cell adhesion and growth on ultrananocrystalline diamond and diamond-like carbon films after different surface modifications

    Energy Technology Data Exchange (ETDEWEB)

    Miksovsky, J. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Voss, A. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Kozarova, R. [Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia (Bulgaria); Kocourek, T.; Pisarik, P. [Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Ceccone, G. [Unit Nanobiosciences, European Commission Joint Research Centre, Ispra (Italy); Kulisch, W. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Jelinek, M. [Institute of Physics ASCR, Prague (Czech Republic); Czech Technical University in Prague, Faculty of Biomedical Engineering, Kladno (Czech Republic); Apostolova, M.D. [Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia (Bulgaria); Reithmaier, J.P. [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany); Popov, C., E-mail: popov@ina.uni-kassel.de [Institute of Nanostructure Technologies and Analytics, Center for Interdisciplinary Nanostructure Science and Technology, University of Kassel (Germany)

    2014-04-01

    Graphical abstract: - Highlights: • UNCD and DLC films were modified by UV/O{sub 3} treatments, O{sub 2} or NH{sub 3}-containing plasmas. • Surface composition, wettability and surface energy change upon modifications. • Higher efficiency of UNCD modifications was observed. • Cell attachment and growth were influenced by the surface termination and roughness. - Abstract: Diamond and diamond-like carbon (DLC) films possess a set of excellent physical and chemical properties which together with a high biocompatibility make them attractive candidates for a number of medical and biotechnological applications. In the current work thin ultrananocrystalline diamond (UNCD) and DLC films were comparatively investigated with respect to cell attachment and proliferation after different surface modifications. The UNCD films were prepared by microwave plasma enhanced chemical vapor deposition, the DLC films by pulsed laser deposition (PLD). The films were comprehensively characterized with respect to their basic properties, e.g. crystallinity, morphology, chemical bonding nature, etc. Afterwards the UNCD and DLC films were modified applying O{sub 2} or NH{sub 3}/N{sub 2} plasmas and UV/O{sub 3} treatments to alter their surface termination. The surface composition of as-grown and modified samples was studied by X-ray photoelectron spectroscopy (XPS). Furthermore the films were characterized by contact angle measurements with water, formamide, 1-decanol and diiodomethane; from the results obtained the surface energy with its dispersive and polar components was calculated. The adhesion and proliferation of MG63 osteosarcoma cells on the different UNCD and DLC samples were assessed by measurement of the cell attachment efficiency and MTT assays. The determined cell densities were compared and correlated with the surface properties of as-deposited and modified UNCD and DLC films.

  4. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

    Directory of Open Access Journals (Sweden)

    Claire M Elkins

    Full Text Available Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS, borate buffered saline (BBS, or Sensitive Eyes Plus Saline Solution (Sensitive Eyes, either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

  5. 3-hydroxykynurenine-mediated modification of human lens proteins: structure determination of a major modification using a monoclonal antibody.

    Science.gov (United States)

    Staniszewska, Magdalena M; Nagaraj, Ram H

    2005-06-10

    Tryptophan can be oxidized in the eye lens by both enzymatic and non-enzymatic mechanisms. Oxidation products, such as kynurenines, react with proteins to form yellow-brown pigments and cause covalent cross-linking. We generated a monoclonal antibody against 3-hydroxykynurenine (3OHKYN)-modified keyhole limpet hemocyanin and characterized it using 3OHKYN-modified amino acids and proteins. This monoclonal antibody reacted with 3OHKYN-modified N(alpha)-acetyl lysine, N(alpha)-acetyl histidine, N(alpha)-acetyl arginine, and N(alpha)-acetyl cysteine. Among the several tryptophan oxidation products tested, 3OHKYN produced the highest concentration of antigen when reacted with human lens proteins. A major antigen from the reaction of 3OHKYN and N(alpha)-acetyl lysine was purified by reversed phase high pressure liquid chromatography, which was characterized by spectroscopy and identified as 2-amino-3-hydroxyl-alpha-((5S)-5-acetamino-5-carboxypentyl amino)-gamma-oxo-benzene butanoic acid. Enzyme-digested cataractous lens proteins displayed 3OHKYN-derived modifications. Immunohistochemistry revealed 3OHKYN modifications in proteins associated with the lens fiber cell plasma membrane. The low molecular products (<10,000 Da) isolated from normal lenses after reaction with glucosidase followed by incubation with proteins generated 3OHKYN-derived products. Human lens epithelial cells incubated with 3OHKYN showed intense immunoreactivity. We also investigated the effect of glycation on tryptophan oxidation and kynurenine-mediated modification of lens proteins. The results showed that glycation products failed to oxidize tryptophan or generate kynurenine modifications in proteins. Our studies indicate that 3OHKYN modifies lens proteins independent of glycation to form products that may contribute to protein aggregation and browning during cataract formation.

  6. Tailored Poly(2-oxazoline) Polymer Brushes to Control Protein Adsorption and Cell Adhesion

    KAUST Repository

    Zhang, Ning

    2012-05-18

    POx bottle-brush brushes (BBBs) are synthesized by SIPGP of 2-isopropenyl-2-oxazoline and consecutive LCROP of 2-oxazolines on 3-aminopropyltrimethoxysilane-modified silicon substrates. The side chain hydrophilicity and polarity are varied. The impact of the chemical composition and architecture of the BBB upon protein (fibronectin) adsorption and endothelial cell adhesion are investigated and prove extremely low protein adsorption and cell adhesion on BBBs with hydrophilic side chains such as poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline). The influence of the POx side chain terminal function upon adsorption and adhesion is minor but the side chain length has a significant effect on bioadsorption. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry

    Science.gov (United States)

    Zhang, Han; Ge, Ying

    2012-01-01

    Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular

  8. Photo-lysine captures proteins that bind lysine post-translational modifications.

    Science.gov (United States)

    Yang, Tangpo; Li, Xiao-Meng; Bao, Xiucong; Fung, Yi Man Eva; Li, Xiang David

    2016-02-01

    Post-translational modifications (PTMs) have key roles in regulating protein-protein interactions in living cells. However, it remains a challenge to identify these PTM-mediated interactions. Here we develop a new lysine-based photo-reactive amino acid, termed photo-lysine. We demonstrate that photo-lysine, which is readily incorporated into proteins by native mammalian translation machinery, can be used to capture and identify proteins that recognize lysine PTMs, including 'readers' and 'erasers' of histone modifications.

  9. Impact of protein modification on the protein corona on nanoparticles and nanoparticle-cell interactions.

    Science.gov (United States)

    Treuel, Lennart; Brandholt, Stefan; Maffre, Pauline; Wiegele, Sarah; Shang, Li; Nienhaus, G Ulrich

    2014-01-28

    Recent studies have firmly established that cellular uptake of nanoparticles is strongly affected by the presence and the physicochemical properties of a protein adsorption layer around these nanoparticles. Here, we have modified human serum albumin (HSA), a serum protein often used in model studies of protein adsorption onto nanoparticles, to alter its surface charge distribution and investigated the consequences for protein corona formation around small (radius ∼5 nm), dihydrolipoic acid-coated quantum dots (DHLA-QDs) by using fluorescence correlation spectroscopy. HSA modified by succinic anhydride (HSAsuc) to generate additional carboxyl groups on the protein surface showed a 3-fold decreased binding affinity toward the nanoparticles. A 1000-fold enhanced affinity was observed for HSA modified by ethylenediamine (HSAam) to increase the number of amino functions on the protein surface. Remarkably, HSAsuc formed a much thicker protein adsorption layer (8.1 nm) than native HSA (3.3 nm), indicating that it binds in a distinctly different orientation on the nanoparticle, whereas the HSAam corona (4.6 nm) is only slightly thicker. Notably, protein binding to DHLA-QDs was found to be entirely reversible, independent of the modification. We have also measured the extent and kinetics of internalization of these nanoparticles without and with adsorbed native and modified HSA by HeLa cells. Pronounced variations were observed, indicating that even small physicochemical changes of the protein corona may affect biological responses.

  10. A cohort of new adhesive proteins identified from transcriptomic analysis of mussel foot glands.

    Science.gov (United States)

    DeMartini, Daniel G; Errico, John M; Sjoestroem, Sebastian; Fenster, April; Waite, J Herbert

    2017-06-01

    The adaptive attachment of marine mussels to a wide range of substrates in a high-energy, saline environment has been explored for decades and is a significant driver of bioinspired wet adhesion research. Mussel attachment relies on a fibrous holdfast known as the byssus, which is made by a specialized appendage called the foot. Multiple adhesive and structural proteins are rapidly synthesized, secreted and moulded by the foot into holdfast threads. About 10 well-characterized proteins, namely the mussel foot proteins (Mfps), the preCols and the thread matrix proteins, are reported as representing the bulk of these structures. To explore how robust this proposition is, we sequenced the transcriptome of the glandular tissues that produce and secrete the various holdfast components using next-generation sequencing methods. Surprisingly, we found around 15 highly expressed genes that have not previously been characterized, but bear key similarities to the previously defined mussel foot proteins, suggesting additional contribution to byssal function. We verified the validity of these transcripts by polymerase chain reaction, cloning and Sanger sequencing as well as confirming their presence as proteins in the byssus. These newly identified proteins greatly expand the palette of mussel holdfast biochemistry and provide new targets for investigation into bioinspired wet adhesion. © 2017 The Author(s).

  11. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    NARCIS (Netherlands)

    Liebscher, Ines; Ackley, Brian; Araç, Demet; Ariestanti, Donna M.; Aust, Gabriela; Bae, Byoung-Il; Bista, Bigyan R.; Bridges, James P.; Duman, Joseph G.; Engel, Felix B.; Giera, Stefanie; Goffinet, André M.; Hall, Randy A.; Hamann, Jörg; Hartmann, Nicole; Lin, Hsi-Hsien; Liu, Mingyao; Luo, Rong; Mogha, Amit; Monk, Kelly R.; Peeters, Miriam C.; Prömel, Simone; Ressl, Susanne; Schiöth, Helgi B.; Sigoillot, Séverine M.; Song, Helen; Talbot, William S.; Tall, Gregory G.; White, James P.; Wolfrum, Uwe; Xu, Lei; Piao, Xianhua

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane alpha-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular

  12. Proteins Play Important Role in Intercellular Adhesion Affecting on Fruit Textural Quality

    DEFF Research Database (Denmark)

    Bahadur Adhikari, Khem; Shomer, Ilan

    2012-01-01

    Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA strength...

  13. Comparison of the adhesive performances of soy meal, water washed meal fractions, and protein isolates

    Science.gov (United States)

    Adhesive bonding of wood plays an increasing role in the forest products industry and is a key factor for efficiently utilizing timber and other lignocellulosic resources. In this work, we obtained five soy meal products through commercial sources or in-house preparations. The protein content was 49...

  14. Protein kinase C beta mediates CD40 ligand-induced adhesion of monocytes to endothelial cells.

    Directory of Open Access Journals (Sweden)

    Zeyu Wu

    Full Text Available Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs to clinically relevant concentrations of CD40L (20 to 80 ng/mL dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1 mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

  15. Post-translational modifications of hepatitis C viral proteins and their biological significance.

    Science.gov (United States)

    Hundt, Jana; Li, Zhubing; Liu, Qiang

    2013-12-21

    Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.

  16. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus

    Science.gov (United States)

    Foster, Timothy J.; Geoghegan, Joan A.; Ganesh, Vannakambadi K.; Höök, Magnus

    2014-01-01

    Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is ‘decorated’ with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. PMID:24336184

  17. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus.

    Science.gov (United States)

    Foster, Timothy J; Geoghegan, Joan A; Ganesh, Vannakambadi K; Höök, Magnus

    2014-01-01

    Staphylococcus aureus is an important opportunistic pathogen and persistently colonizes about 20% of the human population. Its surface is 'decorated' with proteins that are covalently anchored to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections.

  18. Mussel inspired protein-mediated surface modification to electrospun fibers and their potential biomedical applications.

    Science.gov (United States)

    Xie, Jingwei; Michael, Praveesuda Lorwattanapongsa; Zhong, Shaoping; Ma, Bing; MacEwan, Matthew R; Lim, Chwee Teck

    2012-04-01

    Mussel inspired proteins have been demonstrated to serve as a versatile biologic adhesive with numerous applications. The present study illustrates the use of such Mussel inspired proteins (polydopamine) in the fabrication of functionalized bio-inspired nanomaterials capable of both improving cell response and sustained delivery of model probes. X-ray photoelectron spectroscopy analysis confirmed the ability of dopamine to polymerize on the surface of plasma-treated, electrospun poly(ε-caprolactone) (PCL) fiber mats to form polydopamine coating. Transmission electron microscopy images demonstrated that self-polymerization of dopamine was induced by pH shift and that the thickness of polydopamine coating was readily modulated by adjusting the concentration of dopamine and reaction time. Polydopamine coatings were noted to affect the mechanical properties of underlying fiber mats, as mechanical testing demonstrated a decrease in elasticity and increase in stiffness of polydopamine-coated fiber mats. Polydopamine coatings were also utilized to effectively immobilize extracellular matrix proteins (i.e., fibronectin) on the surface of polydopamine-coated, electrospun fibers, resulting in enhancement of NIH3T3 cell attachment, spreading, and cytoskeletal development. Comparison of release rates of rhodamine 6G encapsulated in coated and uncoated PCL fibers also confirmed that polydopamine coatings modulate the release rate of loaded payloads. The authors further demonstrate the significant difference of rhodamine 6G adsorption kinetics in water between PCL fibers and polydopamine-coated PCL fibers. Taken together, polydopamine-mediated surface modification to electrospun fibers may be an effective means of fabricating a wide range of bio-inspired nanomaterials with unique properties for use in tissue engineering, drug delivery, and advanced biomedical applications. Copyright © 2012 Wiley Periodicals, Inc.

  19. Activation of focal adhesion kinase enhances the adhesion of Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway.

    Science.gov (United States)

    Pan, Xiaojing; Wang, Ye; Zhou, Qingjun; Chen, Peng; Xu, Yuanyuan; Chen, Hao; Xie, Lixin

    2011-03-05

    To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs). After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0-24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment. Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani. The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation. Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F. solani via the tyrosine-specific protein kinase signaling pathway.

  20. Spatial and Temporal Effects in Protein Post-translational Modification Distributions in the Developing Mouse Brain

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Edwards, Gregory J; Schwämmle, Veit

    2014-01-01

    Protein post-translational modification (PTM) is a powerful way to modify the behavior of cellular proteins and thereby cellular behavior. Multiple recent studies of evolutionary trends have shown that certain pairs of protein post-translational modifications tend to occur closer to each other than...... for observations of increasingly frequent and diverse protein modification in cell biology. In this study, we use mass spectrometry and proteomic strategies to present biological data showing spatiotemporal PTM co-localization across multiple PTM categories, which display changes over development of the brain...

  1. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking.

    Science.gov (United States)

    Jeon, Eun Young; Hwang, Byeong Hee; Yang, Yun Jung; Kim, Bum Jin; Choi, Bong-Hyuk; Jung, Gyu Yong; Cha, Hyung Joon

    2015-10-01

    Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine crosslinking chemistry. LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

    Science.gov (United States)

    Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

    2013-07-01

    To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Multiple conserved cell adhesion protein interactions mediate neural wiring of a sensory circuit in C. elegans.

    Science.gov (United States)

    Kim, Byunghyuk; Emmons, Scott W

    2017-09-13

    Nervous system function relies on precise synaptic connections. A number of widely-conserved cell adhesion proteins are implicated in cell recognition between synaptic partners, but how these proteins act as a group to specify a complex neural network is poorly understood. Taking advantage of known connectivity in C. elegans, we identified and studied cell adhesion genes expressed in three interacting neurons in the mating circuits of the adult male. Two interacting pairs of cell surface proteins independently promote fasciculation between sensory neuron HOA and its postsynaptic target interneuron AVG: BAM-2/neurexin-related in HOA binds to CASY-1/calsyntenin in AVG; SAX-7/L1CAM in sensory neuron PHC binds to RIG-6/contactin in AVG. A third, basal pathway results in considerable HOA-AVG fasciculation and synapse formation in the absence of the other two. The features of this multiplexed mechanism help to explain how complex connectivity is encoded and robustly established during nervous system development.

  4. CUB domain containing protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells.

    Science.gov (United States)

    Orchard-Webb, David J; Lee, Thong Chuan; Cook, Graham P; Blair, G Eric

    2014-10-09

    Deregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated. CDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation. Engineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates. A number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell

  5. Barnacle settlement and the adhesion of protein and diatom microfouling to xerogel films with varying surface energy and water wettability.

    Science.gov (United States)

    Finlay, John A; Bennett, Stephanie M; Brewer, Lenora H; Sokolova, Anastasiya; Clay, Gemma; Gunari, Nikhil; Meyer, Anne E; Walker, Gilbert C; Wendt, Dean E; Callow, Maureen E; Callow, James A; Detty, Michael R

    2010-08-01

    Previous work has shown that organosilica-based xerogels have the potential to control biofouling. In this study, modifications of chemistry were investigated with respect to their resistance to marine slimes and to settlement of barnacle cyprids. Adhesion force measurements of bovine serum albumin (BSA)-coated atomic force microscopy (AFM) tips to xerogel surfaces prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl-containing precursors, indicated that adhesion was significantly less on the xerogel surfaces in comparison to a poly(dimethylsiloxane) elastomer (PDMSE) standard. The strength of adhesion of BSA on the xerogels was highest on surfaces with the highest and the lowest critical surface tensions, gamma(C) and surface energies, gamma(S), and duplicated the 'Baier curve'. The attachment to and removal of cells of the diatom Navicula perminuta from a similar series of xerogel surfaces were examined. Initial attachment of cells was comparable on all of the xerogel surfaces, but the percentage removal of attached cells by hydrodynamic shear stress increased with gamma(C) and increased wettability as measured by the static water contact angle, theta(Ws), of the xerogel surfaces. The percentage removal of cells of Navicula was linearly correlated with both properties (R(2) = 0.74 for percentage removal as a function of theta(Ws) and R(2) = 0.69 for percentage removal as a function of gamma(C)). Several of the aminopropylsilyl-containing xerogels showed significantly greater removal of Navicula compared to a PDMSE standard. Cypris larvae of the barnacle B. amphitrite showed preferred settlement on hydrophilic/higher energy surfaces. Settlement was linearly correlated with theta(Ws) (R(2) = 0.84) and gamma(C) (R(2) = 0.84). Hydrophilic xerogels should prove useful as coatings for boats in regions where fouling is dominated by microfouling (protein and diatom slimes).

  6. The role of cytoskeleton and adhesion proteins in the resistance to photodynamic therapy. Possible therapeutic interventions.

    Science.gov (United States)

    Di Venosa, Gabriela; Perotti, Christian; Batlle, Alcira; Casas, Adriana

    2015-08-01

    It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

  7. The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

    DEFF Research Database (Denmark)

    Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

    2015-01-01

    Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...... intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded...... that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer....

  8. The functional properties, modification and utilization of whey proteins

    Directory of Open Access Journals (Sweden)

    B. G. Venter

    1986-03-01

    Full Text Available Whey protein has an excellent nutritional value and exhibits a functional potential. In comparison with certain other food proteins, the whey protein content of essential amino acids is extremely favourable for human consumption. Depending on the heat-treatment history thereof, soluble whey proteins with utilizable functional properties, apart from high biological value, true digestibility, protein efficiency ratio and nett protein utilization, can be recovered. Various technological and chemical recovery processes have been designed. Chemically and enzymatically modified whey protein is manufactured to obtain technological and functional advantages. The important functional properties of whey proteins, namely hydration, gelation, emulsifying and foaming properties, are reviewed.

  9. Protein conformational modifications and kinetics of water-protein interactions in milk protein concentrate powder upon aging: effect on solubility.

    Science.gov (United States)

    Haque, Enamul; Bhandari, Bhesh R; Gidley, Michael J; Deeth, Hilton C; Møller, Sandie M; Whittaker, Andrew K

    2010-07-14

    Protein conformational modifications and water-protein interactions are two major factors believed to induce instability of protein and eventually affect the solubility of milk protein concentrate (MPC) powder. To test these hypotheses, MPC was stored at different water activities (a(w) 0.0-0.85) and temperatures (25 and 45 degrees C) for up to 12 weeks. Samples were examined periodically to determine solubility, change in protein conformation by Fourier transform infrared (FTIR) spectroscopy and principal component analysis (PCA), and water status (interaction of water with the protein molecule/surface) by measuring the transverse relaxation time (T(2)) with proton nuclear magnetic resonance ((1)H NMR). The solubility of MPC decreased significantly with aging, and this process was enhanced by increasing water activity (a(w)) and storage temperature. Minor changes in protein secondary structure were observed with FTIR, which indicated some degree of unfolding of protein molecules. PCA of the FTIR data was able to discriminate samples according to moisture content and storage period. Partial least-squares (PLS) analysis showed some correlation between FTIR spectral feature and solubility. The NMR T(2) results indicated the presence of three distinct populations of water molecules, and the proton signal intensity and T(2) values of proton fractions varied with storage conditions (humidity, temperature) and aging. Results suggest that protein/protein interactions may be initiated by unfolding of protein molecules that eventually affects solubility.

  10. BioJava-ModFinder: identification of protein modifications in 3D structures from the Protein Data Bank.

    Science.gov (United States)

    Gao, Jianjiong; Prlic, Andreas; Bi, Chunxiao; Bluhm, Wolfgang F; Dimitropoulos, Dimitris; Xu, Dong; Bourne, Philip E; Rose, Peter W

    2017-07-01

    We developed a new software tool, BioJava-ModFinder, for identifying protein modifications observed in 3D structures archived in the Protein Data Bank (PDB). Information on more than 400 types of protein modifications were collected and curated from annotations in PDB, RESID, and PSI-MOD. We divided these modifications into three categories: modified residues, attachment modifications, and cross-links. We have developed a systematic method to identify these modifications in 3D protein structures. We have integrated this package with the RCSB PDB web application and added protein modification annotations to the sequence diagram and structure display. By scanning all 3D structures in the PDB using BioJava-ModFinder, we identified more than 30 000 structures with protein modifications, which can be searched, browsed, and visualized on the RCSB PDB website. BioJava-ModFinder is available as open source (LGPL license) at ( https://github.com/biojava/biojava/tree/master/biojava-modfinder ). The RCSB PDB can be accessed at http://www.rcsb.org . pwrose@ucsd.edu.

  11. Pseudouridine and N6-methyladenosine modifications weaken PUF protein/RNA interactions.

    Science.gov (United States)

    Vaidyanathan, Pavanapuresan P; AlSadhan, Ishraq; Merriman, Dawn K; Al-Hashimi, Hashim M; Herschlag, Daniel

    2017-05-01

    RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications-pseudouridine (Ψ) and N6-methyladenosine (m6A)-affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure. Here, we test the hypothesis that RNA modifications directly affect interactions between RNA-binding proteins and target RNA. We show that Ψ and m6A weaken the binding of the human single-stranded RNA binding protein Pumilio 2 (hPUM2) to its consensus motif, with individual modifications having effects up to approximately threefold and multiple modifications giving larger effects. While there are likely to be some cases where RNA modifications essentially fully ablate protein binding, here we see modest responses that may be more common. Such modest effects could nevertheless profoundly alter the complex landscape of RNA:protein interactions, and the quantitative rather than qualitative nature of these effects underscores the need for quantitative, systems-level accounting of RNA:protein interactions to understand post-transcriptional regulation. © 2017 Vaidyanathan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Electrochemical methods for detection of post-translational modifications of proteins.

    Science.gov (United States)

    Shumyantseva, Victoria V; Suprun, Elena V; Bulko, Tatiana V; Archakov, Alexander I

    2014-11-15

    Post-translational modifications of proteins play a key role in the regulation of various cellular processes. The analysis and identification of post-translational modifications are probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This review focuses on the electroactivity of amino acids as a tool for analysis of post-translational modifications of proteins. The most attention is paid to the electrochemical detection of phosphorylation/dephosphorylation and glycosylation of proteins, to the best-studied and functionally-significant modifications, and, also, to the electrochemical analysis of activity of enzymes responsible for carrying out phosphorylation/dephosphorylation of proteins. Recent advances in electrochemistry with special references to proteomics are outlined and innovative technologies for protein detection are highlighted. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Mussel adhesive protein provides cohesive matrix for collagen type-1α.

    Science.gov (United States)

    Martinez Rodriguez, Nadine R; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N; Waite, J Herbert

    2015-05-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Unrestricted Mass Spectrometric Data Analysis for Identification, Localization, and Quantification of Oxidative Protein Modifications

    DEFF Research Database (Denmark)

    Rykær, Martin; Svensson, Birte; Davies, Michael J

    2017-01-01

    Oxidation generates multiple diverse post-translational modifications resulting in changes in protein structure and function associated with a wide range of diseases. Of these modifications, carbonylations have often been used as hallmarks of oxidative damage. However, accumulating evidence...... modifications based on so-called "dependent peptides". The strategy involves unrestricted database searches with rigorous filtering focusing on oxidative modifications. The approach was applied to bovine serum albumin and human serum proteins subjected to metal ion-catalyzed oxidation, resulting...... in the identification of a wide range of different oxidative modifications. The most common modification in the oxidized samples is hydroxylation, but carbonylation, decarboxylation, and dihydroxylation are also abundant, while carbonylation showed the largest increase in abundance relative to nonoxidized samples. Site...

  15. Protein-protein binding before and after photo-modification of albumin

    Science.gov (United States)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  16. Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

    Science.gov (United States)

    Polo, Sophie E.; Jackson, Stephen P.

    2011-01-01

    Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed the DNA damage response (DDR). A common feature of DDR proteins is their mobilization in response to genotoxic stress. Here, we outline how the development of various complementary methodologies has provided valuable insights into the spatiotemporal dynamics of DDR protein assembly/disassembly at sites of DNA strand breaks in eukaryotic cells. Considerable advances have also been made in understanding the underlying molecular mechanisms for these events, with post-translational modifications of DDR factors being shown to play prominent roles in controlling the formation of foci in response to DNA-damaging agents. We review these regulatory mechanisms and discuss their biological significance to the DDR. PMID:21363960

  17. Structural basis of the tensile strength of protein complexes mediating cell adhesion

    Science.gov (United States)

    Bayas, Marco Vinicio

    This study explores the behaviour of adhesive complexes of cell adhesion molecules undergoing forced detachment. Molecular-forces measurements combined with Steered Molecular Dynamic (SMD) simulations were used to investigate the mechanical response of the CD2 C58 and hemophilic C-cadherin bonds. The CD2-CD58 adhesive complex, important for the adaptive immune response, contains several salt-bridges in the adhesive interface. SMD simulations showed that these inter-protein salt bridges contribute independently to the tensile strength of the complex. Consistent with this, force measurements with the Surface Force Apparatus (SFA) demonstrated that the elimination of single salt bridges weakens the bond. The corresponding loss in adhesion energy of the CD2-CD58 complex correlates with the importance of the salt bridges observed in the simulations. These findings correlate closely with the effect of the elimination of single salt bridges observed in cell aggregation assays and binding measurements. On the other hand, the hemophilic C-cadherin interaction determines specific cell-cell adhesion during development in Xenopus laevis . Single molecule force spectroscopy was used to characterize the multiple bound states between C-cadherin ectodomains. The experiments showed two short-lived bound states associated with the two outermost ectodomains and two long-lived states associated with the full ectodomain. It is likely that the two short-lived states are involved in the specificity of the interaction since previous studies showed that the corresponding states in E-cadherin have different lifetimes. In addition, SMD simulations of the forced dissociation of the strand dieter of C-cadherin suggested a mechanism for the specificity of cadherin interactions.

  18. Characterization of the in vitro binding and inhibition kinetics of primary amine oxidase/vascular adhesion protein-1 by glucosamine.

    LENUS (Irish Health Repository)

    Olivieri, Aldo

    2012-04-01

    Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium.

  19. Down regulation of NO signaling in Trypanosoma cruzi upon parasite-extracellular matrix interaction: changes in protein modification by nitrosylation and nitration.

    Directory of Open Access Journals (Sweden)

    Milton Pereira

    2015-04-01

    Full Text Available Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas' disease in humans, to components of the extracellular matrix (ECM is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling.Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins or by the biotin switch method (S-nitrosylated proteins and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation.For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.

  20. Protein Adhesion and Ion Substitution (on/in)to Minerals

    Science.gov (United States)

    Charlet, L.; Fernandez Martinez, A.; Chapron, Y.; Sahai, N.; Cuello, G.; Brendle, J.; Marichal, C.

    2008-12-01

    Arsenic and pathogenic prion protein-scrapie (PrPsc) are important contaminants which may soil and water for decades, unless they are removed by sorption. Two sorption mechanisms will be discussed, namely the organics (Prp and single aminoacid) adsorption on clay and the arsenic substitution in gypsum. The elucidation of these contrasted mechanisms will be shown to request complementary molecular-mechanical simulations with experimental spectroscopic investigations. As first example, structural studies performed at ILL/ESRF on As-doped gypsum (CaSO4 2H2O) using neutron and X-ray diffraction data and EXAFS were performed to determine how As fits into the bulk of gypsum structure. The combined Rietveld analysis of neutron and X-ray diffraction data shows an expansion of the unit cell volume proportional to the As concentration within the samples. to-sulfate substitution mechanisms were used as simulation starting hypotheses. DFT-based simulations (Mulliken analysis) were used to interpret charge distribution and to show that among the possible mechanisms, a sulphate substitution by either protonated, or fully deprotonated, arsenate ion, only the protonated arsenate substitution could best fit the EXAFS data. In the second example, we used Molecular Dynamics to understand the mechanism of strong binding of the pathogenic PrP peptide with clay mineral surfaces. We modeled only the infectious moiety, PrP92-138, of the whole PrPsc structure, with explicitly solvating water molecules in contact with the cleavage plane of pyrophillite, as a model for montmorillonite without any cationic substitution. Partial residual negative charges on the cleavage plane were balanced with K+ ions. The peptide anchored to the clay surface via up to 10 hydrogen bonds from lysine and histidine residues to oxygen atoms of the siloxane cavities, and a total adsorption energy of 3465 KJ.mol-1 was obtained. Our results were compared to the one obtained by chemical and thermal analysis, 23Na, 1H

  1. Application of tung oil to improve adhesion strength and water resistance of cottonseed meal and protein adhesives on maple veneer

    Science.gov (United States)

    Cottonseed meal-based products show promise in serving as environment-friendly wood adhesives. However, their practical utilization is currently limited due to low durability and water resistant properties. In this research, we tested the improvement of adhesion strength and water resistance of cott...

  2. Interrogating the Roles of Post-Translational Modifications of Non-Histone Proteins.

    Science.gov (United States)

    Buuh, Zakey Yusuf; Lyu, Zhigang; Wang, Rongsheng E

    2017-05-24

    Post-translational modifications (PTMs) allot versatility to the biological functions of highly conserved proteins. Recently, modifications to non-histone proteins such as methylation, acetylation, phosphorylation, glycosylation, ubiquitination, and many more have been linked to the regulation of pivotal pathways related to cellular response and stability. Due to the roles these dynamic modifications assume, their dysregulation has been associated with cancer and many other important diseases such as inflammatory disorders and neurodegenerative diseases. For this reason, we present a review and perspective on important post-translational modifications on non-histone proteins, with emphasis on their roles in diseases and small molecule inhibitors developed to target PTM writers. Certain PTMs' contribution to epigenetics has been extensively expounded; yet more efforts will be needed to systematically dissect their roles on non-histone proteins, especially for their relationships with nononcological diseases. Finally, current research approaches for PTM study will be discussed and compared, including limitations and possible improvements.

  3. Connection between markers of cholestasis and intensity of oxidative modification of proteins in patients with choledocholithiasis

    Directory of Open Access Journals (Sweden)

    Zoran Damnjanović

    2014-03-01

    Full Text Available The aim of this study was to examine the connection between cholestatic markers and the oxidative protein modification intensity in patients with choledocholithiasis. All the participants were subjected to clinical, laboratory and ultrasonic check-up at the Internal Department of the Military Hospital in Niš, Serbia. The parameters of oxidative stress: carbonyl groups, a measure of oxidative protein modification, and biochemical markers of cholestasis were determined by standard biochemical methods. The concentration of total (r=0.41, p<0.05, direct (r=0.49, p<+0.01 and indirect (r=0.41, p<0.05 bilirubin was in statistically significant positive linear correlation with the intensity of oxidative modification of proteins, while the other biochemical markers of cholestasis did not show such correlation. Total, direct and indirect bilirubins showed a significant positive correlation with oxidative protein modification, assessed through the levels of carbonyl groups in patients with choledocholithiasis.

  4. Cell Cycle Dynamics of Proteins and Post-translational Modifications Using Quantitative Immunofluorescence.

    Science.gov (United States)

    Akopyan, Karen; Lindqvist, Arne; Müllers, Erik

    2016-01-01

    Immunofluorescence can be a powerful tool to detect protein levels, intracellular localization, and post-translational modifications. However, standard immunofluorescence provides only a still picture and thus lacks temporal information. Here, we describe a method to extract temporal information from immunofluorescence images of fixed cells. In addition, we provide an optional protocol that uses micropatterns, which increases the accuracy of the method. These methods allow assessing how protein levels, intracellular localization, and post-translational modifications change through the cell cycle.

  5. Synaptic Cell Adhesion Proteins and Synaptogenesis in the Mammalian Central Nervous System

    Science.gov (United States)

    Brose, N.

    Synapses are asymmetric cell-cell contacts, typically formed between the presynaptic axon terminal of a "sending" nerve cell and the postsynaptic dendrite, the soma or - in some cases - the axon of a "receiving" one. The presynaptic axon terminal is specialized for the complex membrane trafficking mechanisms that underlie regulated secretion of neurotransmitter, while the postsynapse is uniquely specialized for signal transduction. Synaptogenesis, the formation of functional synapses, is the final step in the development of the central nervous system. In the mammalian brain it results in the establishment of a neural network, connecting some 1012 nerve cells with up to 1015 synapses. In principle, synaptogenesis takes place in two consecutive steps that are most likely mediated by cell adhesion molecules. First, an arriving axonal growth cone identifies its appropriate partner cell, creating an initial contact, and, second, specific axonal and dendritic protein components are recruited to this initial contact site, forming a functional synapse. Three cell adhesion systems have recently been shown to be specifically enriched at synaptic contacts: the cadherin/catenin system, the cadherinlike neuronal receptors, and the β-neurexin/neuroligin system. Components of all three cell adhesion systems have been localized to synaptic contacts using immunogold electron microscopy but are also present outside of synapses. The present short review discusses the possible role of these synaptic cell adhesion molecules in synaptogenesis.

  6. A comprehensive resource for integrating and displaying protein post-translational modifications

    Directory of Open Access Journals (Sweden)

    Wang Ting-Yuan

    2009-06-01

    Full Text Available Abstract Background Protein Post-Translational Modification (PTM plays an essential role in cellular control mechanisms that adjust protein physical and chemical properties, folding, conformation, stability and activity, thus also altering protein function. Findings dbPTM (version 1.0, which was developed previously, aimed on a comprehensive collection of protein post-translational modifications. In this update version (dbPTM2.0, we developed a PTM database towards an expert system of protein post-translational modifications. The database comprehensively collects experimental and predictive protein PTM sites. In addition, dbPTM2.0 was extended to a knowledge base comprising the modified sites, solvent accessibility of substrate, protein secondary and tertiary structures, protein domains, protein intrinsic disorder region, and protein variations. Moreover, this work compiles a benchmark to construct evaluation datasets for computational study to identifying PTM sites, such as phosphorylated sites, glycosylated sites, acetylated sites and methylated sites. Conclusion The current release not only provides the sequence-based information, but also annotates the structure-based information for protein post-translational modification. The interface is also designed to facilitate the access to the resource. This effective database is now freely accessible at http://dbPTM.mbc.nctu.edu.tw/.

  7. Mitogen-activated protein kinase (MAPK pathway regulates branching by remodeling epithelial cell adhesion.

    Directory of Open Access Journals (Sweden)

    Anneliis Ihermann-Hella

    2014-03-01

    Full Text Available Although the growth factor (GF signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.

  8. Posttranslational modifications of proteins in type 1 diabetes: the next step in finding the cure?

    Science.gov (United States)

    Dunne, Jessica L; Overbergh, Lut; Purcell, Anthony W; Mathieu, Chantal

    2012-08-01

    The overall role of modification of β-cell antigens in type 1 diabetes has not been elucidated and was the focus of a recent workshop on posttranslational modification of proteins in type 1 diabetes. The prevailing opinion of the workshop attendees was that novel insights into the mechanism of loss of immune tolerance might be gained and that novel diagnostic and therapeutic approaches could be developed for type 1 diabetes if protein modifications were shown to play a critical role in the disease.

  9. Analysis of posttranslational modifications of proteins by tandem mass spectrometry

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Trelle, Morten B; Thingholm, Tine E

    2006-01-01

    -temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful...... for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures....

  10. Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion

    Directory of Open Access Journals (Sweden)

    Pei-Wen Tsai

    2017-12-01

    Full Text Available Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis

  11. Stainless steel modified with poly(ethylene glycol) can prevent protein adsorption but not bacterial adhesion

    DEFF Research Database (Denmark)

    Wei, Jiang; Bagge, Dorthe; Gram, Lone

    2003-01-01

    The surface of AISI 316 grade stainless steel (SS) was modified with a layer of poly(ethylene glycol) (PEG) (molecular weight 5000) with the aim of preventing protein adsorption and bacterial adhesion. Model SS substrates were first modified to introduce a very high density of reactive amine grou...... adsorption is not a prerequisite stopping bacterial adhesion, and that other mechanisms most likely play a role. (C) 2003 Elsevier B.V. All rights reserved.......The surface of AISI 316 grade stainless steel (SS) was modified with a layer of poly(ethylene glycol) (PEG) (molecular weight 5000) with the aim of preventing protein adsorption and bacterial adhesion. Model SS substrates were first modified to introduce a very high density of reactive amine groups...... by the adsorption of branched poly(ethylenimine) (PEI) from water. Methoxy-terminated aldehyde-poly(ethylene glycol) (M-PEG-CHO) was then grafted onto the PEI layers using reductive amination at the lower critical solution temperature (LCST) of the PEG in order to optimize the graft density of the linear PEG chains...

  12. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1

    Science.gov (United States)

    Chang, Chuang-Rung; Blackstone, Craig

    2017-01-01

    Mitochondria in cells comprise a tubulovesicular network shaped continuously by complementary fission and fusion events. The mammalian Drp1 protein plays a key role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in the balance between these opposing processes can occur rapidly, indicating that modifications to these proteins may regulate mitochondrial membrane dynamics. We highlight posttranslational modifications of the mitochondrial fission protein Drp1, for which these regulatory mechanisms are best characterized. This dynamin-related GTPase undergoes a number of steps to mediate mitochondrial fission, including translocation from cytoplasm to the mitochondrial outer membrane, higher-order assembly into spirals, GTP hydrolysis associated with a conformational change and membrane deformation, and ultimately disassembly. Many of these steps may be influenced by covalent modification of Drp1. We discuss the dynamic nature of Drp1 modifications and how they contribute not only to the normal regulation of mitochondrial division, but also to neuropathologic processes. PMID:20649536

  13. Cucurbit[6]uril-Promoted Click Chemistry for Protein Modification.

    Science.gov (United States)

    Finbloom, Joel A; Han, Kenneth; Slack, Clancy C; Furst, Ariel L; Francis, Matthew B

    2017-07-19

    Azide-alkyne cycloaddition is a powerful reaction for the formation of bioconjugates. When catalyzed by Cu(I) or strain promotion, this cycloaddition is considered to be a "click" reaction with many applications in chemical biology and materials science. We report a new type of azide-alkyne click chemistry for the synthesis of protein conjugates using cucurbit[6]uril (CB6) supramolecular chemistry. CB6-promoted azide-alkyne cycloaddition has been previously used for the synthesis of rotaxanes but has not been applied to the development of complex bioconjugates. By developing new substrates for CB6 click that do not contain any cross-reactive functional groups and by optimizing reaction conditions, we converted CB6 click chemistry from a rotaxane synthesis tool into a useful bioconjugation technique. Using these new parameters, we synthesized a series of protein conjugates including protein-peptide, protein-DNA, protein-polymer, and protein-drug conjugates. We further demonstrated that CB6 click can be used in conjunction with strain-promoted azide-alkyne cycloaddition to generate distinct bioconjugates in protein mixtures. CB6 click is a promising new reaction for the development of protein conjugates and can be applied toward the synthesis of complex biomaterials for a wide range of applications.

  14. Double-Layer Surface Modification of Polyamide Denture Base Material by Functionalized Sol-Gel Based Silica for Adhesion Improvement.

    Science.gov (United States)

    Hafezeqoran, Ali; Koodaryan, Roodabeh

    2017-09-21

    Limited surface treatments have been proposed to improve the bond strength between autopolymerizing resin and polyamide denture base materials. Still, the bond strength of autopolymerizing resins to nylon polymer is not strong enough to repair the fractured denture effectively. This study aimed to introduce a novel method to improve the adhesion of autopolymerizing resin to polyamide polymer by a double layer deposition of sol-gel silica and N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (AE-APTMS). The silica sol was synthesized by acid-catalyzed hydrolysis of tetraethylorthosilicate (TEOS) as silica precursors. Polyamide specimens were dipped in TEOS-derived sol (TS group, n = 28), and exposed to ultraviolet (UV) light under O2 flow for 30 minutes. UV-treated specimens were immersed in AE-APTMS solution and left for 24 hours at room temperature. The other specimens were either immersed in AE-APTMS solution (AP group, n = 28) or left untreated (NT group, n = 28). Surface characterization was investigated by fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Two autopolymerizing resins (subgroups G and T, n = 14) were bonded to the specimens, thermocycled, and then tested for shear bond strength with a universal testing machine. Data were analyzed with one-way ANOVA followed by Tukey's HSD (α = 0.05). FTIR spectra of treated surfaces confirmed the chemical modification and appearance of functional groups on the polymer. One-way ANOVA revealed significant differences in shear bond strength among the study groups. Tukey's HSD showed that TST and TSG groups had significantly higher shear bond strength than control groups (p = 0.001 and p < 0.001, respectively). Moreover, bond strength values of APT were statistically significant compared to controls (p = 0.017). Amino functionalized TEOS-derived silica coating is a simple and cost-effective method for improving the bond strength between the autopolymerizing resin and polyamide denture

  15. Preferential adsorption of cell adhesive proteins from complex media on self-assembled monolayers and its effect on subsequent cell adhesion.

    Science.gov (United States)

    Arima, Yusuke; Iwata, Hiroo

    2015-10-01

    We examined the effect of surface chemistry on adsorption of fibronectin (Fn) and vitronectin (Vn) and subsequent cell adhesion, employing self-assembled monolayers (SAMs) of alkanethiols carrying terminal methyl (CH3), hydroxyl groups (OH), carboxylic acid (COOH), and amine (NH2). More Fn and Vn adsorbed to COOH- and NH2-SAMs than to CH3- and OH-SAMs from a mixture with bovine serum albumin (BSA) and from 2% fetal bovine serum. Adhesion of human umbilical vein endothelial cells (HUVECs) on CH3- and OH-SAMs preadsorbed with Fn and BSA decreased with decreasing adsorbed Fn; however, HUVECs adhered to COOH- and NH2-SAMs even in the presence of BSA at 1000-fold more than Fn in a mixture because of the preferential adsorption of Fn and/or displacement of preadsorbed BSA with Fn and Vn in a serum-containing medium. SAMs coated with a mixture of Vn and BSA exhibited adhesion of HUVECs regardless of surface functional groups. A well-organized focal adhesion complex and actin stress fibers were observed only for COOH- and NH2-SAMs when SAMs were preadsorbed with Vn and BSA. These results suggest that COOH- and NH2-SAMs allow for both cell adhesion and cell spreading because of the high density of cell-binding domains derived from adsorbed Vn. Adsorption of cell adhesive proteins including fibronectin (Fn) and vitronectin (Vn) plays an important role in cell adhesion to artificial materials. However, for the development of biomaterials that contact with biological fluids, it is important to understand adsorption of Fn and Vn in complex media containing many kinds of proteins. Here, we focused on adsorption of Fn and Vn from complex media including mixed solution with albumin and fetal bovine serum, and its role on cell adhesion using self-assembled monolayers (SAMs). Our result demonstrates that SAMs carrying carboxylic acid or amine allow for both cell adhesion and cell spreading because of preferentially adsorbed Vn. The result provides insights into surface design of

  16. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    Science.gov (United States)

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Expression profile of the entire family of Adhesion G protein-coupled receptors in mouse and rat

    Directory of Open Access Journals (Sweden)

    Ebendal Ted

    2008-04-01

    Full Text Available Abstract Background The Adhesion G protein-coupled receptors (GPCRs are membrane-bound receptors with long N termini. This family has 33 members in humans. Several Adhesion GPCRs are known to have important physiological functions in CNS development and immune system response mediated by large cell surface ligands. However, the majority of Adhesion GPCRs are still poorly studied orphans with unknown functions. Results In this study we performed the extensive tissue localization analysis of the entire Adhesion GPCR family in rat and mouse. By applying the quantitative real-time PCR technique we have produced comparable expression profile for each of the members in the Adhesion family. The results are compared with literature data and data from the Allen Brain Atlas project. Our results suggest that the majority of the Adhesion GPCRs are either expressed in the CNS or ubiquitously. In addition the Adhesion GPCRs from the same phylogenetic group have either predominant CNS or peripheral expression, although each of their expression profile is unique. Conclusion Our findings indicate that many of Adhesion GPCRs are expressed, and most probably, have function in CNS. The related Adhesion GPCRs are well conserved in their structure and interestingly have considerable overlap in their expression profiles, suggesting similarities among the physiological roles for members within many of the phylogenetically related clusters.

  18. Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco-adhesion in the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    Muriel Mercier-Bonin

    2017-11-01

    Full Text Available Food and probiotic bacteria, in particular lactic acid bacteria, are ingested in large amounts by humans and are part of the transient microbiota which is increasingly considered to be able to impact the resident microbiota and thus possibly the host health. The lactic acid bacterium Lactococcus lactis is extensively used in starter cultures to produce dairy fermented food. Also because of a generally recognized as safe status, L. lactis has been considered as a possible vehicle to deliver in vivo therapeutic molecules with anti-inflammatory properties in the gastrointestinal tract. One of the key factors that may favor health effects of beneficial bacteria to the host is their capacity to colonize transiently the gut, notably through close interactions with mucus, which covers and protects the intestinal epithelium. Several L. lactis strains have been shown to exhibit mucus-binding properties and bacterial surface proteins have been identified as key determinants of such capacity. In this review, we describe the different types of surface proteins found in L. lactis, with a special focus on mucus-binding proteins and pili. We also review the different approaches used to investigate the adhesion of L. lactis to mucus, and particularly to mucins, one of its major components, and we present how these approaches allowed revealing the role of surface proteins in muco-adhesion.

  19. Focal adhesion protein FAP52 self-associates through a sequence conserved among the members of the PCH family proteins.

    Science.gov (United States)

    Nikki, Marko; Meriläinen, Jari; Lehto, Veli-Pekka

    2002-05-21

    FAP52 is a recently described focal adhesion-associated protein. It is a member of an emerging PCH (pombe Cdc15 homology) family of proteins characterized by a common domain organization and involvement in actin cytoskeleton organization, cytokinesis, and vesicular trafficking. Using gel filtration, surface plasmon resonance, and native polyacrylamide gel electrophoresis analysis, combined with chemical cross-linking of both native and recombinant protein, we show that FAP52 self-associates in vitro and suggest that it occurs predominantly as a trimer also in vivo. Analysis of the various domains of FAP52 by surface plasmon resonance showed that the highly alpha-helical region in the N-terminal half of the protein provides the self-association interface. Overexpression of the oligomerization domain in cultured cells was accompanied by major alterations in cellular morphology, actin organization, and the structure of focal adhesions, suggesting that an orderly coming together of FAP52 molecules is crucial for a proper actin filament organization and cytoskeletal structure. Comparison of the primary structures shows that all of the members of the PCH family have, in their N-terminal halves, a similar, highly alpha-helical region, suggesting that they all have a capacity to self-associate.

  20. Modification of carbon nanotubes by amphiphilic glycosylated proteins.

    Science.gov (United States)

    Fang, W; Linder, M B; Laaksonen, P

    2018-02-15

    Precise organization of nanomaterials with functional biomolecules play a key role in many natural materials. In this work, single-walled carbon nanotubes were functionalized by a self-assembling amphiphilic protein that enabled their dispersion into nanofibrillated cellulose matrix. The protein contained a hydrophobic patch and a glycosylated domain and due to its dual functionality, it was able to assemble at the interface of the carbon nanotubes and the nanofibrillated cellulose and thus enhance the interactions between them. The electrical conductivity of the nanocellulose/carbon nanotube composites was improved by approximately 5-fold when the protein modified nanotubes where applied. Also improvement of the mechanical properties due to the proteins was observed. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Protein Modification by Dicarbonyl Molecular Species in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Wesley M. Williams

    2011-01-01

    Full Text Available Neurodegeneration results from abnormalities in cerebral metabolism and energy balance within neurons, astrocytes, microglia, or microvascular endothelial cells of the blood-brain barrier. In Alzheimer's disease, -amyloid is considered the primary contributor to neuropathology and neurodegeneration. It now is believed that certain systemic diseases, such as diabetes mellitus, can contribute to neurodegeneration through the effects of chronic hyperglycemia/insulin resistance resulting in protein glycation, oxidative stress and inflammation within susceptible brain regions. Here, we present an overview of research focusing on the role of protein glycation, oxidative stress, and inflammation in the neurodegenerative process. Of special interest in this paper is the effect of methylglyoxal (MGO, a cytotoxic byproduct of glucose metabolism, elevated in neurodegenerative disease, and diabetes mellitus, on cerebral protein function and oxidative stress. How MGO interacts with amino acid residues within -amyloid, and small peptides within the brain, is also discussed in terms of the affect on protein function.

  2. [New derivatives of azobenzene for the directed modification of proteins].

    Science.gov (United States)

    Khien, Le Tkhi; Shirling, B; Riazanova, A Iu; Zatsepin, T S; Volkov, E M; Kubareva, E A; Velichko, T I; Pingoud, A; Oretskaia, T S

    2009-01-01

    Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

  3. Influence of subtilisin on the adhesion of a marine bacterium which produces mainly proteins as extracellular polymers.

    Science.gov (United States)

    Leroy, C; Delbarre, C; Ghillebaert, F; Compere, C; Combes, D

    2008-09-01

    The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin. The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells. Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp. D41 adhesion and confirm the high antifouling potential of subtilisin. This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.

  4. Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

    Directory of Open Access Journals (Sweden)

    Allan Olspert

    2016-06-01

    Full Text Available Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV and murine norovirus (MNV. These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

  5. The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins.

    Science.gov (United States)

    Chaplin, M F

    1976-01-01

    A simple technique was developed for the specific reversible modification of guanidino groups in proteins involving reaction with ninhydrin. The extent of the reaction is easily determined non-destructively by spectrophotometric analysis. The reagent can also be used for the titration of sterically unhindered thiol groups in proteins. PMID:938493

  6. Structural modifications of serum transthyretin in rats during protein-energy malnutrition.

    Science.gov (United States)

    Henze, Andrea; Rohn, Sascha; Gericke, Beate; Raila, Jens; Schweigert, Florian J

    2008-10-01

    Transthyretin (TTR) is a sensitive marker of protein-energy malnutrition and changes in serum and expression levels during protein and energy deficiency are well described. However, little is known about structural modifications of TTR during protein and/or energy deprivation. Therefore, the aim of this study was to determine the effects of protein inadequacies on post-translational modifications of TTR. For this purpose, male Wistar rats were fed a diet with either casein or gelatine as sole protein source subsequent to a protein wash-out period. Changes in TTR serum levels as well as other markers of nutritional status as body weight, food consumption, total serum protein and serum RBP4 levels as well as antioxidative capacity were determined. Post-translational modifications of TTR were examined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) analysis. The rats from the gelatine group revealed a marked change in the post-translational modification pattern of TTR which was reflected by a significant elevation of sulfonated TTR and which was inversely correlated to the antioxidative capacity. Additionally, the elevation of sulfonated TTR was accompanied by a decrease in body weight and food consumption, low antioxidative capacity as well as a deprivation of serum TTR, RBP4 and total serum protein levels in the animals of the gelatine group. Protein-energy malnutrition leads therefore next to changes in TTR serum concentration, also to changes in the post-translational modification pattern of TTR. Such changes are probably induced by protein-energy malnutrition-driven oxidative stress and might be linked to alterations in protein function and stability.

  7. Intrinsic protein fluorescence assays for GEF, GAP and post-translational modifications of small GTPases.

    Science.gov (United States)

    Goody, Philip R

    2016-12-15

    Evidence and arguments are summarized that suggest that intrinsic (tryptophan) protein fluorescence provides an excellent and convenient signal for monitoring both GEF (guanine nucleotide exchange factor) and GAP (GTPase activating protein) activity of a large number of small GTPases. In addition, post-translational modifications of Rab proteins occurring in a region known to be a hot spot for such modifications also lead to fluorescence changes that can be accurately monitored in a time-dependent manner. It is suggested that intrinsic fluorescence should be the first method chosen for monitoring such reactions of tryptophan-containing small GTPases. Copyright © 2016. Published by Elsevier Inc.

  8. Multienzyme Modification of Hemp Protein for Functional Peptides Synthesis

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    Ranjana Das

    2015-01-01

    Full Text Available Functional foods and nutraceuticals are of special importance, particularly for their impact on human health and prevention of certain chronic diseases. Consequently, the production and properties of bioactive peptides have received an increasing scientific interest over past few years. Present work intends to compare the competence of metalloendopeptidases (“Protease N” and “Protease A” with papain for getting functional peptides from hemp seed meal, which is an obligatory waste of hemp fiber production industry. As a measure of the functional potential hemp protein hydrolysates were analyzed for their antiradical properties in DPPH system. “Protease N” modified protein hydrolysate exhibited comparatively superior radical scavenging activity in DPPH system. Overall findings represent the importance of “Protease N,” as endopeptidase in getting peptides of good antiradical properties from various protein sources.

  9. Small heat shock proteins in cellular adhesion and migration: evidence from Plasmodium genetics.

    Science.gov (United States)

    Montagna, Georgina N; Matuschewski, Kai; Buscaglia, Carlos A

    2012-01-01

    Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells.

  10. Antiadhesive Properties of Arabinogalactan Protein from Ribes nigrum Seeds against Bacterial Adhesion of Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Jutta Messing

    2014-03-01

    Full Text Available Fruit extracts from black currants (Ribes nigrum L. are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2 was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. 125I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects.

  11. Differential effect of actomyosin relaxation on the dynamic properties of focal adhesion proteins.

    Directory of Open Access Journals (Sweden)

    Irena Lavelin

    Full Text Available Treatment of cultured cells with inhibitors of actomyosin contractility induces rapid deterioration of stress fibers, and disassembly of the associated focal adhesions (FAs. In this study, we show that treatment with the Rho kinase inhibitor Y-27632, which blocks actomyosin contractility, induces disarray in the FA-associated actin bundles, followed by the differential dissociation of eight FA components from the adhesion sites. Live-cell microscopy indicated that the drug triggers rapid dissociation of VASP and zyxin from FAs (τ values of 7-8 min, followed by talin, paxillin and ILK (τ ~16 min, and then by FAK, vinculin and kindlin-2 (τ = 25-28 min. Examination of the molecular kinetics of the various FA constituents, using Fluorescence Recovery After Photobleaching (FRAP, in the absence of or following short-term treatment with the drug, revealed major changes in the kon and koff values of the different proteins tested, which are in close agreement with their differential dissociation rates from the adhesion sites. These findings indicate that mechanical, actomyosin-generated forces differentially regulate the molecular kinetics of individual FA-associated molecules, and thereby modulate FA composition and stability.

  12. Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

    Science.gov (United States)

    Messing, Jutta; Niehues, Michael; Shevtsova, Anna; Borén, Thomas; Hensel, Andreas

    2014-03-24

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²⁵I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects.

  13. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    Science.gov (United States)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  14. Recombinant Probiotic Expressing Listeria Adhesion Protein Attenuates Listeria monocytogenes Virulence In Vitro

    Science.gov (United States)

    Koo, Ok Kyung; Amalaradjou, Mary Anne Roshni; Bhunia, Arun K.

    2012-01-01

    Background Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection. Methodology/Principal Findings The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (LbpLAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to LbpLAP for 1, 4, 15, or 24 h significantly (Pmonocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, LbpLAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. LbpLAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, LbpLAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h. Conclusions/Significance Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, LbpLAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high

  15. Adipocyte protein modification by Krebs cycle intermediates and fumarate ester-derived succination.

    Science.gov (United States)

    Manuel, Allison M; Frizzell, Norma

    2013-11-01

    Protein succination, the non-enzymatic modification of cysteine residues by fumarate, is distinguishable from succinylation, an enzymatic reaction forming an amide bond between lysine residues and succinyl-CoA. Treatment of adipocytes with 30 mM glucose significantly increases protein succination with only a small change in succinylation. Protein succination may be significantly increased intracellularly after treatment with fumaric acid esters, however, the ester must be removed by saponification to permit 2SC-antibody detection of the fumarate adduct.

  16. The effect of surface modification on the retention strength of polyetheretherketone crowns adhesively bonded to dentin abutments.

    Science.gov (United States)

    Uhrenbacher, Julia; Schmidlin, Patrick R; Keul, Christine; Eichberger, Marlis; Roos, Malgorzata; Gernet, Wolfgang; Stawarczyk, Bogna

    2014-12-01

    The mechanical properties of polyetheretherketone (PEEK) make it an ideal material for fixed dental prostheses; however, insufficient information is available about the cementation of these restorations. The purpose of this study was to evaluate the retention strength of differently pretreated and conditioned PEEK crowns luted to dental abutments. Human teeth were prepared in a standardized manner, and PEEK crowns were milled (N=160, n=10 per group) and conditioned as follows: airborne-particle abrasion, sulfuric etching, piranha etching, and no conditioning. These groups were divided into adhesive systems: visio.link, Signum PEEK Bond, Ambarino P60, and no adhesive and luted to dentin abutments. After water storage (60 days) and thermocycling (5000 cycles, 5°C/55°C), the retention strength of the crowns was determined with a pull-off test, and failure types were classified. The data were analyzed with the Kruskal-Wallis, 1-way ANOVA, and χ(2) test (α=.05). Crowns that were unconditioned and piranha etched and/or adhesively untreated or pretreated with Ambarino P60 had the lowest retention strength. The highest values were found for the airborne-particle abrasion and sulfuric etched groups and/or crowns adhesively pretreated with Signum PEEK Bond and visio.link. Composite resin cement that remained on dentin was observed more frequently for unconditioned groups in combination with Ambarino P60 and no adhesive pretreatment. Mixed failure types were found more frequently in the airborne-particle abrasion group in combination with visio.link, Signum PEEK Bond, and no adhesive pretreatment, in the sulfuric acid etched group combined with Ambarino P60 and no adhesive pretreatment, and after the piranha acid pretreatment in combination with visio.link or Signum PEEK Bond. The adhesion of the tested PEEK crowns to dentin was satisfactory after treatment with airborne-particle abrasion or etching with sulfuric acid and/or when additional adhesive systems such as visio

  17. Post-Translational Protein Modifications of Rare and Unconventional Types: Implications in Functions and Diseases.

    Science.gov (United States)

    Basak, Sarmistha; Lu, Chunyu; Basak, Ajoy

    2016-01-01

    Protein post-translational modification (PTM) occurs following their biosynthesis and is a key cellular event that defines their ultimate functional properties. It is an important control mechanism for display of biological functions of proteins often in a profound manner. It may switch on or off a protein's function. Several studies have been conducted to understand their mechanisms, physiological pathways and functional properties. PTMs have been shown to alter structural, conformational and physicochemical properties of proteins. So far a variety of protein modifications have been detected in physiological systems. These involve covalent modifications of amino acids via their side chains, backbone peptide bonds and terminal moieties. Following PTM, proteins may become (a) pathologically toxic, (b) biologically active or inactive, (c) more or less susceptible to proteolytic processing, (d) increasingly/decreasingly bound to its partner protein/s, or (e) modified with altered protease activities. These changes may affect pathways linked to cell signaling/transduction, trafficking, storing, expression, binding and/or affinity. Any of these events may be linked to metabolic, growth and/or chronic dysfunctions with serious health consequences that may include cancer, cardiovascular disease, stroke, viral/bacterial/parasite infections, inflammation, thrombosis, diabetes; central nervous system related conditions. Some of the modifications are more prevalent physiologically and widely studied. However, in recent years additional PTMs have been described that are less common. These include glypiation, neddylayion, siderophorylation, sumoylation, AMPylation, Cholesteroylation and others which are also important. This manuscript provides a comprehensive review of these rare and unconventional types of protein modifications and their functional implications to health, metabolism and disease conditions.

  18. Early Lung Cancer Detection via Global Protein Modification Profiles

    Science.gov (United States)

    2013-12-01

    human papillomavirus . Arch. Pathol. Lab. Med. 127: 978-83. 2. Jin B, Robertson KD., (2013). DNA methyltransferases, DNA damage repair, and cancer., Adv...shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number...modalities in individual patients. Our methodology overcomes several limitations of current technologies by targeting ubiquitous proteins that control

  19. Chemical reporter for visualizing metabolic cross-talk between carbohydrate metabolism and protein modification.

    Science.gov (United States)

    Zaro, Balyn W; Chuh, Kelly N; Pratt, Matthew R

    2014-09-19

    Metabolic chemical reporters have been largely used to study posttranslational modifications. Generally, it was assumed that these reporters entered one biosynthetic pathway, resulting in labeling of one type of modification. However, because they are metabolized by cells before their addition onto proteins, metabolic chemical reporters potentially provide a unique opportunity to read-out on both modifications of interest and cellular metabolism. We report here the development of a metabolic chemical reporter 1-deoxy-N-pentynyl glucosamine (1-deoxy-GlcNAlk). This small-molecule cannot be incorporated into glycans; however, treatment of mammalian cells results in labeling of a variety proteins and enables their visualization and identification. Competition of this labeling with sodium acetate and an acetyltransferase inhibitor suggests that 1-deoxy-GlcNAlk can enter the protein acetylation pathway. These results demonstrate that metabolic chemical reporters have the potential to isolate and potentially discover cross-talk between metabolic pathways in living cells.

  20. Protein Nanosheet Mechanics Controls Cell Adhesion and Expansion on Low-Viscosity Liquids.

    Science.gov (United States)

    Kong, Dexu; Megone, William; Nguyen, Khai D Q; Di Cio, Stefania; Ramstedt, Madeleine; Gautrot, Julien E

    2018-02-13

    Adherent cell culture typically requires cell spreading at the surface of solid substrates to sustain the formation of stable focal adhesions and assembly of a contractile cytoskeleton. However, a few reports have demonstrated that cell culture is possible on liquid substrates such as silicone and fluorinated oils, even displaying very low viscosities (0.77 cSt). Such behavior is surprising as low viscosity liquids are thought to relax much too fast (adhesions (with lifetimes on the order of minutes to hours). Here we show that cell spreading and proliferation at the surface of low viscosity liquids are enabled by the self-assembly of mechanically strong protein nanosheets at these interfaces. We propose that this phenomenon results from the denaturation of globular proteins, such as albumin, in combination with the coupling of surfactant molecules to the resulting protein nanosheets. We use interfacial rheology and atomic force microscopy indentation to characterize the mechanical properties of protein nanosheets and associated liquid-liquid interfaces. We identify a direct relationship between interfacial mechanics and the association of surfactant molecules with proteins and polymers assembled at liquid-liquid interfaces. In addition, our data indicate that cells primarily sense in-plane mechanical properties of interfaces, rather than relying on surface tension to sustain spreading, as in the spreading of water striders. These findings demonstrate that bulk and nanoscale mechanical properties may be designed independently, to provide structure and regulate cell phenotype, therefore calling for a paradigm shift for the design of biomaterials in regenerative medicine.

  1. Parallel Control over Surface Charge and Wettability Using Polyelectrolyte Architecture: Effect on Protein Adsorption and Cell Adhesion.

    Science.gov (United States)

    Guo, Shanshan; Zhu, Xiaoying; Li, Min; Shi, Liya; Ong, June Lay Ting; Jańczewski, Dominik; Neoh, Koon Gee

    2016-11-09

    Surface charge and wettability, the two prominent physical factors governing protein adsorption and cell adhesion, have been extensively investigated in the literature. However, a comparison between these driving forces in terms of their independent and cooperative effects in affecting adhesion is rarely explored on a systematic and quantitative level. Herein, we formulate a protocol that features two-dimensional control over both surface charge and wettability with limited cross-parameter influence. This strategy is implemented by controlling both the polyion charge density in the layer-by-layer (LbL) assembly process and the polyion side-chain chemical structures. The 2D property matrix spans surface isoelectric points ranging from 5 to 9 and water contact angles from 35 to 70°, with other interferential factors (e.g., roughness) eliminated. The interplay between these two surface variables influences protein (bovine serum albumin, lysozyme) adsorption and 3T3 fibroblast cell adhesion. For proteins, we observe the presence of thresholds for surface wettability and electrostatic driving forces necessary to affect adhesion. Beyond these thresholds, the individual effects of electrostatic forces and wettability are observed. For fibroblast, both surface charge and wettability have an effect on its adhesion. The combined effects of positive charge and hydrophilicity lead to the highest cell adhesion, whereas negative charge and hydrophobicity lead to the lowest cell adhesion. Our design strategy can potentially form the basis for studying the distinct behaviors of electrostatic force or wettability driven interfacial phenomena and serve as a reference in future studies assessing protein adsorption and cell adhesion to surfaces with known charge and wettability within the property range studied here.

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

    DEFF Research Database (Denmark)

    Brown, Alan; Turner, Louise; Christoffersen, Stig

    2013-01-01

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria...

  3. Modifications of the organic and mineral fractions of dental tissues following conditioning by self-etching adhesives.

    Science.gov (United States)

    Dieng-Sarr, Farimata; Sharrock, Patrick; Dabsie, Firas; Grégoire, Geneviève

    2011-02-01

    Our objective was to analyse the acid strengths and concentrations in contemporary self-etch adhesives and test whether the adhesion/decalcification concept functions the same way for all products. The self-etching adhesives were dissolved in a 50% water-ethanol solvent, these were reacted with biological apatite (HA) in the form of powder of human dentine in order to quantify calcium release and study the reaction products as a function of acid strengths and concentrations. The four self-etching adhesives investigated were AdheSE One (Ivoclar Vivadent, Schaan, Liechtenstein), Adper Easy Bond (3M ESPE, St Paul, MN, USA), Optibond All-In-One (KERR, Orange, CA, USA), Xeno V (Dentsply De Trey, Konstanz, Germany). Acid concentrations were found to span the range from 1 to 2 mmol/l, and the acid dissociation constants varied between apparent pKa values of 3.4 and 4.2. The pH values changed with time from values near 2.8 to 3.6, confirming the buffering action of HA. The stronger acids dissolved more calcium ions but left less organic matter attached to the tissue particles. Thermogravimetric and infrared analysis demonstrated that the weaker acids tended to bind to HA surfaces and increased significantly the organic to mineral ratios of the powders. Self-etching adhesives can be differentiated and classified in two types: weak acids attach to the mineral phase and leach little calcium; strong acids bind to the calcium ions, demineralize more and tend to debond from the dentinal hard tissues by forming more soluble calcium salts. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Significance of Mitochondrial Protein Post-translational Modifications in Pathophysiology of Brain Injury.

    Science.gov (United States)

    Klimova, Nina; Long, Aaron; Kristian, Tibor

    2017-09-21

    Mitochondria are complex organelles that undergo constant fusion and fission in order to adapt to the ever-changing cellular environment. The fusion/fission proteins, localized in the inner and outer mitochondrial membrane, play critical roles under pathological conditions such as acute brain injury and neurodegenerative diseases. Post-translational modifications of these proteins tightly regulate their function and activity, ultimately impacting mitochondrial dynamics and their efficiency to generate ATP. The individual post-translational modifications that are known to affect mitochondrial dynamics include SUMOylation, ubiquitination, phosphorylation, S-nitrosylation, acetylation, O-linked N-acetyl-glucosamine glycosylation, ADP-ribosylation, and proteolytic cleavage. Under stress or pathologic conditions, several of these modifications are activated leading to a complex regulatory mechanism that shifts the state of the mitochondrial network. The main goal is to accommodate and adapt the cellular bioenergetics metabolism to the energetic demand of the new extra- and/or intracellular environment. Understanding the complex relationship between these modifications on fusion and fission proteins in particular pathologic stress or diseases can provide new promising therapeutic targets and treatment approaches. Here, we discuss the specific post-translational modifications of mitochondrial fusion/fission proteins under pathologic conditions and their impact on mitochondrial dynamics.

  5. Effect of interfacial serum proteins on melanoma cell adhesion to biodegradable poly(l-lactic acid) microspheres coated with hydroxyapatite.

    Science.gov (United States)

    Shinto, Hiroyuki; Hirata, Takuya; Fukasawa, Tomonori; Fujii, Syuji; Maeda, Hayata; Okada, Masahiro; Nakamura, Yoshinobu; Furuzono, Tsutomu

    2013-08-01

    We have measured the interaction forces between a murine melanoma cell and a poly(l-lactic acid) (PLLA) microsphere coated with/without hydroxyapatite (HAp) nanoparticles (i.e., an HAp/PLLA or a bare PLLA microsphere) in a serum-free culture medium, using atomic force microscopy (AFM) with colloid probe technique, in order to investigate how the HAp-nanoparticle coating as well as interfacial serum proteins influence the cell-microsphere adhesion. The cell adhesion force of the HAp/PLLA microspheres was 1.4-fold stronger than that of the bare PLLA microspheres. When the microspheres were pretreated with a culture medium supplemented with 10% fetal bovine serum, the cell adhesion force of the HAp/PLLA microspheres was increased by a factor of 2.1; in contrast, no change was observed in the cell adhesion force of the bare PLLA microspheres before/after the pretreatment. Indeed, the cell adhesion force of the HAp/PLLA was 2.8-fold larger than that of the bare PLLA after the pretreatment. Additionally, we have investigated the effect of interfacial serum proteins on the zeta potentials of these microspheres. On the basis of the obtained results, possible mechanism of cell adhesion to the HAp/PLLA and bare PLLA microspheres in the presence/absence of the interfacial serum proteins is discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry.

    Science.gov (United States)

    Souda, Puneet; Ryan, Christopher M; Cramer, William A; Whitelegge, Julian

    2011-12-01

    Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Siderophores and mussel foot proteins: the role of catechol, cations, and metal coordination in surface adhesion.

    Science.gov (United States)

    Maier, Greg P; Butler, Alison

    2017-07-01

    Metal coordination, hydrogen bonding, redox reactions, and covalent crosslinking are seemingly disparate chemical and physicochemical processes that are all accomplished in natural materials by the catechol functional group. This review focuses on the reactivity of catechols in tris-2,3-dihydroxybenzoyl-containing microbial siderophores and synthetic analogs, as well as Dopa-(3,4-dihydroxyphenylalanine)-containing mussel foot proteins that adhere to surfaces in aqueous conditions. Mussel foot proteins with a high content of Dopa and cationic amino acids, Lys and Arg, adhere strongly to mica, an aluminosilicate mineral, in aqueous conditions. The siderophore cyclic trichrysobactin, tris-(2,3-dihydroxybenzoyl-D-Lys-L-Ser) and related synthetic analogs in which the tri-Ser macrolactone is replaced by Tren, tris-(2-aminoethyl)amine, also adheres strongly to mica. Variation in the nature of the catechol and cationic groups in synthetic analogs reveals a synergism between the cationic amino acid and the catechol, required for strong aqueous adhesion. Autoxidation and iron(III)-catalyzed oxidation of 2,3-dihydroxy and 3,4-dihydroxy catechols are also considered. These siderophore analogs provide a platform to understand catechol interactions and reactivity on surfaces, which may ultimately improve the design of synthetic materials that address diverse challenges in medicine, materials science, as well as other disciplines, in which surface adhesion in aqueous conditions is important.

  8. Post-translational modifications of intermediate filament proteins: mechanisms and functions.

    Science.gov (United States)

    Snider, Natasha T; Omary, M Bishr

    2014-03-01

    Intermediate filaments (IFs) are cytoskeletal and nucleoskeletal structures that provide mechanical and stress-coping resilience to cells, contribute to subcellular and tissue-specific biological functions, and facilitate intracellular communication. IFs, including nuclear lamins and those in the cytoplasm (keratins, vimentin, desmin, neurofilaments and glial fibrillary acidic protein, among others), are functionally regulated by post-translational modifications (PTMs). Proteomic advances highlight the enormous complexity and regulatory potential of IF protein PTMs, which include phosphorylation, glycosylation, sumoylation, acetylation and prenylation, with novel modifications becoming increasingly appreciated. Future studies will need to characterize their on-off mechanisms, crosstalk and utility as biomarkers and targets for diseases involving the IF cytoskeleton.

  9. Dysbiosis may trigger autoimmune diseases via inappropriate posttranslational modification of host proteins

    Directory of Open Access Journals (Sweden)

    Aaron eLerner

    2016-02-01

    Full Text Available The gut ecosystem with myriads of microorganisms and the high concentration of immune system cells can be considered as a separate organ on its own. The balanced interaction between the host and microbial cells has been shaped during the long co-evolutionary process. In dysbiotic conditions, however, this balance is compromised and results in abnormal interaction between the host and microbiota. It is hypothesize here that the changed spectrum of microbial enzymes involved in posttranslational modification of proteins may contribute to the aberrant modification of host proteins thus generating autoimmune responses by the host, resulting in autoimmune diseases.

  10. Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

    Energy Technology Data Exchange (ETDEWEB)

    Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju; Kim, Hye-Lee; Park, Jong-Chul [Cellbiocontrol Laboratory, Department of Medical Engineering, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Jin Lee, Seung [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Bong-Jin; Wang, Kang-Kyun; Kim, Yong-Rok [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-Gu, Seoul 120-749 (Korea, Republic of)

    2013-08-21

    Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion and maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH{sub 2} (399.70 eV) was increased significantly and –N=CH (400.80 eV) and –NH{sub 3}{sup +} (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic.

  11. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region....... In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF...... recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs....

  12. Bacterial lipid modification of proteins requires appropriate secretory signals even for expression - implications for biogenesis and protein engineering.

    Science.gov (United States)

    Kumar, Subramani; Balamurali, M M; Sankaran, Krishnan

    2014-09-01

    Sec- and Tat-mediated bacterial lipid modification of proteins are important posttranslational processes owing to their vital roles in cellular functions, membrane targeting and biotechnological applications like ELISA, biosensor, adjuvant-free vaccines, liposomal drug delivery etc. However a better understanding of the tight coupling of secretory and lipid modification machineries and the processes associated will help unravel this essential biological event and utilize it for engineering applications. Further, there is a need for a systematic and convincing investigation into membrane targeting, solubilization and ease-of-purification of engineered lipoproteins to facilitate scientists in readily applying this new protein engineering tool. Therefore, in this study, we have investigated systematically recombinant expression, translocation, solubilization and purification of three White Spot Syndrome Viral (WSSV) proteins, ICP11, VP28 and VP281. Our study shows that the lipid modification and secretion processes are tightly coupled to the extent that mismatch between folding kinetics and signal sequence of target proteins could lead to transcriptional-translational uncoupling or aborted translation. The proteins expressed as lipoproteins through Tat-pathway were targeted to the inner membrane achieving considerable enrichment. These His-tagged proteins were then purified to apparent homogeneity in detergent-free form using single-step Immobilized Metal Affinity Chromatography. This study has interesting findings in lipoprotein biogenesis enhancing the scope of this unique post-translational protein engineering tool for obtaining pure detergent-free, membrane or hydrophobic surface-associating diagnostic targets and vaccine candidates for WSSV.

  13. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

    Science.gov (United States)

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

  14. Cholesterol Modification of Hedgehog Signaling Proteins in Animal Development

    Science.gov (United States)

    Porter, Jeffrey A.; Young, Keith E.; Beachy, Philip A.

    1996-10-01

    To determine the function of the pS2 trefoil protein, which is normally expressed in the gastric mucosa, the mouse pS2 (mpS2) gene was inactivated. The antral and pyloric gastric mucosa of mpS2-null mice was dysfunctional and exhibited severe hyperplasia and dysplasia. All homozygous mutant mice developed antropyloric adenoma, and 30 percent developed multifocal intraepithelial or intramucosal carcinomas. The small intestine was characterized by enlarged villi and an abnormal infiltrate of lymphoid cells. These results indicate that mpS2 is essential for normal differentiation of the antral and pyloric gastric mucosa and may function as a gastric-specific tumor suppressor gene.

  15. The effect of titanium implant surface modification on the dynamic process of initial microbial adhesion and biofilm formation

    NARCIS (Netherlands)

    Han, A.; Li, X.; Huang, B.; Tsoi, J.K.-H.; Matinlinna, J.P.; Chen, Z.; Deng, D.M.

    2016-01-01

    Purpose: The aim of the study was to investigate the dynamic process of biofilm adhesion on titanium implant with two surface treatments, either pickled (PT) or moderately roughened by sandblasting with large grits and acid-etched (SLA). Materials and methods: Two types of titanium disks with

  16. Importance of post-translational modifications on the function of key haemostatic proteins.

    Science.gov (United States)

    Karlaftis, Vasiliki; Perera, Sachin; Monagle, Paul; Ignjatovic, Vera

    2016-01-01

    Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.

  17. Polysialic acid modification of the synaptic cell adhesion molecule SynCAM 1 in human embryonic stem cell-derived oligodendrocyte precursor cells.

    Science.gov (United States)

    Werneburg, Sebastian; Buettner, Falk F R; Mühlenhoff, Martina; Hildebrandt, Herbert

    2015-05-01

    Oligodendrocyte precursor cells (OPCs) are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia) is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs. Copyright © 2015. Published by Elsevier B.V.

  18. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines*

    Science.gov (United States)

    Müller, Alexandra; Eller, Jakob; Albrecht, Frank; Prochnow, Pascal; Kuhlmann, Katja; Bandow, Julia Elisabeth; Slusarenko, Alan John

    2016-01-01

    Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines. PMID:27008862

  19. Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

    Science.gov (United States)

    Müller, Alexandra; Eller, Jakob; Albrecht, Frank; Prochnow, Pascal; Kuhlmann, Katja; Bandow, Julia Elisabeth; Slusarenko, Alan John; Leichert, Lars Ingo Ole

    2016-05-27

    Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Structural and functional characterization of the proteins responsible for N6-methyladenosine modification and recognition.

    Science.gov (United States)

    Liu, Ke; Ding, Yumin; Ye, Weiyuan; Liu, Yanli; Yang, Jihong; Liu, Jinlin; Qi, Chao

    2015-06-22

    More than 100 types of RNA modifications have been identified so far, which are involved in a variety of cellular processes. N6-methyladenosine (m6A), as one most abundant RNA modification, is found in several RNA species, and mainly located in the stop codons, long internal exons as well as 3'UTR. It was reported that m6A modification is preferred after G in the conserved sequence RRm6ACH (R = A/G and H = A/C/U). There are two families of enzymes responsible for maintaining the balance of m6A methylation: RNA methyltransferases and demethylases, which add and remove methyl marks from RNA, respectively. METTL3 complex, the m6A RNA methyltransferase, has been identified, and two kinds of demethylases are characterized thus far, including Fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5). Besides the "writers" and "erasers" for m6A, m6A specific recognizing protein, such as the YTH domain, also has attracted significant attention. Herein, we will focus on the recent progress in understanding biological/biochemical functions and structures of proteins responsible for the m6A RNA modification and recognition. Detailed analysis of these important proteins will guide us in designing target-specific small molecule chemical probes and inhibitors.

  1. Engineering adhesive proteins enhanced by self-assembling peptide and enzyme cross-linking

    OpenAIRE

    JAMIE JOSEPH CASTILLO

    2017-01-01

    Conventional adhesives efficiently work on dry surfaces but fail in the presence of water. Currently, there is a great need for adhesives in surgical procedures and underwater construction. Nature gives a hint on how to develop wet adhesives. Mussels survive harsh underwater conditions by secreting an adhesive which attaches to different surfaces. However, natural extraction was found uneconomical. Recombinant engineering approach is a promising alternative method for large-scale adhesive pro...

  2. Effect of anticoagulants on the protein corona-induced reduced drug carrier adhesion efficiency in human blood flow.

    Science.gov (United States)

    Sobczynski, Daniel J; Eniola-Adefeso, Omolola

    2017-01-15

    Plasma proteins rapidly coat the surfaces of particulate drug carriers to form a protein corona upon their injection into the bloodstream. The high presence of immunoglobulins in the corona formed on poly(lactic-co-glycolic acid) (PLGA) vascular-targeted carrier (VTC) surfaces was recently shown to negatively impact their adhesion to activated endothelial cells (aECs) in vitro. Here, we characterized the influence of anticoagulants, or their absence, on the binding efficiency of VTCs of various materials via modulation of their protein corona. Specifically, we evaluated the adhesion of PLGA, poly(lactic acid) (PLA), polycaprolactone (PCL), silica, and polystyrene VTCs to aECs in heparinized, citrated, and non-anticoagulated (serum and whole) blood flows relative to buffer control. Particle adhesion is substantially reduced in non-anticoagulated blood flows regardless of the material type while only moderate to minimal reduction is observed for VTCs in anticoagulant-containing blood flow depending on the anticoagulant and material type. The substantial reduction in VTC adhesion in blood flows was linked to a high presence of immunoglobulin-sized proteins in the VTC corona via SDS-PAGE analysis. Of all the materials evaluated, PLGA was the most sensitive to plasma protein effects while PCL was the most resistant, suggesting particle hydrophobicity is a critical component of the observed negative plasma protein effects. Overall, this work demonstrates that anticoagulant positively alters the effect of plasma proteins in prescribing VTC adhesion to aECs in human blood flow, which has implication in the use of in vitro blood flow assays for functional evaluation of VTCs for in vivo use. This study addresses the impact of anticoagulant on altering the extent of the previously observed protein corona-induced adhesion reduction of vascular-targeted drug carriers in human blood flows. Specifically, serum blood flow (no anticoagulant) magnifies the negative effect of the

  3. Prediction of human protein function from post-translational modifications and localization features

    DEFF Research Database (Denmark)

    Jensen, Lars Juhl; Gupta, Ramneek; Blom, Nikolaj

    2002-01-01

    We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from...... a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects...

  4. Post-Translational Modifications of Desulfovibrio vulgaris Hildenborough Sulfate Reduction Pathway Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gaucher, S.P.; Redding, A.M.; Mukhopadhyay, A.; Keasling, J.D.; Singh, A.K.

    2008-03-01

    Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications(PTMs). Although PTMs are a critical aspectof cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit(DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium

  5. Adhesion of nitrile rubber (NBR) to polyethylene terephthalate (PET) fabric. Part 1: PET surface modification by methylenediphenyl di-isocyanate (MDI)

    Energy Technology Data Exchange (ETDEWEB)

    Razavizadeh, Mahmoud; Jamshidi, Masoud, E-mail: mjamshidi@iust.ac.ir

    2016-01-01

    Graphical abstract: - Highlights: • Glutaric anhydride peroxide (GAP) was grafted on PET surface by UV irradiation method. Then MDI was attached to GAP on PET surface. • The fabric was vulcanized by nitrile rubber. • Peet test was performed after each stage of surface modification. • Curing temperature was increased and the tests were repeated. • Effect of MDI coating on PET without carboxylation was evaluated. Effect of vulcanizing temperature on this product was also studied. - Abstract: Fiber to rubber adhesion is an important subject in rubber composite industry. It is well known that surface physical, mechanical and chemical treatments are effective methods to improve interfacial bonding. Ultra violet (UV) light irradiation is an efficient method which is used to increase interfacial interactions. In this research UV assisted chemical modification of PET fabric was used to increase its bonding to nitrile rubber (NBR). NBR is perfect selection to produce fuel and oil resistant rubber parts but it has weak bonding to fabrics. For this purpose at first, the PET fabric was carboxylated under UV irradiation and then methylenediphenyl diisocyanate (MDI) was reacted and grafted to carboxylated PET. T-peel test was used to evaluate PET fabric to NBR bonding strength. Attenuated total reflectance-Fourier transform infrared spectroscopy (FTIR-AT) was used to assess surface modifications of the PET fabrics. The chemical composition of the PET surfaces before and after carboxylation and MDI grafting was investigated by X-ray photoelectron spectroscopy (XPS). It was found that at vulcanizing temperature of 150 °C, carboxylation in contrary to MDI grafting, improved considerably PET to NBR adhesion. Finally effect of curing temperature on PET to NBR bonding strength was determined. It was found that increasing vulcanizing temperature to 170 °C caused considerable improvement (about 134%) in bonding strength.

  6. The roles of post-translational modifications in the context of protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Guangyou Duan

    2015-02-01

    Full Text Available Among other effects, post-translational modifications (PTMs have been shown to exert their function via the modulation of protein-protein interactions. For twelve different main PTM-types and associated subtypes and across 9 diverse species, we investigated whether particular PTM-types are associated with proteins with specific and possibly "strategic" placements in the network of all protein interactions by determining informative network-theoretic properties. Proteins undergoing a PTM were observed to engage in more interactions and positioned in more central locations than non-PTM proteins. Among the twelve considered PTM-types, phosphorylated proteins were identified most consistently as being situated in central network locations and with the broadest interaction spectrum to proteins carrying other PTM-types, while glycosylated proteins are preferentially located at the network periphery. For the human interactome, proteins undergoing sumoylation or proteolytic cleavage were found with the most characteristic network properties. PTM-type-specific protein interaction network (PIN properties can be rationalized with regard to the function of the respective PTM-carrying proteins. For example, glycosylation sites were found enriched in proteins with plasma membrane localizations and transporter or receptor activity, which generally have fewer interacting partners. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic PIN properties. By integrating global protein interaction networks and specific PTMs, our study offers a novel approach to unraveling the role of PTMs in cellular processes.

  7. The roles of post-translational modifications in the context of protein interaction networks.

    Science.gov (United States)

    Duan, Guangyou; Walther, Dirk

    2015-02-01

    Among other effects, post-translational modifications (PTMs) have been shown to exert their function via the modulation of protein-protein interactions. For twelve different main PTM-types and associated subtypes and across 9 diverse species, we investigated whether particular PTM-types are associated with proteins with specific and possibly "strategic" placements in the network of all protein interactions by determining informative network-theoretic properties. Proteins undergoing a PTM were observed to engage in more interactions and positioned in more central locations than non-PTM proteins. Among the twelve considered PTM-types, phosphorylated proteins were identified most consistently as being situated in central network locations and with the broadest interaction spectrum to proteins carrying other PTM-types, while glycosylated proteins are preferentially located at the network periphery. For the human interactome, proteins undergoing sumoylation or proteolytic cleavage were found with the most characteristic network properties. PTM-type-specific protein interaction network (PIN) properties can be rationalized with regard to the function of the respective PTM-carrying proteins. For example, glycosylation sites were found enriched in proteins with plasma membrane localizations and transporter or receptor activity, which generally have fewer interacting partners. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic PIN properties. By integrating global protein interaction networks and specific PTMs, our study offers a novel approach to unraveling the role of PTMs in cellular processes.

  8. Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding.

    Science.gov (United States)

    Gassner, H L; Esco, M; Smithson, M W; Kurpakus, M A

    1997-04-01

    The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing. Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1. A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGF alpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing. Both TGF beta 1 and beta 2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex. However, in our model TGF beta isoforms did not have identical functions. TGF beta 2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGF beta 1. TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

  9. Site‐Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

    OpenAIRE

    Kuan, Seah Ling; Wang, Tao; Weil, Tanja

    2016-01-01

    Abstract The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site‐directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site‐selective modifications since most proteins contain disulfide bonds. In this Revie...

  10. Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications

    OpenAIRE

    Scott, Anthony; Song, Jing; Ewing, Rob; Wang, Zhenghe

    2014-01-01

    DNA methylation is an important epigenetic mechanism that ensures correct gene expression and maintains genetic stability. DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation during replication. Dysregulation of DNMT1 is implicated in a variety of diseases. DNMT1 protein stability is regulated via various post-translational modifications, such as acetylation and ubiquitination, but also through protein–protein interactions. These mechanisms ensure DNMT1 is pro...

  11. Protein Modification: A Proposed Mechanism for the Long-Term Pathogenesis of Traumatic Brain Injury

    Science.gov (United States)

    2015-06-04

    carbonylated protein, followed by immobilization on a membrane and immunoblotting for the second modification; and (3) investigation of specific...6 94. Murinson B, Guarnaccia J. 2008. Stiff-person syndrome with amphiphysin antibodies: Distinctive features of a rare disease. Neurology 71:1955

  12. Understanding Marine Mussel Adhesion

    Energy Technology Data Exchange (ETDEWEB)

    H. G. Silverman; F. F. Roberto

    2007-12-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are waterimpervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.

  13. Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

    Directory of Open Access Journals (Sweden)

    Revati eWani

    2014-10-01

    Full Text Available The perception of reactive oxygen species (ROS has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g. cancer. New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically-distinct alterations to the protein (e.g. sulfenic/sulfinic/sulfonic acid, disulfides. These post-translational modifications (PTM are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

  14. Effects of Heterologous tRNA Modifications on the Production of Proteins Containing Noncanonical Amino Acids

    Directory of Open Access Journals (Sweden)

    Ana Crnković

    2018-02-01

    Full Text Available Synthesis of proteins with noncanonical amino acids (ncAAs enables the creation of protein-based biomaterials with diverse new chemical properties that may be attractive for material science. Current methods for large-scale production of ncAA-containing proteins, frequently carried out in Escherichia coli, involve the use of orthogonal aminoacyl-tRNA synthetases (o-aaRSs and tRNAs (o-tRNAs. Although o-tRNAs are designed to be orthogonal to endogenous aaRSs, their orthogonality to the components of the E. coli metabolism remains largely unexplored. We systematically investigated how the E. coli tRNA modification machinery affects the efficiency and orthogonality of o-tRNASep used for production of proteins with the ncAA O-phosphoserine (Sep. The incorporation of Sep into a green fluorescent protein (GFP in 42 E. coli strains carrying deletions of single tRNA modification genes identified several genes that affect the o-tRNA activity. Deletion of cysteine desulfurase (iscS increased the yield of Sep-containing GFP more than eightfold, while overexpression of dimethylallyltransferase MiaA and pseudouridine synthase TruB improved the specificity of Sep incorporation. These results highlight the importance of tRNA modifications for the biosynthesis of proteins containing ncAAs, and provide a novel framework for optimization of o-tRNAs.

  15. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation.

    Directory of Open Access Journals (Sweden)

    Wenjun Deng

    2015-12-01

    Full Text Available Post-transcriptional modifications of transfer RNAs (tRNAs have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5 and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2 modifications catalyzed by tRNA methyltransferase 9 (Trm9 in tRNAArg(UCU and tRNAGlu(UUC and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

  16. Post-Translational Modifications of Cardiac Mitochondrial Proteins in Cardiovascular Disease: Not Lost in Translation.

    Science.gov (United States)

    Marquez, Jubert; Lee, Sung Ryul; Kim, Nari; Han, Jin

    2016-01-01

    Protein post-translational modifications (PTMs) are crucial in regulating cellular biology by playing key roles in processes such as the rapid on and off switching of signaling network and the regulation of enzymatic activities without affecting gene expressions. PTMs lead to conformational changes in the tertiary structure of protein and resultant regulation of protein function such as activation, inhibition, or signaling roles. PTMs such as phosphorylation, acetylation, and S-nitrosylation of specific sites in proteins have key roles in regulation of mitochondrial functions, thereby contributing to the progression to heart failure. Despite the extensive study of PTMs in mitochondrial proteins much remains unclear. Further research is yet to be undertaken to elucidate how changes in the proteins may lead to cardiovascular and metabolic disease progression in particular. We aimed to summarize the various types of PTMs that occur in mitochondrial proteins, which might be associated with heart failure. This study will increase the understanding of cardiovascular diseases through PTM.

  17. Platelet adhesion and plasma protein adsorption control of collagen surfaces by He{sup +} ion implantation

    Energy Technology Data Exchange (ETDEWEB)

    Kurotobi, K. E-mail: kurotobi@postman.riken.go.jp; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M

    2003-05-01

    He{sup +} ion implanted collagen-coated tubes with a fluence of 1 x 10{sup 14} ions/cm{sup 2} were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2}. Platelet adhesion (using platelet rich plasma) was inhibited on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Platelet activation with washed platelets was observed on untreated collagen and He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was inhibited with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He{sup +} ion implanted collagen over a fluence of 1 x 10{sup 13} ions/cm{sup 2}. On the 1 x 10{sup 14} ions/cm{sup 2} implanted collagen, no platelet activation was observed due to the influence of plasma proteins. >From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and that plasma protein adsorption took an important role repairing the graft surface.

  18. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    Science.gov (United States)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  19. Evaluation of Serum Vascular Adhesion Protein-1 as a Potential Biomarker in Thyroid Cancer

    Directory of Open Access Journals (Sweden)

    Zhigang Hu

    2016-01-01

    Full Text Available Vascular adhesion protein-1 (VAP-1 is a glycoprotein that mediates tissue-selective lymphocyte adhesion. The prognostic value of VAP-1 has been determined in gastric cancer. The aim of this study was to evaluate the changes and the predictive value of serum VAP-1 in patients with thyroid cancer. A total of 126 patients with thyroid nodules and 53 healthy controls participated in this study. The patients were further divided into subgroup 1 (69 cases with benign thyroid nodules and subgroup 2 (57 cases with thyroid cancer. Serum VAP-1 was measured by time-resolved immunofluorometric assay. Diagnostic value of presurgical VAP-1 for thyroid cancer was conducted by receiver operating characteristic (ROC curves. Serum levels of VAP-1 were significantly lower in thyroid cancer group than in healthy control and benign thyroid nodule groups. VAP-1 concentrations negatively correlated with serum thyroglobulin (Tg levels in thyroid cancer patients (r=-0.81; p<0.001. The optimum cut-off value of VAP-1 was 456.6 ng/mL with a 77.4% specificity and 66.7% sensitivity for thyroid cancer diagnosis. Serum VAP-1 decreased in thyroid cancer patients and VAP-1 could be a potential useful adjunct biomarker in the diagnosis of thyroid cancer.

  20. Lysine-Directed Post-translational Modifications of Tau Protein in Alzheimer's Disease and Related Tauopathies

    Directory of Open Access Journals (Sweden)

    Christiana Kontaxi

    2017-08-01

    Full Text Available Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.

  1. Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins

    Science.gov (United States)

    Raborn, Erinn S.; Jamerson, Melissa; Marciano-Cabral, Francine; Cabral, Guy A.

    2014-01-01

    Aims The aim of this study was to assess the effect of select cannabinoids on human immunodeficiency virus type 1 (HIV-1) transactivating (Tat) protein-enhanced monocyte-like cell adhesion to proteins of the extracellular matrix (ECM). Main Methods Collagen IV, laminin, or an ECM gel were used to construct extracellular matrix layers. Human U937 monocyte-like cells were exposed to Tat in the presence of Δ9-tetrahydrocannabinol (THC), CP55,940, and other select cannabinoids. Cell attachment to ECM proteins was assessed using an adhesion assay. Key findings THC and CP55,940 inhibited Tat-enhanced attachment of U937 cells to ECM proteins in a mode that was linked to the cannabinoid receptor type 2 (CB2R). The cannabinoid treatment of Tat-activated U937 cells was associated with altered β1-integrin expression and distribution of polymerized actin, suggesting a modality by which these cannabinoids inhibited adhesion to the ECM. Significance The blood-brain barrier (BBB) is a complex structure that is composed of cellular elements and an extracellular matrix (ECM). HIV-1 Tat promotes transmigration of monocytes across this barrier, a process that includes interaction with ECM proteins. The results indicate that cannabinoids that activate the CB2R inhibit the ECM adhesion process. Thus, this receptor has potential to serve as a therapeutic agent for ablating neuroinflammation associated with HIV-elicited influx of monocytes across the BBB. PMID:24742657

  2. Facile immobilization of heparin on bioabsorbable iron via mussel adhesive protein (MAPs

    Directory of Open Access Journals (Sweden)

    Xuchen Xu

    2014-10-01

    Full Text Available Motivated by adhesive proteins in mussels, strategies using dopamine to modified surface have become particularly attractive. In the present work, we developed a novel and convenient method to modify the biodegradable Fe plates with heparin. Iron was first treated by a facile one-step pH-induced polymerization of dopamine, and then a high density heparin was successfully grafted onto the surface via coupling with polydopamine (PDA active layer. Heparin immobilization contributed much longer blood clotting coagulation time than the pure Fe sample, and hence reduced the risk of thrombosis. Cell viability tests suggested that the heparin modified Fe plates were more favorable to the proliferation of ECV304 cells. In summary, the heparin modified Fe plates with good anti-thrombus properties and inhibiting the proliferation of VSMC cells provide great prospects for biodegradable iron.

  3. Protein Footprinting by the Combined Use of Reversible and Irreversible Lysine Modifications

    Science.gov (United States)

    Hanai, Ryo; Wang, James C.

    1994-12-01

    A two-step lysine-modification procedure has been devised to chemically footprint protein surfaces involved in macromolecular interactions. A protein tagged at one particular end, in the free state or in a complex, is first treated lightly with a reversible lysine-modifying reagent. The protein is then unfolded and treated extensively with an irreversible lysine reagent to block those lysines that did not react previously; next, the first lysine modification is reversed, and a lysine-specific endoproteinase is used to cleave the tagged polypeptide at the deblocked lysines. Separation of the proteolytic products by size and identification of the tagged fragments map the positions of these lysines. In this procedure, the reversible lysine reagent serves as the chemical footprinting agent, as cleavage of the polypeptide ensues only at the sites of reaction with this reagent. Lysines involved in macromolecular contacts are identified from differences in proteolytic patterns of the tagged protein when the first lysine modification is done with the protein in the free form and in a complex. Application of the method to vaccinia virus topoisomerase identifies a number of lysines that are involved in its binding to DNA.

  4. Oxidative modification of native protein residues using cerium(IV) ammonium nitrate.

    Science.gov (United States)

    Seim, Kristen L; Obermeyer, Allie C; Francis, Matthew B

    2011-10-26

    A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.

  5. E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan); Funahashi, Tohru; Shimomura, Iichiro [Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka (Japan); Kihara, Shinji, E-mail: skihara@sahs.med.osaka-u.ac.jp [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan)

    2016-02-05

    Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

  6. Impact of Enzymatic and Microbial Bioprocessing on Protein Modification and Nutritional Properties of Wheat Bran.

    Science.gov (United States)

    Arte, Elisa; Rizzello, Carlo G; Verni, Michela; Nordlund, Emilia; Katina, Kati; Coda, Rossana

    2015-10-07

    Besides providing dietary fiber, wheat bran is a recognized source of protein and is considered a very valuable substitute for other protein-rich sources in the food and feed industry. Nonetheless, several factors affect protein bioavailability, including bran's layered structure. This study showed the influence on the release and protein modification of wheat bran of different bioprocessing methods involving the activation of endogenous enzymes of bran, the addition of an enzyme mixture having carbohydrase activity, and microbial fermentation. Bioprocessing in acidic conditions significantly enhanced the solubilization of protein from wheat bran, reaching the highest value in the treatment where the sole endogenous protease activity was activated. Bioprocessing through controlled fermentation allowed a more intense proteolysis and strongly impacted the in vitro digestibility of proteins. The combined use of starter cultures and cell-wall-degrading enzymes was characterized by the highest increase of phytase activity and total phenols.

  7. PTM-SD: a database of structurally resolved and annotated posttranslational modifications in proteins.

    Science.gov (United States)

    Craveur, Pierrick; Rebehmed, Joseph; de Brevern, Alexandre G

    2014-01-01

    Posttranslational modifications (PTMs) define covalent and chemical modifications of protein residues. They play important roles in modulating various biological functions. Current PTM databases contain important sequence annotations but do not provide informative 3D structural resource about these modifications. Posttranslational modification structural database (PTM-SD) provides access to structurally solved modified residues, which are experimentally annotated as PTMs. It combines different PTM information and annotation gathered from other databases, e.g. Protein DataBank for the protein structures and dbPTM and PTMCuration for fine sequence annotation. PTM-SD gives an accurate detection of PTMs in structural data. PTM-SD can be browsed by PDB id, UniProt accession number, organism and classic PTM annotation. Advanced queries can also be performed, i.e. detailed PTM annotations, amino acid type, secondary structure, SCOP class classification, PDB chain length and number of PTMs by chain. Statistics and analyses can be computed on a selected dataset of PTMs. Each PTM entry is detailed in a dedicated page with information on the protein sequence, local conformation with secondary structure and Protein Blocks. PTM-SD gives valuable information on observed PTMs in protein 3D structure, which is of great interest for studying sequence-structure- function relationships at the light of PTMs, and could provide insights for comparative modeling and PTM predictions protocols. Database URL: PTM-SD can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/PTM-SD/. © The Author(s) 2014. Published by Oxford University Press.

  8. Removable Backbone Modification Method for the Chemical Synthesis of Membrane Proteins.

    Science.gov (United States)

    Li, Jia-Bin; Tang, Shan; Zheng, Ji-Shen; Tian, Chang-Lin; Liu, Lei

    2017-05-16

    Chemical synthesis can produce water-soluble globular proteins bearing specifically designed modifications. These synthetic molecules have been used to study the biological functions of proteins and to improve the pharmacological properties of protein drugs. However, the above advances notwithstanding, membrane proteins (MPs), which comprise 20-30% of all proteins in the proteomes of most eukaryotic cells, remain elusive with regard to chemical synthesis. This difficulty stems from the strong hydrophobic character of MPs, which can cause considerable handling issues during ligation, purification, and characterization steps. Considerable efforts have been made to improve the solubility of transmembrane peptides for chemical ligation. These methods can be classified into two main categories: the manipulation of external factors and chemical modification of the peptide. This Account summarizes our research advances in the development of chemical modification especially the two generations of removable backbone modification (RBM) strategy for the chemical synthesis of MPs. In the first RBM generation, we install a removable modification group at the backbone amide of Gly within the transmembrane peptides. In the second RBM generation, the RBM group can be installed into all primary amino acid residues. The second RBM strategy combines the activated intramolecular O-to-N acyl transfer reaction, in which a phenyl group remains unprotected during the coupling process, which can play a catalytic role to generate the activated phenyl ester to assist in the formation of amide. The key feature of the RBM group is its switchable stability in trifluoroacetic acid. The stability of these backbone amide N-modifications toward TFA can be modified by regulating the electronic effects of phenol groups. The free phenol group is acylated to survive the TFA deprotection step, while the acyl phenyl ester will be quantitatively hydrolyzed in a neutral aqueous solution, and the free

  9. Protein Modifiers Generally Provide Limited Improvement in Wood Bond Strength of Soy Flour Adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda Lorenz

    2013-01-01

    Soy flour adhesives using a polyamidoamine-epichlorohydrin (PAE) polymeric coreactant are used increasingly as wood adhesives for interior products. Although these adhesives give good performance, higher bond strength under wet conditions is desirable. Wet strength is important for accelerated tests involving the internal forces generated by the swelling of wood and...

  10. Effect of dispersion method and CNT loading on the quality and performance of nanocomposite soy protein/CNTs adhesive for wood application

    Science.gov (United States)

    Afolabi, Ayo Samuel; Oluwafolakemi Sadare, Olawumi; Olawale Daramola, Michael

    2016-09-01

    In this article the effect of dispersion method and carbon nanotubes (CNTs) loading on the quality and performance of a nanocomposite adhesive is reported. The nanocomposite soy protein isolate adhesive was successfully developed by incorporating CNTs into the soy protein isolate (SPI) for enhanced bond strength and water resistance. Dispersion methods, namely mechanical (shear) mixing and mechanical/sonication were employed to aid good dispersion and interfacial interaction between soy protein matrix and the carbon nanofillers during the preparation of the adhesive. The concentration of the CNT was varied from 0.1-0.7 wt% in the nanocomposite adhesive. The morphology and the surface chemistry of the adhesives were checked with SEM and FTIR, respectively. The shear strength of the developed adhesives was investigated according to European standard (EN-204) for interior wood application on a tensile testing machine. The morphological structure of the nanocomposite adhesive obtained from SEM images showed homogeneous dispersion of CNTs in SPI using the two dispersion methods; shear mixing and sonication/shear mixing. Fourier transform infrared spectra showed chemical functionalities and successful interaction between CNTs and SPI adhesive. Thermogravimetric profile of the adhesive samples showed that the newly developed nanocomposite adhesive was thermally stable at a temperature up to about 600 °C at a higher percentage loading of 0.5 wt% CNTs. The result showed that sonication method of dispersion of CNTs into the SPI adhesive had a higher shear strength compared to the mechanical method of dispersion both at dry and wet state.

  11. Construction of a cell-surface display system based on the N-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins.

    Science.gov (United States)

    Bao, S; Yu, S; Guo, X; Zhang, F; Sun, Y; Tan, L; Duan, Y; Lu, F; Qiu, X; Ding, C

    2015-07-01

    To construct and demonstrate a surface display system that could be used to identify mycoplasma adhesion proteins. Using the N-terminal domain of InaZ (InaZN) as the anchoring motif and the enhanced green fluorescent protein (EGFP) as the reporter, the surface display system pET-InaZN-EGFP was constructed. Then, the mgc2 gene which encodes an adhesin and the holB gene which encodes DNA polymerase III subunit delta' (nonadhesin, negative control) of Mycoplasma gallisepticum were cloned into the pET-InaZN-EGFP respectively. The fusion proteins were expressed in Escherichia coli BL21 (DE3). The distribution of the fusion proteins in E. coli cells was determined using SDS-PAGE followed by Western blotting, based on cell fractionation. Escherichia coli cell surface display of the fusion protein was confirmed by immunofluorescence microscopy. The results indicated that the fusion proteins were not only anchored to the outer membrane fraction but also were successfully displayed on the surface of E. coli cells. Adhesion analysis of E. coli harbouring InaZN-EGFP-mgc2 to host cells showed that the MGC2-positive E. coli cells can effectively adhere to the surfaces of DF-1 cells. A surface display system using the InaZN as the anchoring motif and EGFP as the reporter was developed to identify putative adhesins of mycoplasma. Results indicated that adhesion by the cytadhesin-like protein MGC2 of mycoplasma can be reproduced using this surface display system. This is the first construction of surface display system which could be used to identify the adhesion proteins of mycoplasma. The method developed in this study can even be used to select and identify the adhesion proteins of other pathogens. © 2015 The Society for Applied Microbiology.

  12. Surface modification of textile fibers and cords by plasma polymerization for improvement of adhesion to polymeric matrices

    Science.gov (United States)

    Luo, Shijian

    In this work, aramid and polyester fibers and cords were treated in a continuous or pulsed DC plasma containing organic monomer vapor in custom-built reactors. Characterization indicated that, with one monomer under different deposition conditions, plasma-polymerized films were formed on the surface of fibers and cords with a variety of properties. The films significantly improve the adhesion performance of fibers and cords to polymeric matrices. The pull-out forces of treated samples were increased up to 90% compared to untreated ones. The lower power/high pressure (LW/HP) condition consistently gave better pull-out results than high power/low pressure (HW/LP) condition, no matter what monomer was used. Pulsing the input power also affected the adhesion performance positively. The effect of plasma polymerization on fiber strength is minimal. Plasma-polymerized films were thoroughly characterized by a range of analytical techniques. All the plasma polymer films were found highly unsaturated. The films prepared in LW/HP conditions were more unsaturated and less crosslinked than those deposited in HW/LP conditions. Some pyrrole ring structures were maintained in the plasma-polymerized pyrrole (PPy) films, while more rings remained intact in the mild LW/HP conditions. During atmospheric exposure, oxidation was found in both PPy and PAc (plasma-polymerized acetylene) films. It finished quickly in PPy films but occurred gradually in PAc films. Thin film interferometry and nano-indentation test showed that films deposited in LW/HP conditions had lower hardness, modulus and density. Partial rupture of the plasma film on the fiber surface was found in SFM after the fiber was pulled out from epoxy matrix. Based on the extensive data and failure interface analysis, a mechanical penetrating network mechanism for adhesion improvement was proposed. Besides the effect of oxygen-containing functional groups on the plasma-polymerized films, their hardness, modulus and crosslinking

  13. Protein Interactions, Post-translational Modifications and Topologies in Human Cells*

    Science.gov (United States)

    Chavez, Juan D.; Weisbrod, Chad R.; Zheng, Chunxiang; Eng, Jimmy K.; Bruce, James E.

    2013-01-01

    The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Excitingly, these results demonstrate the applicability of cross-linking to make direct topological measurements on post-translationally modified proteins. The results presented here provide new details on the structures of known multi-protein complexes as well as evidence for new protein-protein interactions. PMID:23354917

  14. Ageing-induced solubility loss in milk protein concentrate powder: effect of protein conformational modifications and interactions with water.

    Science.gov (United States)

    Haque, Enamul; Bhandari, Bhesh R; Gidley, Michael J; Deeth, Hilton C; Whittaker, Andrew K

    2011-11-01

    Protein conformational modifications and water-protein interactions are two major factors believed to induce instability of protein and eventually affect the solubility of milk protein concentrate (MPC) powder. To test these hypotheses, MPC was stored at different water activities (a(w) 0.0-0.85) and temperatures (25 and 45 °C) for up to 12 weeks. Samples were examined periodically to determine solubility, change in protein conformation by Fourier transform infrared (FTIR) spectroscopy and water status (interaction of water with the protein molecule/surface) by measuring the transverse relaxation time (T(2) ) with proton nuclear magnetic resonance ((1) H NMR). The solubility of MPC decreased significantly with ageing and this process was enhanced by increasing water activity (a(w) ) and temperature. Minor changes in protein secondary structure were observed with FTIR which indicated some degree of unfolding of protein molecules. The NMR T(2) results indicated the presence of three distinct populations of water molecules and the proton signal intensity and T(2) values of proton fractions varied with storage condition (humidity) and ageing. Results suggest that protein/protein interactions may be initiated by unfolding of protein molecules that eventually affects solubility. Copyright © 2011 Society of Chemical Industry.

  15. Physicochemical modifications accompanying UV laser induced surface structures on poly(ethylene terephthalate) and their effect on adhesion of mesenchymal cells.

    Science.gov (United States)

    Rebollar, Esther; Pérez, Susana; Hernández, Margarita; Domingo, Concepción; Martín, Margarita; Ezquerra, Tiberio A; García-Ruiz, Josefa P; Castillejo, Marta

    2014-09-07

    This work reports on the formation of different types of structures on the surface of polymer films upon UV laser irradiation. Poly(ethylene terephthalate) was irradiated with nanosecond UV pulses at 193 and 266 nm. The polarization of the laser beam and the irradiation angle of incidence were varied, giving rise to laser induced surface structures with different shapes and periodicities. The irradiated surfaces were topographically characterized by atomic force microscopy and the chemical modifications induced by laser irradiation were inspected via micro-Raman and fluorescence spectroscopies. Contact angle measurements were performed with different liquids, and the results evaluated in terms of surface free energy components. Finally, in order to test the influence of surface properties for a potential application, the modified surfaces were used for mesenchymal stem cell culture assays and the effect of nanostructure and surface chemistry on cell adhesion was evaluated.

  16. The recognition of adsorbed and denatured proteins of different topographies by β2 integrins and effects on leukocyte adhesion and activation

    DEFF Research Database (Denmark)

    Brevig, T.; Holst, B.; Ademovic, Z.

    2005-01-01

    Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography...

  17. Mining Proteomic Data to Expose Protein Modifications in Methanosarcina mazei strain Gö1

    Directory of Open Access Journals (Sweden)

    Deborah eLeon

    2015-03-01

    Full Text Available Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to mine information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling. Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more.This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.

  18. Adsorption and adhesion of common serum proteins to nanotextured gallium nitride.

    Science.gov (United States)

    Bain, Lauren E; Hoffmann, Marc P; Bryan, Isaac; Collazo, Ramón; Ivanisevic, Albena

    2015-02-14

    As the broader effort towards device and material miniaturization progresses in all fields, it becomes increasingly important to understand the implications of working with functional structures that approach the size scale of molecules, particularly when considering biological systems. It is well known that thin films and nanostructures feature different optical, electrical, and mechanical properties from their bulk composites; however, interactions taking place at the interface between nanomaterials and their surroundings are less understood. Here, we explore interactions between common serum proteins - serum albumin, fibrinogen, and immunoglobulin G - and a nanotextured gallium nitride surface. Atomic force microscopy with a carboxyl-terminated colloid tip is used to probe the 'activity' of proteins adsorbed onto the surface, including both the accessibility of the terminal amine to the tip as well as the potential for protein extension. By evaluating the frequency of tip-protein interactions, we can establish differences in protein behaviour on the basis of both the surface roughness as well as morphology, providing an assessment of the role of surface texture in dictating protein-surface interactions. Unidirectional surface features - either the half-unit cell steppes of as-grown GaN or those produced by mechanical polishing - appear to promote protein accessibility, with a higher frequency of protein extension events taking place on these surfaces when compared with less ordered surface features. Development of a full understanding of the factors influencing surface-biomolecule interactions can pave the way for specific surface modification to tailor the bio-material interface, offering a new path for device optimization.

  19. Vienna-PTM web server: a toolkit for MD simulations of protein post-translational modifications.

    Science.gov (United States)

    Margreitter, Christian; Petrov, Drazen; Zagrovic, Bojan

    2013-07-01

    Post-translational modifications (PTMs) play a key role in numerous cellular processes by directly affecting structure, dynamics and interaction networks of target proteins. Despite their importance, our understanding of protein PTMs at the atomistic level is still largely incomplete. Molecular dynamics (MD) simulations, which provide high-resolution insight into biomolecular function and underlying mechanisms, are in principle ideally suited to tackle this problem. However, because of the challenges associated with the development of novel MD parameters and a general lack of suitable computational tools for incorporating PTMs in target protein structures, MD simulations of post-translationally modified proteins have historically lagged significantly behind the studies of unmodified proteins. Here, we present Vienna-PTM web server (http://vienna-ptm.univie.ac.at), a platform for automated introduction of PTMs of choice to protein 3D structures (PDB files) in a user-friendly visual environment. With 256 different enzymatic and non-enzymatic PTMs available, the server performs geometrically realistic introduction of modifications at sites of interests, as well as subsequent energy minimization. Finally, the server makes available force field parameters and input files needed to run MD simulations of modified proteins within the framework of the widely used GROMOS 54A7 and 45A3 force fields and GROMACS simulation package.

  20. Post-translational Modifications and Protein Quality Control in Motor Neuron and Polyglutamine Diseases.

    Science.gov (United States)

    Sambataro, Fabio; Pennuto, Maria

    2017-01-01

    Neurodegenerative diseases, including motor neuron and polyglutamine (polyQ) diseases, are a broad class of neurological disorders. These diseases are characterized by neuronal dysfunction and death, and by the accumulation of toxic aggregation-prone proteins in the forms of inclusions and micro-aggregates. Protein quality control is a cellular mechanism to reduce the burden of accumulation of misfolded proteins, a function that results from the coordinated actions of chaperones and degradation systems, such as the ubiquitin-proteasome system (UPS) and autophagy-lysosomal degradation system. The rate of turnover, aggregation and degradation of the disease-causing proteins is modulated by post-translational modifications (PTMs), such as phosphorylation, arginine methylation, palmitoylation, acetylation, SUMOylation, ubiquitination, and proteolytic cleavage. Here, we describe how PTMs of proteins linked to motor neuron and polyQ diseases can either enhance or suppress protein quality control check and protein aggregation and degradation. The identification of molecular strategies targeting these modifications may offer novel avenues for the treatment of these yet incurable diseases.

  1. Surface-modified nanoparticles as a new, versatile, and mechanically robust nonadhesive coating : Suppression of protein adsorption and bacterial adhesion

    NARCIS (Netherlands)

    Holmes, P. F.; Currie, E. P. K.; Thies, J. C.; van der Mei, H. C.; Busscher, H. J.; Norde, W.

    2009-01-01

    The synthesis of surface-modified silica nanoparticles, chemically grafted with acrylate and poly(ethylene glycol) (PEG) groups, and the ability of the resulting crosslinked coatings to inhibit protein adsorption and bacterial adhesion are explored. Water contact angles, nanoindentation, and atomic

  2. Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: Over-expression, purification and biophysical characterization

    NARCIS (Netherlands)

    Zeev-Ben-Mordehai, Tzviya; Paz, Aviv; Peleg, Yoav; Toker, Lilly; Wolf, Sharon G.; Rydberg, Edwin H.; Sussman, Joel L.; Silman, Israel

    Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote

  3. In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins

    Directory of Open Access Journals (Sweden)

    Giugliano Loreny

    2004-04-01

    Full Text Available Abstract Background Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries. These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes. Breastfeeding plays a very important role in protecting infants from intestinal infections. Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro. The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S. flexneri and S. sonnei. To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography. Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays. Results Our revealed showed that both adhesion and invasion of Shigella species were inhibited by low concentration of secretory immunoglobulin A, lactoferrin and free secretory component. This work also showed that, these proteins bind to superficial and whole-cell Shigella proteins. Conclusions Our findings suggest that human milk may act inhibiting adhesion and, consequently, invasion of Shigella, thereafter preventing shigellosis in infants.

  4. Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

    NARCIS (Netherlands)

    Xu, C.P.; Boks, N.P.; Vries, de J.; Kaper, H.J.; Norde, W.; Busscher, H.J.; Mei, van der H.C.

    2008-01-01

    Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn

  5. Improved protein-adsorption resistance of digital microfluidic device via surface coating and structure modification

    Science.gov (United States)

    Chen, Shun-Yuan; Hu, Heng-Cang; Yu, Chih-Sheng; Hu, Yi-Chiuen

    2007-12-01

    The surface of solid indium tin oxide (ITO) glass supports for samples in electrowetting system needs to be protein-resistant. Since Teflon is the most conventional coating material used to improve the contact angle between the glass and solvent, it still unable to prevent nonspecific proteins adsorption absolutely under the applied potential. In this paper, we described a feasible method that could minimize non-specific proteins adsorption most probably during droplet processing. A regular micro-scale structure was patterned by photolithography, and dielectric layer was covered on the electrodes. Finally a thin layer of Sigmacoat® was coated by physical vapor deposition. The surface characteristic of our chip was analyzed by atomic force microscopy and Contact Angle Analyzer. We found that the adhesion of bio-molecule was efficiently decreased by this modified processing, and could prevent electrolysis more efficiently.

  6. Post-translational modifications in mitochondria: protein signaling in the powerhouse.

    Science.gov (United States)

    Stram, Amanda R; Payne, R Mark

    2016-11-01

    There is an intimate interplay between cellular metabolism and the pathophysiology of disease. Mitochondria are essential to maintaining and regulating metabolic function of cells and organs. Mitochondrial dysfunction is implicated in diverse diseases, such as cardiovascular disease, diabetes and metabolic syndrome, neurodegeneration, cancer, and aging. Multiple reversible post-translational protein modifications are located in the mitochondria that are responsive to nutrient availability and redox conditions, and which can act in protein-protein interactions to modify diverse mitochondrial functions. Included in this are physiologic redox signaling via reactive oxygen and nitrogen species, phosphorylation, O-GlcNAcylation, acetylation, and succinylation, among others. With the advent of mass proteomic screening techniques, there has been a vast increase in the array of known mitochondrial post-translational modifications and their protein targets. The functional significance of these processes in disease etiology, and the pathologic response to their disruption, are still under investigation. However, many of these reversible modifications act as regulatory mechanisms in mitochondria and show promise for mitochondrial-targeted therapeutic strategies. This review addresses the current knowledge of post-translational processing and signaling mechanisms in mitochondria, and their implications in health and disease.

  7. Immunolocalization of specific keratin associated beta-proteins (beta-keratins) in the adhesive setae of Gekko gecko.

    Science.gov (United States)

    Alibardi, Lorenzo

    2013-08-01

    The previous identification of 21 proteins in the digital setae transcriptome of Gekko gecko, 2 alpha-keratins of 52-53kDa and 19 beta-proteins (beta-keratins) of 10-21kDa, has indicated that most of setal corneous proteins are cysteine-rich. The production of specific antibodies for two of the main beta-protein subfamilies expressed in gecko setae has allowed the ultrastructural localization of two beta-proteins indicated as Ge-cprp-9 (cysteine-rich) and Ge-gprp-6 (glycine-rich). Only Ge-cprp-9, representing most of the 16 cysteine-rich beta-proteins, is present in the oberhautchen, setae and in the terminal spatula where adhesion takes place, supporting the previous expression study. Instead, the glycine-rich beta-proteins (Ge-gprp-6), representing the 3 glycine-rich beta-proteins of digital epidermis is only present in the stiff beta-layer of the digital scales and in the thin beta layer of the pad lamella sustaining the setae. Ge-cprp-9 is representative for most of the remaining 15 cys-rich proteins (Ge-cprp 1-16) and may have a structural and functional role in the process of adhesion. Most of the cysteine-rich setal proteins have a net positive charge and it is here hypothesized that these proteins may induce the formation of dipoles at the surface interface between the spatula and the substrate, enhancing the van der Waals forces and therefore adhesion to the substrate. The selection and improvement of these proteins during the evolution of geckos may have represented a successful factor for the survival and ecological adaptations of these climbing lizards. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

    Science.gov (United States)

    Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

    2012-03-01

    Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

  9. B-cell receptor-associated protein 31 regulates human embryonic stem cell adhesion, stemness, and survival via control of epithelial cell adhesion molecule.

    Science.gov (United States)

    Kim, Won-Tae; Seo Choi, Hong; Min Lee, Hyun; Jang, Young-Joo; Ryu, Chun Jeih

    2014-10-01

    B-Cell receptor-associated protein 31 (BAP31) regulates the export of secreted membrane proteins from the endoplasmic reticulum (ER) to the downstream secretory pathway. Previously, we generated a monoclonal antibody 297-D4 against the surface molecule on undifferentiated human embryonic stem cells (hESCs). Here, we found that 297-D4 antigen was localized to pluripotent hESCs and downregulated during early differentiation of hESCs and identified that the antigen target of 297-D4 was BAP31 on the hESC-surface. To investigate the functional role of BAP31 in hESCs, BAP31 expression was knocked down by small interfering RNA. BAP31 depletion impaired hESC self-renewal and pluripotency and drove hESC differentiation into multicell lineages. BAP31 depletion hindered hESC proliferation by arresting cell cycle at G0/G1 phase and inducing caspase-independent cell death. Interestingly, BAP31 depletion reduced hESC adhesion to extracellular matrix (ECM). Analysis of cell surface molecules showed decreased expression of epithelial cell adhesion molecule (EpCAM) in BAP31-depleted hESCs, while ectopic expression of BAP31 elevated the expression of EpCAM. EpCAM depletion also reduced hESC adhesion to ECM, arrested cell cycle at G0/G1 phase and induced cell death, producing similar effects to those of BAP31 depletion. BAP31 and EpCAM were physically associated and colocalized at the ER and cell surface. Both BAP31 and EpCAM depletion decreased cyclin D1 and E expression and suppressed PI3K/Akt signaling, suggesting that BAP31 regulates hESC stemness and survival via control of EpCAM expression. These findings provide, for the first time, mechanistic insights into how BAP31 regulates hESC stemness and survival via control of EpCAM expression. © 2014 AlphaMed Press.

  10. Overview of xeroderma pigmentosum proteins architecture, mutations and post-translational modifications.

    Science.gov (United States)

    Feltes, Bruno César; Bonatto, Diego

    2015-01-01

    The xeroderma pigmentosum complementation group proteins (XPs), which include XPA through XPG, play a critical role in coordinating and promoting global genome and transcription-coupled nucleotide excision repair (GG-NER and TC-NER, respectively) pathways in eukaryotic cells. GG-NER and TC-NER are both required for the repair of bulky DNA lesions, such as those induced by UV radiation. Mutations in genes that encode XPs lead to the clinical condition xeroderma pigmentosum (XP). Although the roles of XPs in the GG-NER/TC-NER subpathways have been extensively studied, complete knowledge of their three-dimensional structure is only beginning to emerge. Hence, this review aims to summarize the current knowledge of mapped mutations and other structural information on XP proteins that influence their function and protein-protein interactions. We also review the possible post-translational modifications for each protein and the impact of these modifications on XP protein functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. PTMOracle: A Cytoscape App for Covisualizing and Coanalyzing Post-Translational Modifications in Protein Interaction Networks.

    Science.gov (United States)

    Tay, Aidan P; Pang, Chi Nam Ignatius; Winter, Daniel L; Wilkins, Marc R

    2017-05-05

    Post-translational modifications of proteins (PTMs) act as key regulators of protein activity and of protein-protein interactions (PPIs). To date, it has been difficult to comprehensively explore functional links between PTMs and PPIs. To address this, we developed PTMOracle, a Cytoscape app for coanalyzing PTMs within PPI networks. PTMOracle also allows extensive data to be integrated and coanalyzed with PPI networks, allowing the role of domains, motifs, and disordered regions to be considered. For proteins of interest, or a whole proteome, PTMOracle can generate network visualizations to reveal complex PTM-associated relationships. This is assisted by OraclePainter for coloring proteins by modifications, OracleTools for network analytics, and OracleResults for exploring tabulated findings. To illustrate the use of PTMOracle, we investigate PTM-associated relationships and their role in PPIs in four case studies. In the yeast interactome and its rich set of PTMs, we construct and explore histone-associated and domain-domain interaction networks and show how integrative approaches can predict kinases involved in phosphodegrons. In the human interactome, a phosphotyrosine-associated network is analyzed but highlights the sparse nature of human PPI networks and lack of PTM-associated data. PTMOracle is open source and available at the Cytoscape app store: http://apps.cytoscape.org/apps/ptmoracle .

  12. Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: effect on HMG-CoA synthase.

    Science.gov (United States)

    Andringa, Kelly K; Bajt, Mary Lynn; Jaeschke, Hartmut; Bailey, Shannon M

    2008-04-01

    Acetaminophen (APAP) overdose is the leading cause of drug related liver failure in many countries. N-acetyl-p-benzoquinone imine (NAPQI) is a reactive metabolite that is formed by the metabolism of APAP. NAPQI preferentially binds to glutathione and then cellular proteins. NAPQI binding is considered an upstream event in the pathophysiology, especially when binding to mitochondrial proteins and therefore leads to mitochondrial toxicity. APAP caused a significant increase in liver toxicity 3h post-APAP administration as measured by increased serum alanine aminotransferase (ALT) levels. Using high-resolution mitochondrial proteomics techniques to measure thiol and protein changes, no significant change in global thiol levels was observed. However, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMG-CoA synthase) had significantly decreased levels of reduced thiols and activity after APAP treatment. HMG-CoA synthase is a key regulatory enzyme in ketogenesis and possesses a number of critical cysteines in the active site. Similarly, catalase, a key enzyme in hydrogen peroxide metabolism, also showed modification in protein thiol content. These data indicate post-translational modifications of a few selected proteins involved in mitochondrial and cellular regulation of metabolism during liver toxicity after APAP overdose. The pathophysiological relevance of these limited changes in protein thiols remains to be investigated.

  13. Surface modification of argon/oxygen plasma treated vulcanized ethylene propylene diene polymethylene surfaces for improved adhesion with natural rubber

    Energy Technology Data Exchange (ETDEWEB)

    Basak, Ganesh C. [Rubber Technology Centre, Indian Institute of Technology, Kharagpur 721302 (India); Bandyopadhyay, Abhijit [Department of Polymer Science and Technology, University of Calcutta, Calcutta 700 009 (India); Neogi, Sudarsan [Department of Chemical Engineering, Indian Institute of Technology, Kharagpur 721302 (India); Bhowmick, Anil K., E-mail: anilkb@rtc.iitkgp.ernet.in [Rubber Technology Centre, Indian Institute of Technology, Kharagpur 721302 (India)

    2011-01-15

    Vulcanized ethylene propylene diene polymethylene (EPDM) rubber surface was treated in a radio frequency capacitatively coupled low pressure argon/oxygen plasma to improve adhesion with compounded natural rubber (NR) during co-vulcanization. The plasma modified surfaces were analyzed by means of contact angle measurement, surface energy, attenuated total reflection-infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, energy dispersive X-ray sulfur mapping and atomic force microscopy. Several experimental variables such as plasma power, length of exposure time and composition of the argon-oxygen gas mixture were considered. It was delineated that plasma treatment changed both surface composition and roughness, and consequently increased peel strength. The change in surface composition was mainly ascribed to the formation of C-O and -C=O functional groups on the vulcanized surfaces. A maximum of 98% improvement in peel strength was observed after plasma treatment.

  14. Surface modification of argon/oxygen plasma treated vulcanized ethylene propylene diene polymethylene surfaces for improved adhesion with natural rubber

    Science.gov (United States)

    Basak, Ganesh C.; Bandyopadhyay, Abhijit; Neogi, Sudarsan; Bhowmick, Anil K.

    2011-01-01

    Vulcanized ethylene propylene diene polymethylene (EPDM) rubber surface was treated in a radio frequency capacitatively coupled low pressure argon/oxygen plasma to improve adhesion with compounded natural rubber (NR) during co-vulcanization. The plasma modified surfaces were analyzed by means of contact angle measurement, surface energy, attenuated total reflection-infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, energy dispersive X-ray sulfur mapping and atomic force microscopy. Several experimental variables such as plasma power, length of exposure time and composition of the argon-oxygen gas mixture were considered. It was delineated that plasma treatment changed both surface composition and roughness, and consequently increased peel strength. The change in surface composition was mainly ascribed to the formation of C-O and -Cdbnd O functional groups on the vulcanized surfaces. A maximum of 98% improvement in peel strength was observed after plasma treatment.

  15. Non-Specific Protein Modifications by a Phytochemical Induce Heat Shock Response for Self-Defense

    Science.gov (United States)

    Ohnishi, Kohta; Ohkura, Shinya; Nakahata, Erina; Ishisaka, Akari; Kawai, Yoshichika; Terao, Junji; Mori, Taiki; Ishii, Takeshi; Nakayama, Tsutomu; Kioka, Noriyuki; Matsumoto, Shinya; Ikeda, Yasutaka; Akiyama, Minoru; Irie, Kazuhiro; Murakami, Akira

    2013-01-01

    Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities. PMID:23536805

  16. Protein CoAlation: a redox-linked post-translational modification.

    Science.gov (United States)

    Ley, Steven C; de Carvalho, Luiz Pedro S

    2017-08-10

    Regulation of metabolic pathways by signal transduction and transcriptional cascades can alter cellular levels of metabolites. Metabolites themselves can also have regulatory activity as shown in a new study published in the Biochemical Journal Tsuchiya et al. describe a novel antibody and mass spectrometry-based method for identifying proteins that are reversibly modified with Coenzyme A (CoA). Analysis of the 'CoAlated proteome' under conditions of oxidative and metabolic stress revealed a bias towards the modification of metabolic enzymes by CoA. Furthermore, CoAlation was shown to alter the activity of target proteins. These results suggest that CoAlation is a widespread post-translational modification that may have important roles in the metabolic response to stress. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  17. Cloning and sequencing of adhesion protein gene of Trichomonas gallinae from pigeon.

    Science.gov (United States)

    Luo, F; Li, G Q; Su, R Q; Liang, G; Chen, Z H; Hicham, W

    2010-02-26

    The adhesion protein (AP) gene of Trichomonas gallinae from pigeon was cloned and sequenced. The first-strand cDNA of the AP gene of T. gallinae from pigeon was amplified by reverse transcription polymerase chain reaction (RT-PCR) with total RNA extracting kit and cloned in the vector pMD18-T. The recombinant plasmid was identified by PCR and restriction endonuclease, and the positive clone was sequenced and analysed by comparing the sequence similarity with other sequences in the GenBank. The AP gene of T. gallinae had a length of 1032bp, which contained a complete open reading frame (ORF) of 930bp long, coding for 309 amino acids. The sequence analysis revealed that the homology with three AP genes of Trichomonas vaginalis (i.e., TVU87096, TVU87097 and TVU87098) was 94.2%, 92.6% and 92.0%, respectively. It is concluded that the successfully cloned AP gene from T. gallinae will provide the basis for the expression of the AP gene in prokaryotic and eukaryotic cells and the preparation of its recombinant protein.

  18. Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes.

    Science.gov (United States)

    Bacakova, Marketa; Lopot, Frantisek; Hadraba, Daniel; Varga, Marian; Zaloudkova, Margit; Stranska, Denisa; Suchy, Tomas; Bacakova, Lucie

    2015-01-01

    It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  19. Proteinaceous determinants of surface colonization in bacteria: Bacterial adhesion and biofilm formation from a protein secretion perspective

    Directory of Open Access Journals (Sweden)

    Caroline eCHAGNOT

    2013-10-01

    Full Text Available Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative, monoderm (archetypal Gram-positive and diderm-mycolate (archetypal acid-fast bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field.

  20. Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Lerche, Mathilde H.; Poulsen, Flemming Martin

    2001-01-01

    the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type...... in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found...

  1. Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Braga, Thiago Verano; Roepstorff, Peter

    2015-01-01

    The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines...... for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis...

  2. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    Science.gov (United States)

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. © 2011 Blackwell Publishing Ltd.

  3. Post-translational modifications of host proteins by Legionella pneumophila: a sophisticated survival strategy.

    Science.gov (United States)

    Rolando, Monica; Buchrieser, Carmen

    2012-03-01

    Eukaryotic proteins are tightly regulated by post-translational modifications, leading to a very subtle degree of regulation in time and space. Pathogen-mediated post-translational modifications are key strategies to modulate host factors by targeting central signaling pathways in the host cell. Legionella pneumophila, an intracellular pathogen that coevolved with protozoan hosts, encodes a large arsenal of secreted effectors conferring the ability to evade host cellular defenses and to manipulate them to promote invasion and intracellular replication. Conservation of many signaling pathways of protozoa in human macrophages confers the ability of L. pneumophila to infect humans, causing a severe pneumonia called legionnaires' disease. Most of the secreted proteins are delivered by the Dot/Icm type IV secretion system and several of these have been shown to act on different cellular pathways critical for infection. Moreover, multiple effectors target a single host function to orchestrate bacterial survival. In this review, we focus on those effectors in the repertoire of L. pneumophila proteins that target key cellular pathways by specific post-translational modifications.

  4. Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

    2017-01-01

    Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein, we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.

  5. A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

    DEFF Research Database (Denmark)

    Pirone, Lucia; Xolalpa, Wendy; Sigurdsson, Jón Otti

    2017-01-01

    L conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bio......-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates...

  6. Expression and distribution of cell adhesion-related proteins in bovine parthenogenetic embryos: The effects of oocyte vitrification.

    Science.gov (United States)

    Zeng, Yan; Fu, Xiangwei; Zhou, Guangbin; Yue, Mingxing; Zhou, Yanhua; Zhu, Shien

    2013-07-01

    The objective was to investigate expression of cell adhesion-related proteins (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) in bovine parthenogenetic embryos derived from vitrified-warmed oocytes. Bovine oocytes at metaphase II were randomly allocated into three groups: (1) untreated (control); (2) exposed to vitrification solution without freezing (toxicity); and (3) vitrified and warmed by the open-pulled straw method (vitrification). After parthenogenetic activation, in the vitrification group compared with the control, the timing of compaction was delayed in (108-120 vs. 96-108 hours, respectively), and the percentage of blastocysts that developed from eight-cell embryos was lower (32.08% vs. 61.03%; P vitrification delayed embryo compaction by affecting adhesion junction formation and function, immunostaining and quantitative reverse transcription polymerase chain reaction were done to characterize distribution patterns (E-cadherin, β-catenin, and the cytoskeletal protein F-actin) and expression levels of cell adhesion-related proteins (β-catenin). Distribution of β-catenin in eight-cell embryos from the vitrification group changed dramatically compared with the control and toxicity groups. Relative expression of β-catenin at the mRNA and protein levels was lower (P bovine parthenogenetic eight-cell embryos derived from vitrified-warmed oocytes were associated with embryo compaction and reduced competence for subsequent embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases.

    Science.gov (United States)

    Li, S; Kim, M; Hu, Y L; Jalali, S; Schlaepfer, D D; Hunter, T; Chien, S; Shyy, J Y

    1997-11-28

    Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2. Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. DeltamSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK.

  8. Protein modification and replicative senescence of WI-38 human embryonic fibroblasts

    DEFF Research Database (Denmark)

    Ahmed, Emad K; Rogowska-Wrzesinska, Adelina; Roepstorff, Peter

    2010-01-01

    cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP-stimulated Lon-like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified......Summary Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins...... build up and potentially affect cellular function during replicative senescence of WI-38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis-based proteomic approach coupled with immunodetection of HNE-, AGE-modified and carbonylated...

  9. Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement.

    Science.gov (United States)

    Wells, Evan; Robinson, Anne Skaja

    2017-01-01

    Recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. Avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. Numerous approaches to express recombinant proteins exist, but Escherichia coli, Saccharomyces cerevisiae, and mammalian systems (e.g. Chinese hamster ovary cells, CHO) are the most widely utilized. Improvements to production in these hosts have focused on novel expression cassettes, cell line modifications, engineering secretion pathways, and media design. Here, we describe recent developments for improving protein production in E. coli, S. cerevisiae, and CHO systems and compare recent advancements to previous knowledge in the field. With the expanding importance and prevalence of protein therapeutics, these improvements will serve as the framework for future discoveries. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. [Effects of different surface modifications on micro-structure and adhesion of zirconia ceramic: an in vitro study].

    Science.gov (United States)

    Siwen, Li; Shishi, Li; Yanhong, Wang; Hongmei, Ma

    2017-02-01

    This study evaluated the effect of different mechanical-chemical surface treatments on the characteristics, microstructure, and composition of zirconia ceramics and on the zirconia-dentin adhesion. The sintered commercial zirconia blocks (IPS e.max ZirCAD) were sectioned into 126 beams (6 mm×6 mm×5 mm) and randomly assigned to seven experimental groups (n=18). The zirconia block specimens were further treated as follows: (A) untreated, as control; (B) sandblasted with 50 μm Al2O3; (C) sandblasted with 50 μm Al2O3+30 μm silica powder; (D) sandblasted with 50 μm Al2O3+30% silica-sol coating; (E) sandblasted with 110 μm Al2O3; (F) sandblasted with 110 μm Al2O3+30 μm silica powder; and (G) sandblasted with 110 μm Al2O3+30% silica-sol coating. The surface roughness (Ra) of zirconia ceramics using X-ray diffraction (XRD), field emission scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) after seven surface treatments was analyzed. Seventy specimens of dentin surfaces were prepared. A dual-cure resin cement was applied into zirconia surfaces with its corresponding adhesive components to dentin. Shear bond strength (SBS) of each sample was measured using a universal testing machine. The data were analyzed by ANOVA using SPSS 17.0 software. Ra of zirconia were significantly different compared with the control group (Psilica-coating methods. Air abrasion significantly increased the micro-cracks in the ceramic surface and caused the grain boundaries to disappear. A serious shrinkage of the thin silica film can be observed after sintering procedure. This process induced cracks and the film to strip away slightly from the surfaces of Y-TZP substrate. The SBS values from large to small were F and G groups, C and D groups, B and E groups (Psilica powder abrasion, and silica-sol coating affect the surface morphology, structure, and composition of the tooth. This effect can also achieve the improved micro-mechanical interlocking or chemical

  11. Silk protein as a new optically transparent adhesion layer for an ultra-smooth sub-10 nm gold layer

    Science.gov (United States)

    Min, Kyungtaek; Umar, Muhammad; Ryu, Shinyoung; Lee, Soonil; Kim, Sunghwan

    2017-03-01

    Ultra-thin and ultra-smooth gold (Au) films are appealing for photonic applications including surface plasmon resonances and transparent contacts. However, poor adhesion at the Au-dielectric interface prohibits the formation of a mechanically stable, ultra-thin, and ultra-smooth Au film. A conventional solution is to use a metallic adhesion layer, such as titanium and chromium, however such layers cause the optical properties of pure Au to deteriorate. Here we report the use of silk protein to enhance the adhesion at the Au-dielectric interface, thus obtaining ultra-smooth sub-10 nm Au films. The Au films that were deposited onto the silk layer exhibited superior surface roughness to those deposited on SiO2, Si, and poly(methyl methacrylate), along with improved adhesion, electrical conductivity, and optical transparency. Additionally, we confirm that a metal-insulator-metal optical resonator can be successfully generated using a silk insulating layer without the use of a metallic adhesion layer.

  12. Adhesion of Trypanosoma cruzi trypomastigotes to fibronectin or laminin modifies tubulin and paraflagellar rod protein phosphorylation.

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    Eliciane C Mattos

    Full Text Available BACKGROUND: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM, as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. CONCLUSIONS/SIGNIFICANCE: Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

  13. Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1.

    Science.gov (United States)

    Schaffrath, Raffael; Klassen, Roland

    2017-01-02

    Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm5s2U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm5s2U and Ψ38 in tRNAGlnUUG was shown to mediate efficient synthesis of the Q/N rich [PIN+] prion forming protein Rnq1. 1 In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNAGlnUUG, but not its isoacceptor tRNAGlnCUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm5s2U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.

  14. EXPERIMENTAL STUDY ON THE MODIFICATIONS PRODUCED AT THE INTERFACE BETWEEN THE PERIODONTAL ADHESIVE SPLINTS AND THE DENTAL SURFACE

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    Bogdan VÂSCU

    2016-03-01

    Full Text Available As the market offer for bioadhesive materials is constantly increasing, while the dental surfaces on which they are applied show specific features, different from those commonly resulting from the preparation of carious processes, knowledge on their behavioral characteristics is absolutely necessary for their utilization under optimum conditions, through methods assuming prolongued clinical performances, assured by dimensional and colouristic stability and by a reduced cure contraction, for diminishing as much as possible the space of marginal percolation and fracture of the free enamel-free margins, as well as for delamination of immobilization from the afferent dental structure. Selection of the type of material for periodonthic teeth immobilization and of the technique to be applied is decided on the basis of a systematic, clinical and radiological analysis meant at establishing: the number of affected teeth, the type of occlusion and the possible parafunctions, oral hygiene, the aesthetic requirements of the patient, his/her age and motivation for a periodical monitorization. Numerous modern materials employed in the immobilization of periodonthic teeth are closely related not only to their physical properties and long-term stability, but also to the oral environment in which they are functioning. Modern adhesive materials are well-suited for dental recovery of the remaining healthy structures, due to their capacity of chemically and micromechanically adhering onto them.

  15. ProteomeScout: a repository and analysis resource for post-translational modifications and proteins.

    Science.gov (United States)

    Matlock, Matthew K; Holehouse, Alex S; Naegle, Kristen M

    2015-01-01

    ProteomeScout (https://proteomescout.wustl.edu) is a resource for the study of proteins and their post-translational modifications (PTMs) consisting of a database of PTMs, a repository for experimental data, an analysis suite for PTM experiments, and a tool for visualizing the relationships between complex protein annotations. The PTM database is a compendium of public PTM data, coupled with user-uploaded experimental data. ProteomeScout provides analysis tools for experimental datasets, including summary views and subset selection, which can identify relationships within subsets of data by testing for statistically significant enrichment of protein annotations. Protein annotations are incorporated in the ProteomeScout database from external resources and include terms such as Gene Ontology annotations, domains, secondary structure and non-synonymous polymorphisms. These annotations are available in the database download, in the analysis tools and in the protein viewer. The protein viewer allows for the simultaneous visualization of annotations in an interactive web graphic, which can be exported in Scalable Vector Graphics (SVG) format. Finally, quantitative data measurements associated with public experiments are also easily viewable within protein records, allowing researchers to see how PTMs change across different contexts. ProteomeScout should prove useful for protein researchers and should benefit the proteomics community by providing a stable repository for PTM experiments. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array.

    Science.gov (United States)

    Partolina, Marina; Thoms, Hazel C; MacLeod, Kenneth G; Rodriguez-Blanco, Giovanny; Clarke, Matthew N; Venkatasubramani, Anuroop V; Beesoo, Rima; Larionov, Vladimir; Neergheen-Bhujun, Vidushi S; Serrels, Bryan; Kimura, Hiroshi; Carragher, Neil O; Kagansky, Alexander

    2017-01-01

    The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2-24 h after inhibition of HDACs in different cancer cell lines

  17. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    Science.gov (United States)

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. [Intensity of lipid peroxidation and protein oxidative modification of goat and cow milk].

    Science.gov (United States)

    Vysokogorskiĭ, V E; Gavrilova, N B; Arkhipenko, Iu A

    2014-01-01

    Indices of free-radical peroxidation have been estimated: intensity of lipid per- oxidation and protein oxidative modification of goat and cow milk of specific breeds of forest-steppe zone of Omsk region. The obtained results indicate that processes of lipid peroxidation and protein oxidative destruction in goat and cow milk of different breeds occur with different gradation. The content of carbonile derivatives in goat milk of Saan breed 1.4 (0.95; 1.5) u/ml was lower than in cow's milk of black-and-white breed 4.6 (1.1; 6.0) u/ml (p = 0.005) what could be caused by large content of protein thiol groups of this kind of milk and lower quantity of amino acid residues that are available for carbonylation. This kind of milk is characterized by higher SH-group content than cow milk for 31% and Switzerland goat milk for 20% (p = 0.005). The content of cetodiens and attached triens in isopropanol phase of the lipid extract of goat milk of Swiss breed is lower by 30% than in cow milk. In isopropanol phase of the milk lipid extracts contain- ing phospholipids the level of Schiff grounding did not differ. The results obtained prove that goat milk contain less protein subjected to oxidative modification.

  19. Protein modifications in cooked pork products investigated by a proteomic approach.

    Science.gov (United States)

    Di Luccia, Aldo; la Gatta, Barbara; Nicastro, Annarita; Petrella, Giovanni; Lamacchia, Carmela; Picariello, Gianluca

    2015-04-01

    To evaluate process-induced protein modifications in cooked ham and emulsion sausages, the proteomes of whole-cut (Parma and "Praga" cooked hams) and comminuted pork (mortadella and würstel) products were compared to raw pork using two-dimensional gel electrophoresis (2-DE) coupled to image analysis and mass spectrometry (MS). Other than heat-induced breakdown of part of the myosin heavy chains, the 2-DE pattern of cooked ham was substantially similar to that of raw pork. However, the MS-based analysis showed minor modifications, including the extensive oxidation of methionines. In contrast, likely due to emulsification, comminuted sausages were characterized by an abundant insoluble protein fraction (IPF). Interestingly, tropomyosin and myosin light chains in comminuted sausages were exclusively found in the IPF. Our results indicate that the protein aggregation systems of cooked hams and emulsion sausages reflect the processing conditions and are definitely different, the former being characterized mainly by disulphide bridges and the latter by additional covalent inter-protein links. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

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    Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.; Matzke, Melissa M.; Nair, Parameswaran; Bigelow, Diana J.; Pounds, Joel G.; Zangar, Richard C.

    2014-01-31

    Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, we found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from

  1. Mussel adhesive protein coating: A potential therapeutic method for self-healing of cracked teeth

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    Li Bo-Lin

    2015-01-01

    Full Text Available Introduction: Nowadays, cracked tooth syndrome is the third main cause of tooth extraction, following caries and periodontal diseases, done in almost all the dental clinics. Nevertheless, the diagnosis and treatment of this condition remain controversial. All candidate therapeutics, such as occlusal adjustment, preventive filling, root canal therapy (RCT, and crown restoration, provide unpredictable outcomes. As such, methods to prevent further crack development and to induce crack self-healing must be developed. The Hypothesis: Mussels secreting adhesive foot protein (Mafp can attach to various surfaces under aqueous conditions. In nature, mussels adhere to stones and deposit layer by layer through mineralization, thereby forming mussel-stone composites with excellent mechanical property. Given the natural process of mussel-stone complex formation, we hypothesize that application of Mafp coating at the crack interface may mineralize the cracks by capturing calcium and phosphate ions from the saliva. This process consequently leads to crack self-healing and complete restoration of the tooth structure. Evaluation of the Hypothesis: To test our hypothesis, we need to develop a model in vivo. Cracked teeth disks are adhered together using Mafp solution. Then, the tooth disks are sutured on the interior side of the cheeks. After regular intervals, the disks are removed and characterized. Scanning electron microscopy is performed to evaluate the morphology of the crack interface. Microhardness and shear bond strength are used to evaluate the mechanical property of the healing cracked zone. Transmission electron microscopy is also conducted to evaluate the crystallinity of the crack interface.

  2. Plasmodium vivax thrombospondin related adhesion protein: immunogenicity and protective efficacy in rodents and Aotus monkeys

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    Angélica Castellanos

    2007-06-01

    Full Text Available The thrombospondin related adhesion protein (TRAP is a malaria pre-erythrocytic antigen currently pursued as malaria vaccine candidate to Plasmodium falciparum. In this study, a long synthetic peptide (LSP representing a P. vivax TRAP fragment involved in hepatocyte invasion was formulated in both Freund and Montanide ISA 720 adjutants and administered by IM and subcutaneous routes to BALB/c mice and Aotus monkeys. We measured specific humoral immune responses in both animal species and performed a sporozoite challenge in Aotus monkeys to assess the protective efficacy of the vaccine. After immunization both mice and Aotus seroconverted as shown by ELISA, and the specific anti-peptide antibodies cross reacted with the parasite in IFAT assays. Only two out of six immunized animals became infected after P. vivax sporozoite challenge as compared with four out of six animals from the control group. These results suggest that this TRAP fragment has protective potential against P. vivax malaria and deserves further studies as vaccine candidate.

  3. Posttranslational modification of bioaerosol protein by common gas pollutants: NO2 and O3

    Science.gov (United States)

    Abdullahi Mahmood, Marliyyah; Bloss, William; Pope, Francis

    2016-04-01

    Air pollution can exacerbate several medical conditions, for example, hay fever and asthma. The global incidence of hay fever has been rising for decades; however, the underlying reasons behind this rise remain unclear. It is hypothesized that the exposure of pollen to common gas phase pollutants, such as nitrogen dioxide (NO2) and ozone (O3), increases the allergenicity of the pollen and thus increases hay fever incidence (Reinmuth-Selzle et al., 2014, Franze, et al., 2005). Since atmospheric pollutants often have greater concentrations within urban areas (in particular NO2) the hypothesis suggests that greater allergenicity should occur in urban areas. Certainly, several studies do suggest higher hay fever incidence within urban areas compared to rural areas (Schröder et al., 2015). Previous published work suggests a link between increased allergies and changes in the chemical composition of pollen protein via posttranslational modification of the protein (Reinmuth-Selzle et al., 2014). This study investigates the posttranslational modification of two highly allergenic pollen species (Birch and Ragweed) that are common in Europe. Within the laboratory, we expose pollen grains to atmospherically relevant exposures of gas phase NO2, O3 and other common gas phase oxidants under a range of environmentally relevant conditions. The effects of the exposures on the biochemistry of the pollen grains were probed using a proteomic approach (liquid chromatography coupled ultra-high resolution spectrometer). Our findings indicate the interaction between gas phase pollutants and pollen cause protein specific modifications; in particular nitration that occurs upon tyrosine residues and nitrosylation on cysteine residues. These modifications may affect human immune response to the pollen protein, which may suggest a possible reason for increased allergies in reaction to such chemically altered protein. Quantification of the relative degree of PTMs, from a variety of

  4. Enhanced adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions.

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    Sheiam Sulaeman

    Full Text Available Campylobacter jejuni is responsible for the major foodborne bacterial enteritis in humans. In contradiction with its fastidious growth requirements, this microaerobic pathogen can survive in aerobic food environments, suggesting that it must employ a variety of protection mechanisms to resist oxidative stress. For the first time, C. jejuni 81-176 inner and outer membrane subproteomes were analyzed separately using two-dimensional protein electrophoresis (2-DE of oxygen-acclimated cells and microaerobically grown cells. LC-MS/MS analyses successfully identified 42 and 25 spots which exhibited a significantly altered abundance in the IMP-enriched fraction and in the OMP-enriched fraction, respectively, in response to oxidative conditions. These spots corresponded to 38 membrane proteins that could be grouped into different functional classes: (i transporters, (ii chaperones, (iii fatty acid metabolism, (iv adhesion/virulence and (v other metabolisms. Some of these proteins were up-regulated at the transcriptional level in oxygen-acclimated cells as confirmed by qRT-PCR. Downstream analyses revealed that adhesion of C. jejuni to inert surfaces and swarming motility were enhanced in oxygen-acclimated cells or paraquat-stressed cells, which could be explained by the higher abundance of membrane proteins involved in adhesion and biofilm formation. The virulence factor CadF, over-expressed in the outer membrane of oxygen-acclimated cells, contributes to the complex process of C. jejuni adhesion to inert surfaces as revealed by a reduction in the capability of C. jejuni 81-176 ΔCadF cells compared to the isogenic strain.Taken together, these data demonstrate that oxygen-enriched conditions promote the over-expression of membrane proteins involved in both the biofilm initiation and virulence of C. jejuni.

  5. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

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    Veronika N Bade

    Full Text Available The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls, ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation. ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no

  6. Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A.

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    Shelby Calkins

    Full Text Available Focal adhesions (FAs are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM and the cell's cytoskeleton. FAs consist of anchor membrane protein (integrins, scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin, signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH, and signaling kinases (e.g. focal adhesion kinase (FAK and Src kinase. While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these

  7. Effect of surface modification on the In vitro protein adsorption and cell cytotoxicity of vinorelbine nanoparticles

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    Nandhakumar Sathyamoorthy

    2017-01-01

    Full Text Available Context: Nanocarriers possessing long-circulating abilities could take advantage of the pathophysiology of tumor vasculature to achieve spatial placement. To attain such qualities, the drug carriers should possess suitable physicochemical properties such as size and surface hydrophilicity. Aim: The aim of this study was to prepare poly(ε-caprolactone nanoparticles (NPs loaded with vinorelbine bitartrate (VB and to modify its steric properties using polyethylene glycol and poloxamer. Furthermore, the influence of surface modification of NPs on their physicochemical and cell interactive properties was evaluated. Materials and Methods: NPs were prepared by double emulsion solvent extraction–evaporation technique. The prepared NPs were evaluated for their physicochemical properties, in vitro protein adsorption and cell cytotoxicity. Results and Discussion: The NPs were <250 nm with an entrapment efficiency ranging between 40% and 52%. The zeta potential of the NPs varied from −7.52 mV to −1.27 mV depending on the surface modification. The in vitro release studies exhibited a biphasic pattern with an initial burst release followed by controlled release of the drug over 72 h. The protein adsorption studies revealed that the ability to resist protein adsorption was influenced by the concentration of surface-modifying agents and the amount of proteins available for interaction. The surface-modified NPs produced cell cytotoxicity comparable to free VB at higher concentrations owing to sustained release of the drug into the cellular environment. Conclusion: The results emphasize that surface modification of nanocarriers is an essential and effective tool to dodge opsonization and phagocytosis in the physiological milieu.

  8. The correlation between the adsorption of adhesive proteins and cell behaviour on hydroxyl-methyl mixed self-assembled monolayers.

    Science.gov (United States)

    Barrias, Cristina C; Martins, M Cristina L; Almeida-Porada, Graça; Barbosa, Mário A; Granja, Pedro L

    2009-01-01

    The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH(3) terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under non-competitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cell-binding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH(3))-SAMs.

  9. Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1

    Science.gov (United States)

    Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

    2014-01-01

    T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130 000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion. PMID:24877199

  10. Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types.

    Science.gov (United States)

    Alves, C M; Yang, Y; Marton, D; Carnes, D L; Ong, J L; Sylvia, V L; Dean, D D; Reis, R L; Agrawal, C M

    2008-10-01

    We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface. (c) 2008 Wiley Periodicals, Inc.

  11. Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

    Directory of Open Access Journals (Sweden)

    Michaela Mühlberg

    2015-05-01

    Full Text Available To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

  12. Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55

    NARCIS (Netherlands)

    Karpus, Olga N.; Veninga, Henrike; Hoek, Robert M.; Flierman, Dennis; van Buul, Jaap D.; Vandenakker, Corianne C.; VanBavel, Ed; Medof, M. Edward; van Lier, René A. W.; Reedquist, Kris A.; Hamann, Jörg

    2013-01-01

    Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane β subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is

  13. Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts

    DEFF Research Database (Denmark)

    Couchman, J R; Badley, R A; Rees, D A

    1983-01-01

    The roles of the microfilament-associated proteins vinculin, alpha-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick...... the associated microfilament bundles. Indeed the appearance of small bodies containing alpha-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions. Myosin and filamin...... earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage...

  14. Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack?

    Science.gov (United States)

    Boys, Brian L; Kuprowski, Mark C; Noël, James J; Konermann, Lars

    2009-05-15

    The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI

  15. IL-2 induces beta2-integrin adhesion via a wortmannin/LY294002-sensitive, rapamycin-resistant pathway. Phosphorylation of a 125-kilodalton protein correlates with induction of adhesion, but not mitogenesis

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S

    1996-01-01

    Besides its function as a growth factor, IL-2 induces beta2-integrin-dependent, homotypic adhesion of IL-2R-positive T cells. In this study, we investigated how IL-2R are functionally and biochemically linked to the beta2-integrin adhesion pathway. After a lag period of 15 to 20 min, IL-2 induces...... beta2-integrin-dependent, homotypic adhesion in Ag-specific, human T cell lines. The IL-2 adhesion response is blocked by wortmannin and LY294002, inhibitors of phosphatidylinositol-3 (PI-3) kinase activity. In contrast, rapamycin strongly inhibits IL-2-induced proliferation without inhibiting IL-2...... on mitogenesis. IL-2R ligation rapidly (signal transducer and activator of transcription (Stat) proteins, the p85 subunit of the PI-3 kinase, and an as yet unidentified 125-kDa protein (p125). Wortmannin, LY294002...

  16. STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post-Translational Modifications.

    Science.gov (United States)

    Spencer, Jean L; Bhatia, Vivek N; Whelan, Stephen A; Costello, Catherine E; McComb, Mark E

    2013-12-01

    The identification of protein post-translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post-Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi-sample comparison by collating, scoring, and ranking PTMs and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting.

  17. Posttranslational Protein Modification in the Salivary Glands of Sjögren’s Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Rafael Herrera-Esparza

    2013-01-01

    Full Text Available The present study investigated posttranslational reactions in the salivary glands of patients with Sjögren’s syndrome. We analysed the biopsies of primary Sjögren’s patients using immunohistochemistry and a tag-purified anticyclic citrullinated protein (CCP antibody to detect citrullinated peptides, and the presence of peptidylarginine deiminase 2 (PAD2 was assessed simultaneously. The present work demonstrated the weak presence of the PAD2 enzyme in some normal salivary glands, although PAD2 expression was increased considerably in Sjögren’s patients. The presence of citrullinated proteins was also detected in the salivary tissues of Sjögren’s patients, which strongly supports the in situ posttranslational modification of proteins in this setting. Furthermore, the mutual expression of CCP and PAD2 suggests that this posttranslational modification is enzyme dependent. In conclusion, patients with Sjögren’s syndrome expressed the catalytic machinery to produce posttranslational reactions that may result in autoantigen triggering.

  18. A role for the protein tyrosine phosphatase CD45 in macrophage adhesion through the regulation of paxillin degradation.

    Directory of Open Access Journals (Sweden)

    Joëlle St-Pierre

    Full Text Available CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2 and focal adhesion kinase (FAK, kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.

  19. Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

    Science.gov (United States)

    Fonar, Yuri; Gutkovich, Yoni E.; Root, Heather; Malyarova, Anastasia; Aamar, Emil; Golubovskaya, Vita M.; Elias, Sarah; Elkouby, Yaniv M.; Frank, Dale

    2011-01-01

    Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior–posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK–Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells. PMID:21551070

  20. In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae

    Directory of Open Access Journals (Sweden)

    Atsushi Kurotani

    2015-08-01

    Full Text Available Recent proteome analyses have reported that intrinsically disordered regions (IDRs of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

  1. Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications.

    Directory of Open Access Journals (Sweden)

    Arantza Soler-Cantero

    Full Text Available Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine-protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu(++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters. This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

  2. Modification of the protein corona-nanoparticle complex by physiological factors.

    Science.gov (United States)

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    Science.gov (United States)

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  4. Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress.

    Science.gov (United States)

    Hashiguchi, Akiko; Komatsu, Setsuko

    2016-12-21

    The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs) can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.

  5. Plant-Derived Phenolics Inhibit the Accrual of Structurally Characterised Protein and Lipid Oxidative Modifications

    Science.gov (United States)

    Naudí, Alba; Romero, Maria-Paz; Cassanyé, Anna; Serrano, José C. E.; Arola, Lluis; Valls, Josep; Bellmunt, Maria Josep; Prat, Joan; Pamplona, Reinald; Portero-Otin, Manuel; Motilva, Maria-José

    2012-01-01

    Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine- protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake. PMID:22952663

  6. In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

    Science.gov (United States)

    Kurotani, Atsushi; Sakurai, Tetsuya

    2015-08-20

    Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

  7. Adhesion and degranulation promoting adapter protein (ADAP is a central hub for phosphotyrosine-mediated interactions in T cells.

    Directory of Open Access Journals (Sweden)

    Marc Sylvester

    Full Text Available TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486-783. Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC we identified SLP-76, PLCgamma, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

  8. Adhesive bonding of resin composite to various titanium surfaces using different metal conditioners and a surface modification system

    Directory of Open Access Journals (Sweden)

    Hercules Jorge ALMILHATTI

    2013-12-01

    Full Text Available Objective: This study evaluated the effect of three metal conditioners on the shear bond strength (SBS of a prosthetic composite material to cpTi grade I having three surface treatments. Material and Methods: One hundred sixty eight rivet-shaped specimens (8.0x2.0 mm were cast and subjected to polishing (P or sandblasting with either 50 mm (50SB or 250 mm (250SB Al2O3. The metal conditioners Metal Photo Primer (MPP, Cesead II Opaque Primer (OP, Targis Link (TL, and one surface modification system Siloc (S, were applied to the specimen surfaces, which were covered with four 1-mm thick layers of resin composite. The resin layers were exposed to curing light for 90 s separately. Seven specimens from each experimental group were stored in water at 37ºC for 24 h while the other 7 specimens were subjected to 5,000 thermal cycles consisting of water baths at 4ºC and 60ºC (n=7. All specimens were subjected to SBS test (0.5 mm/min until failure occurred, and further 28 specimens were analyzed using scanning electron microscope (SEM and X-ray energy-dispersive spectroscopy (EDS. Data were analyzed by 3-way ANOVA followed by post-hoc Tukey's test (α=0.05. Results: On 50SB surfaces, OP groups showed higher SBS means than MPP (P<0.05, while no significant difference was found among OP, S, and TL groups. On 250SB surfaces, OP and TL groups exhibited higher SBS than MPP and S (P<0.05. No significant difference in SBS was found between OP and TL groups nor between MPP and S groups. The use of conditioners on 250SB surfaces resulted in higher SBS means than the use of the same products on 50SB surfaces (P<0.05. Conclusion: Sandblasting associated with the use of metal conditioners improves SBS of resin composites to cpTi.

  9. Additive effect of recombinant Mycobacterium tuberculosis ESAT-6 protein and ESAT-6/CFP-10 fusion protein in adhesion of macrophages through fibronectin receptors.

    Science.gov (United States)

    Hemmati, Mina; Seghatoleslam, Atefeh; Rasti, Mozhgan; Ebadat, Saeedeh; Naghibalhossaini, Fakhraddin; Mostafavi-Pour, Zohreh

    2016-04-01

    Tuberculous granulomas are the sites of interaction between the T cells, macrophages, and extracellular matrix (ECM) to control the infection caused by Mycobacterium tuberculosis (M. tuberculosis). A predominant role of RD-1-encoded secretory proteins, early secreted antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) in the formation of granulomas has recently been emphasized. However, the precise molecular events that induce the formation of these granulomatous structures are yet to be elucidated. Macrophages use integrins to adhere to fibronectin (FN) as a major component of the ECM. The major goal of this study was to investigate whether recombinant M. tuberculosis antigens can modulate integrin-mediated macrophage adhesion. Differentiated THP-1 cell line was stimulated with recombinant ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins and evaluated for alterations in the expression levels of α5β1 and α4β1 by semiquantitative real-time polymerase chain reaction. The role of these recombinant antigens in the cytoskeleton rearrangement was determined by adhesion assay and immunofluorescent microscopy. Our data showed that ESAT-6 and ESAT-6/CFP-10 fusion proteins could induce adhesion of macrophages to FN through α4β1 integrin. An increased expression level of α4β1 integrin in comparison with α5β1 integrin in differentiated THP-1 cells was also observed. Results of immunofluorescence studies showed that recombinant proteins-treated THP-1 cells form well-organized stress fibers and focal contacts containing vinculin compared with untreated THP-1 cells. Increased expression level of α4β1 in differentiated THP-1 cells could suggest the important role of α4β1 integrin in adhesion and focal contact formation of macrophages exposed to M. tuberculosis antigens. Copyright © 2014. Published by Elsevier B.V.

  10. Calcium dobesilate inhibits the alterations in tight junction proteins and leukocyte adhesion to retinal endothelial cells induced by diabetes.

    Science.gov (United States)

    Leal, Ermelindo C; Martins, João; Voabil, Paula; Liberal, Joana; Chiavaroli, Carlo; Bauer, Jacques; Cunha-Vaz, José; Ambrósio, António F

    2010-10-01

    Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood-retinal barrier (BRB) permeability induced by diabetes. Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress.

  11. Modulation of cell adhesion and migration by the histone methyltransferase subunit mDpy-30 and its interacting proteins.

    Directory of Open Access Journals (Sweden)

    Bin Xia

    2010-07-01

    Full Text Available We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT complex, also localizes at the trans-Golgi network (TGN, where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of beta1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of beta1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to beta1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as beta1 integrin.

  12. Modification of the protein corona–nanoparticle complex by physiological factors

    Energy Technology Data Exchange (ETDEWEB)

    Braun, Nicholas J.; DeBrosse, Madeleine C.; Hussain, Saber M. [Molecular Bioeffects Branch, Bioeffects Division, Human Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright Patterson AFB, 2729 R. St, Bldg 837, Dayton, OH, 45433 (United States); Comfort, Kristen K., E-mail: kcomfort1@udayton.edu [Department of Chemical and Materials Engineering, University of Dayton, 524 Kettering Laboratories, 300 College Park, Dayton, OH 45469 (United States)

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona–NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13 nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. - Highlights: • Dynamic flow increased the size of the gold nanoparticle protein corona. • Exposure to biological fluids altered protein corona size and composition. • Interstitial fluid modified the nano-cellular interface and deposition efficiency. • Tannic acid coated nanoparticles induced toxicity in an interstitial environment.

  13. Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications.

    Science.gov (United States)

    Patil, Ashish; Chan, Clement T Y; Dyavaiah, Madhu; Rooney, John P; Dedon, Peter C; Begley, Thomas J

    2012-07-01

    Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm ( 5) U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm ( 5) s ( 2) U), classified as key determinants of translational fidelity. We also show that in the absence of mcm ( 5) U and mcm ( 5) s ( 2) U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways.

  14. Do post-translational beta cell protein modifications trigger type 1 diabetes?

    DEFF Research Database (Denmark)

    Størling, Joachim; Overgaard, Anne Julie; Brorsson, Caroline Anna

    2013-01-01

    Type 1 diabetes is considered an autoimmune disease characterised by specific T cell-mediated destruction of the insulin-producing beta cells. Yet, except for insulin, no beta cell-specific antigens have been discovered. This may imply that the autoantigens in type 1 diabetes exist in modified...... forms capable of specifically triggering beta cell destruction. In other immune-mediated diseases, autoantigens targeted by the immune system have undergone post-translational modification (PTM), thereby creating tissue-specific neo-epitopes. In a similar manner, PTM of beta cell proteins might create...... beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading...

  15. Protein adsorption to graphene surfaces controlled by chemical modification of the substrate surfaces.

    Science.gov (United States)

    Kamiya, Yasutaka; Yamazaki, Kenji; Ogino, Toshio

    2014-10-01

    We have investigated effects of the support substrate surfaces on properties of the attached graphene flakes by observing protein adsorption to the graphene surfaces on SiO2/Si substrates that are modified with self-assembled monolayers to control their hydrophilicity. Using atomic force microscopy operated in aqueous environment, we found that high-density clusters of agglomerated avidin molecules form on the graphene flakes in the areas supported by a hydrophobic substrate surface, whereas very low density of large avidin clusters form at the edge of graphene flakes in the area supported by a hydrophilic surface. These results demonstrate that hydrophilicity of the support surface affects hydrophilicity of the graphene surface also in aqueous environment and that surface modification of the support substrate is a useful technique to control protein adsorption phenomena on graphene surfaces for realization of high sensitive graphene biosensors. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Papillote and Piopio: Drosophila ZP-domain proteins required for cell adhesion to the apical extracellular matrix and microtubule organization.

    Science.gov (United States)

    Bökel, Christian; Prokop, Andreas; Brown, Nicholas H

    2005-02-01

    Adhesion between epithelial cells and extracellular substrates is normally mediated through basal adhesion complexes. However, some cells also possess comparable junctions on their apical surface. Here, we describe two new Drosophila proteins, Piopio and Papillote, that are required for the link between the apical epithelial surface and the overlying apical extracellular matrix (aECM). The two proteins share a zona pellucida (ZP) domain with mammalian aECM components, including the tectorins found in the vertebrate inner ear. Tagged versions of both proteins localized to the apical epithelial surface. Mutations in piopio, papillote and dumpy (another gene encoding a ZP-domain protein) cause defects in the innermost layer of the aECM and its detachment from the epidermis. Loss of Piopio, but not Papillote or Dumpy, causes the absence of specialized microtubule bundles from pupal wings, suggesting that Piopio plays a role in microtubule organization. Thus, ZP domain-containing proteins may have shared functions within the aECM, while also exhibiting specific interactions with the cytoskeleton.

  17. The adhesion modulation protein, AmpA localizes to an endocytic compartment and influences substrate adhesion, actin polymerization and endocytosis in vegetative Dictyostelium cells

    Directory of Open Access Journals (Sweden)

    Noratel Elizabeth F

    2012-11-01

    Full Text Available Abstract Background AmpA is a secreted 24Kd protein that has pleiotropic effects on Dictyostelium development. Null mutants delay development at the mound stage with cells adhering too tightly to the substrate. Prestalk cells initially specify as prespore cells and are delayed in their migration to the mound apex. Extracellular AmpA can rescue these defects, but AmpA is also necessary in a cell autonomous manner for anterior like cells (ALCs to migrate to the upper cup. The ALCs are only 10% of the developing cell population making it difficult to study the cell autonomous effect of AmpA on the migration of these cells. AmpA is also expressed in growing cells, but, while it contains a hydrophobic leader sequence that is cleaved, it is not secreted from growing cells. This makes growing cells an attractive system for studying the cell autonomous function of AmpA. Results In growing cells AmpA plays an environment dependent role in cell migration. Excess AmpA facilitates migration on soft, adhesive surfaces but hinders migration on less adhesive surfaces. AmpA also effects the level of actin polymerization. Knockout cells polymerize less actin while over expressing cells polymerize more actin than wild type. Overexpression of AmpA also causes an increase in endocytosis that is traced to repeated formation of multiple endocytic cups at the same site on the membrane. Immunofluorescence analysis shows that AmpA is found in the Golgi and colocalizes with calnexin and the slow endosomal recycling compartment marker, p25, in a perinuclear compartment. AmpA is found on the cell periphery and is endocytically recycled to the perinuclear compartment. Conclusion AmpA is processed through the secretory pathway and traffics to the cell periphery where it is endocytosed and localizes to what has been defined as a slow endosomal recycling compartment. AmpA plays a role in actin polymerization and cell substrate adhesion. Additionally AmpA influences cell

  18. Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation

    Directory of Open Access Journals (Sweden)

    Cigdem ATALAYIN

    2016-01-01

    Full Text Available Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2. The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

  19. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    The signal transduction pathways associated with neural cell adhesion molecule (NCAM)-induced neuritogenesis are only partially characterized. We here demonstrate that NCAM-induced neurite outgrowth depends on activation of p59(fyn), focal adhesion kinase (FAK), phospholipase Cgamma (PLCgamma......), protein kinase C (PKC), and the Ras-mitogen-activated protein (MAP) kinase pathway. This was done using a coculture system consisting of PC12-E2 cells grown on fibroblasts, with or without NCAM expression, allowing NCAM-NCAM interactions resulting in neurite outgrowth. PC12-E2 cells were transiently...... propose a model of NCAM signaling involving two pathways: NCAM-Ras-MAP kinase and NCAM-FGF receptor-PLCgamma-PKC, and we propose that PKC serves as the link between the two pathways activating Raf and thereby creating the sustained activity of the MAP kinases necessary for neuronal differentiation....

  20. Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

    Science.gov (United States)

    Stoichevska, Violet; Peng, Yong Y; Vashi, Aditya V; Werkmeister, Jerome A; Dumsday, Geoff J; Ramshaw, John A M

    2017-03-01

    Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017. © 2016 Wiley Periodicals, Inc.

  1. A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease.

    Science.gov (United States)

    von Figura, K; Schmidt, B; Selmer, T; Dierks, T

    1998-06-01

    In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.

  2. A novel water-based process produces eco-friendly bio-adhesive made from green cross-linked soybean soluble polysaccharide and soy protein.

    Science.gov (United States)

    Yuan, Cheng; Chen, Mingsong; Luo, Jing; Li, Xiaona; Gao, Qiang; Li, Jianzhang

    2017-08-01

    In this study, an eco-friendly soy protein adhesive was developed that utilized two components from soybean meal without addition of any toxic material. A plant-based, water-soluble and inexpensive soybean soluble polysaccharide was used as the novel renewable material to combine with soy protein to produce a soy protein adhesive. Three-plywood was fabricated with the resulting adhesive, and its wet shear strength was measured. The results showed the wet shear strength of plywood bonded by the adhesive reached 0.99MPa, meeting the water resistance requirement for interior use panels. This improvement was attributed to the following reasons: (1) Combination of cross-linked soybean soluble polysaccharide and soy protein formed an interpenetrating network structure, improving the thermal stability and water resistance of the cured adhesive. (2) Adding CL-SSPS decreased the adhesive viscosity to 15.14Pas, which increased the amount of the adhesive that penetrate the wood's surface and formed more interlocks. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    2009-07-01

    Full Text Available SYT (SYnovial sarcoma Translocated gene or SS18 is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

  4. iPTMnet: an integrated resource for protein post-translational modification network discovery.

    Science.gov (United States)

    Huang, Hongzhan; Arighi, Cecilia N; Ross, Karen E; Ren, Jia; Li, Gang; Chen, Sheng-Chih; Wang, Qinghua; Cowart, Julie; Vijay-Shanker, K; Wu, Cathy H

    2017-11-14

    Protein post-translational modifications (PTMs) play a pivotal role in numerous biological processes by modulating regulation of protein function. We have developed iPTMnet (http://proteininformationresource.org/iPTMnet) for PTM knowledge discovery, employing an integrative bioinformatics approach-combining text mining, data mining, and ontological representation to capture rich PTM information, including PTM enzyme-substrate-site relationships, PTM-specific protein-protein interactions (PPIs) and PTM conservation across species. iPTMnet encompasses data from (i) our PTM-focused text mining tools, RLIMS-P and eFIP, which extract phosphorylation information from full-scale mining of PubMed abstracts and full-length articles; (ii) a set of curated databases with experimentally observed PTMs; and iii) Protein Ontology that organizes proteins and PTM proteoforms, enabling their representation, annotation and comparison within and across species. Presently covering eight major PTM types (phosphorylation, ubiquitination, acetylation, methylation, glycosylation, S-nitrosylation, sumoylation and myristoylation), iPTMnet knowledgebase contains more than 654 500 unique PTM sites in over 62 100 proteins, along with more than 1200 PTM enzymes and over 24 300 PTM enzyme-substrate-site relations. The website supports online search, browsing, retrieval and visual analysis for scientific queries. Several examples, including functional interpretation of phosphoproteomic data, demonstrate iPTMnet as a gateway for visual exploration and systematic analysis of PTM networks and conservation, thereby enabling PTM discovery and hypothesis generation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. The structural and functional signatures of proteins that undergo multiple events of post-translational modification.

    Science.gov (United States)

    Pejaver, Vikas; Hsu, Wei-Lun; Xin, Fuxiao; Dunker, A Keith; Uversky, Vladimir N; Radivojac, Predrag

    2014-08-01

    The structural, functional, and mechanistic characterization of several types of post-translational modifications (PTMs) is well-documented. PTMs, however, may interact or interfere with one another when regulating protein function. Yet, characterization of the structural and functional signatures of their crosstalk has been hindered by the scarcity of data. To this end, we developed a unified sequence-based predictor of 23 types of PTM sites that, we believe, is a useful tool in guiding biological experiments and data interpretation. We then used experimentally determined and predicted PTM sites to investigate two particular cases of potential PTM crosstalk in eukaryotes. First, we identified proteins statistically enriched in multiple types of PTM sites and found that they show preferences toward intrinsically disordered regions as well as functional roles in transcriptional, posttranscriptional, and developmental processes. Second, we observed that target sites modified by more than one type of PTM, referred to as shared PTM sites, show even stronger preferences toward disordered regions than their single-PTM counterparts; we explain this by the need for these regions to accommodate multiple partners. Finally, we investigated the influence of single and shared PTMs on differential regulation of protein-protein interactions. We provide evidence that molecular recognition features (MoRFs) show significant preferences for PTM sites, particularly shared PTM sites, implicating PTMs in the modulation of this specific type of macromolecular recognition. We conclude that intrinsic disorder is a strong structural prerequisite for complex PTM-based regulation, particularly in context-dependent protein-protein interactions related to transcriptional and developmental processes. www.modpred.org. © 2014 The Protein Society.

  6. PTMcode v2: a resource for functional associations of post-translational modifications within and between proteins.

    Science.gov (United States)

    Minguez, Pablo; Letunic, Ivica; Parca, Luca; Garcia-Alonso, Luz; Dopazo, Joaquin; Huerta-Cepas, Jaime; Bork, Peer

    2015-01-01

    The post-translational regulation of proteins is mainly driven by two molecular events, their modification by several types of moieties and their interaction with other proteins. These two processes are interdependent and together are responsible for the function of the protein in a particular cell state. Several databases focus on the prediction and compilation of protein-protein interactions (PPIs) and no less on the collection and analysis of protein post-translational modifications (PTMs), however, there are no resources that concentrate on describing the regulatory role of PTMs in PPIs. We developed several methods based on residue co-evolution and proximity to predict the functional associations of pairs of PTMs that we apply to modifications in the same protein and between two interacting proteins. In order to make data available for understudied organisms, PTMcode v2 (http://ptmcode.embl.de) includes a new strategy to propagate PTMs from validated modified sites through orthologous proteins. The second release of PTMcode covers 19 eukaryotic species from which we collected more than 300,000 experimentally verified PTMs (>1,300,000 propagated) of 69 types extracting the post-translational regulation of >100,000 proteins and >100,000 interactions. In total, we report 8 million associations of PTMs regulating single proteins and over 9.4 million interplays tuning PPIs. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Monocyte Adhesion and Plaque Recruitment During Atherosclerosis Development Is Regulated by the Adapter Protein Chat-H/SHEP1.

    Science.gov (United States)

    Herbin, Olivier; Regelmann, Adam G; Ramkhelawon, Bhama; Weinstein, Erica G; Moore, Kathryn J; Alexandropoulos, Konstantina

    2016-09-01

    The chronic inflammation associated with atherosclerosis is caused by lipid deposition followed by leukocyte recruitment to the arterial wall. We previously showed that the hematopoietic cell-specific adaptor protein Cas- and Hef1-associated signal transducer hematopoietic isoform (Chat-H)/SHEP1 regulated lymphocyte adhesion and migration. In this study, we analyzed the role of Chat-H in atherosclerosis development. Using Chat-H-deficient bone marrow transplantation in low-density lipoprotein receptor-deficient mice, we found that Chat-H regulated atherosclerotic plaque formation. Chat-H deficiency in hematopoietic cells associated with lower plaque complexity and fewer leukocytes in the lesions, whereas myeloid-specific deletion of Chat-H was sufficient for conferring atheroprotection. Chat-H deficiency resulted in reduced recruitment of classical Ly6c(high) and nonclassical Ly6c(low) monocytes to the plaques, which was accompanied by increased numbers of both monocyte subsets in the blood. This associated with defective adhesion of Chat-H-deficient Ly6c(high) and Ly6c(low) monocytes to vascular cell adhesion molecule-1 in vitro and impaired infiltration of fluorescent bead-loaded monocytes to atherosclerotic plaques. In contrast, Chat-H was dispensable for CX3CL1 and CCR1/CCR5-dependent migration of monocytes. Our findings highlight Chat-H as a key protein that regulates atherosclerosis development by controlling monocyte adhesion and recruitment to the plaques and identify a novel target that may be exploited for treating atherosclerosis. © 2016 American Heart Association, Inc.

  8. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    components. These substances may both mediate and stabilize the bacterial biofilm. Finally, several adhesive structures were examined, and a novel physiological biofilm phenotype in E.coli biofilms was characterized, namely cell chain formation. The autotransporter protein, antigen 43, was implicated...

  9. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

    Science.gov (United States)

    Halim, Adnan; Carlsson, Michael C; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L; Wandall, Hans H

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The "Sticky Patch" Model of Crystallization and Modification of Proteins for Enhanced Crystallizability.

    Science.gov (United States)

    Derewenda, Zygmunt S; Godzik, Adam

    2017-01-01

    Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e., crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure, and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular "sticky patch" model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer "sticky patches" which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the "sticky patch" model. We discuss state-of-the-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis.

  11. Differential role of eDNA, proteins, and polysaccharides in cell-cell and cell-substrate adhesion by three Staphylococcus species

    DEFF Research Database (Denmark)

    Meyer, Rikke Louise; Okshevsky, Mira Ursula; Zeng, Guanghong

    affected by DNase and dispersin treatments, hence eDNA and polysaccharides were essential for cell-cell interactions. We showed that proteins, polysaccharides and eDNA contribute differently to the adhesion of three Staphylcococcus species, underlining the need to either tailor biofilm prevention...... valuable for designing new approaches to biofilm prevention. In this study, we combine microfluidic flow-cell studies with single-cell analyses to understand how polysaccharides, extracellular DNA (eDNA), and proteins contribute individually and in concert to mediate bacterial adhesion and aggregation...... on abiotic surfaces. We quantified initial adhesion, cell aggregation, and single-cell adhesion forces of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus xylosus in the presence and absence of DNase, dispersin, or subtilisin, which cleave extracellular DNA, polysaccharides and proteins...

  12. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

    Science.gov (United States)

    Nally, Jarlath E; Grassmann, Andre A; Planchon, Sébastien; Sergeant, Kjell; Renaut, Jenny; Seshu, Janakiram; McBride, Alan J; Caimano, Melissa J

    2017-01-01

    Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p 1.25 or proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and

  13. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

    Directory of Open Access Journals (Sweden)

    Jarlath E. Nally

    2017-08-01

    Full Text Available Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE. Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < −1.25 across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30

  14. Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

    Science.gov (United States)

    Gersbach, Charles A; Gaj, Thomas; Barbas, Carlos F

    2014-08-19

    The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular

  15. Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

    Directory of Open Access Journals (Sweden)

    Celma Cristina C

    2009-09-01

    Full Text Available Abstract Background Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way. Results This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins. Conclusion 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in Ac

  16. Circulating renalase, catecholamines, and vascular adhesion protein 1 in hypertensive patients.

    Science.gov (United States)

    Maciorkowska, Dominika; Zbroch, Edyta; Malyszko, Jolanta

    2015-11-01

    The aim of the study was to estimate and correlate circulating levels of renalase, vascular adhesion protein-1 (VAP-1), catecholamines in patients with primary hypertension. The renalase, VAP-1, and catecholamines concentration was estimated in 121 hypertensive patients. The correlation between renalase, VAP-1 levels and catecholamine concentration in blood, blood pressure control, pharmacological therapy, and medical history were taken in to consideration. The median office blood pressure was 145.5/86 mm Hg and was significantly higher than the median home blood pressure measurement value, which was 135/80 mm Hg, P < .05. Circulating renalase and VAP-1 (Me 9.57 μg/mL and Me = 326.7 ng/mL) levels were significantly higher in patients with hypertension comparing to healthy individuals (3.83 μg/mL and 248.37 ng/mL, P < .05). The correlation between renalase and noradrenalin concentration in blood was observed (r = 0.549; P < .05), also the correlation between VAP-1 and noradrenaline was noticed (r = 0.21, P = .029). Renalase level was higher in patients with coronary artery disease and correlated with decreased ejection fraction. VAP-1 concentration correlated also with left ventricular ejection fraction (r = -0.23, P = .013). Hypertensive patients with diabetes mellitus had almost statistically significant higher VAP-1 concentration compared with hypertensive patients without diabetes mellitus (Me = 403.22 ng/mL vs. Me = 326,68 ng/mL, P = .064). In multiple regression analysis, renalase was predicted by plasma dopamine and norepinephrine as also diastolic office blood pressure and left ventricle ejection fraction. Circulating renalase and VAP-1 levels are elevated in patients with poor blood pressure control. Its correlation with noradrenalin concentration need further studies to find out the role of renalase as also VAP-1 in pathogenesis and treatment of hypertension. Copyright © 2015 American Society of Hypertension. Published by

  17. Diverse post-translational modifications of the pannexin family of channel-forming proteins.

    Science.gov (United States)

    Penuela, Silvia; Lohman, Alexander W; Lai, Wesley; Gyenis, Laszlo; Litchfield, David W; Isakson, Brant E; Laird, Dale W

    2014-01-01

    The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.

  18. Adhesion of endothelial cells and adsorption of serum proteins on gas-plasma treated polytetrafluoroethylene

    NARCIS (Netherlands)

    Dekker, A.; Dekker, A.; Reitsma, K.; Beugeling, T.; Beugeling, T.; Bantjes, A.; Bantjes, A.; Feijen, Jan; van Aken, W.G.

    1991-01-01

    From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon® or Dacron® may be the reason why endothelialization of these grafts does not occur after implantation in humans. We

  19. Atypical ubiquitin chains: new molecular signals. 'Protein Modifications: Beyond the Usual Suspects' review series.

    Science.gov (United States)

    Ikeda, Fumiyo; Dikic, Ivan

    2008-06-01

    Ubiquitin (Ub) is a small protein modifier that regulates many biological processes, including gene transcription, cell-cycle progression, DNA repair, apoptosis, virus budding and receptor endocytosis. Ub can be conjugated to target proteins either as a monomer or as Ub chains that vary in length and linkage type. The various types of Ub modification are linked to distinct physiological functions in cells. MonoUb, for example, regulates DNA repair and receptor endocytosis, whereas lysine 48-linked Ub chains label proteins for proteasomal degradation. More recently, the importance of chains conjugated through the other six lysines in Ub, known as atypical Ub chains, has been revealed. Atypical chains can be homotypic, sequentially using the same lysine residue in Ub for conjugation; mixed-linkage, utilizing several distinct lysines to connect consecutive Ub moieties; or heterologous, connecting Ub with other Ub-like modifiers. Here, we describe recent progress in the understanding of atypical Ub chain assembly and their recognition by Ub-binding domains, and we discuss further their functional roles in vivo.

  20. Oxidative Modification of Blood Serum Proteins in Multiple Sclerosis after Interferon Beta and Melatonin Treatment

    Directory of Open Access Journals (Sweden)

    Monika Adamczyk-Sowa

    2017-01-01

    Full Text Available Multiple sclerosis (MS is a disease involving oxidative stress (OS. This study was aimed at examination of the effect of melatonin supplementation on OS parameters, especially oxidative protein modifications of blood serum proteins, in MS patients. The study included 11 control subjects, 14 de novo diagnosed MS patients with the relapsing-remitting form of MS (RRMS, 36 patients with RRMS receiving interferon beta-1b (250 μg every other day, and 25 RRMS patients receiving interferon beta-1b plus melatonin (5 mg daily. The levels of N′-formylkynurenine, kynurenine, dityrosine, carbonyl groups, advanced glycation products (AGEs, advanced oxidation protein products (AOPP, and malondialdehyde were elevated in nontreated RRSM patients. N′-Formylkynurenine, kynurenine, AGEs, and carbonyl contents were decreased only in the group treated with interferon beta plus melatonin, while dityrosine and AOPP contents were decreased both in the group of patients treated with interferon beta and in the group treated with interferon beta-1b plus melatonin. These results demonstrate that melatonin ameliorates OS in MS patients supporting the view that combined administration of interferon beta-1b and melatonin can be more effective in reducing OS in MS patients than interferon beta-1b alone.

  1. Modification of peptide and protein cysteine thiol groups by conjugation with a degradation product of ascorbate.

    Science.gov (United States)

    Kay, Phyla; Wagner, J Richard; Gagnon, Hugo; Day, Robert; Klarskov, Klaus

    2013-09-16

    Ascorbate is an important water-soluble antioxidant, which when oxidized by reactive oxygen species is converted into dehydroascorbate (DHA). If not rapidly reduced back to ascorbate, DHA decomposes to a reactive 5-carbon compound (DHA*, +130 Da) that can modify reduced cysteinyl residues in peptides and proteins in vitro. The formation of cysteine adducts by DHA* was characterized by mass spectrometry using reduced insulin B-chain, α-lactalbumin, and hemoglobin. Mass spectrometry of DHA* modified insulin B-chain revealed the presence of one and two DHA* adducts. Enzymatic cleavage and tandem mass spectrometry of modified peptides allowed unambiguous localization of DHA* to the two cysteine residues in positions 7 and 19 of the insulin B-chain. Incubations of DHA with α-lactalbumin revealed that approximately 25% of the protein population was in a reduced state and could be modified by DHA*. The adduct was assigned to the N-terminally located cysteinyl residue in position 6. Incubation of hemoglobin with DHA followed by pepsin digestion and electrospray ionization tandem mass spectrometry (ESI-MSMS) of the peptide mixture allowed for the identification of three modified peptides. Tandem mass spectrometry of the modified peptides, two from the hemoglobin A-chain with identical mass and one from the hemoglobin B-chain, gave a complete series of y-type fragment ions, which were assigned to the cysteine containing peptides (100)LLSHCL(105) (A-chain), (101)LSHCLL(106) (A-chain), and (111)VCVLAHHFGKE(121) (B-chain). Although the DHA* adduct was lost from the peptides derived from α-lactalbumin and hemoglobin before fragmentation of the peptide bond, carbamidomethylation of the proteins prior to incubation with DHA abolished the formation of DHA*-protein adducts and confirmed that the target was indeed the cysteine thiol group. Future studies are focused on the modification of proteins by DHA* in cells and in vivo.

  2. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    Directory of Open Access Journals (Sweden)

    Bernadette Sosa-García

    2010-11-01

    Full Text Available The retinoblastoma protein (pRb is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

  3. Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins.

    Science.gov (United States)

    Schwämmle, Veit; Verano-Braga, Thiago; Roepstorff, Peter

    2015-11-03

    The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis, multivariate analysis and data interpretation. We furthermore discuss the potential of future developments that will help to gain deep insight into the PTM-ome and its biological role in cells. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

    Nielsen, Janne; Kulahin, Nikolaj; Walmod, Peter

    2008-01-01

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

  5. Cdc42 Effector Protein 2 (XCEP2 is required for normal gastrulation and contributes to cellular adhesion in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Nelson Richard W

    2004-10-01

    Full Text Available Abstract Background Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis. Results In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis, a gene encoding a Rho GTPase effector protein (Xenopus Cdc42 effector protein 2, or XCEP2 was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell

  6. Serine34 phosphorylation of RHO guanine dissociation inhibitor (RHOGDI{alpha}) links signaling from conventional protein kinase C to RHO GTPase in cell adhesion

    DEFF Research Database (Denmark)

    Dovas, Athanassios; Choi, Youngsil; Yoneda, Atsuko

    2010-01-01

    Protein kinase Calpha (PKCalpha) is an essential serine/threonine kinase regulating many signaling networks. At cell adhesion sites, PKCalpha can impact the actin cytoskeleton through its influence on RhoGTPases but the intermediate steps are not well known. One important regulator of Rho....... Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with FRET microscopy sensing GTP-RhoA levels, the data reveal a common pathway...... in cell adhesion linking two essential mediators, PKCalpha and RhoA....

  7. The structural and functional signatures of proteins that undergo multiple events of post-translational modification

    Science.gov (United States)

    Pejaver, Vikas; Hsu, Wei-Lun; Xin, Fuxiao; Dunker, A Keith; Uversky, Vladimir N; Radivojac, Predrag

    2014-01-01

    The structural, functional, and mechanistic characterization of several types of post-translational modifications (PTMs) is well-documented. PTMs, however, may interact or interfere with one another when regulating protein function. Yet, characterization of the structural and functional signatures of their crosstalk has been hindered by the scarcity of data. To this end, we developed a unified sequence-based predictor of 23 types of PTM sites that, we believe, is a useful tool in guiding biological experiments and data interpretation. We then used experimentally determined and predicted PTM sites to investigate two particular cases of potential PTM crosstalk in eukaryotes. First, we identified proteins statistically enriched in multiple types of PTM sites and found that they show preferences toward intrinsically disordered regions as well as functional roles in transcriptional, posttranscriptional, and developmental processes. Second, we observed that target sites modified by more than one type of PTM, referred to as shared PTM sites, show even stronger preferences toward disordered regions than their single-PTM counterparts; we explain this by the need for these regions to accommodate multiple partners. Finally, we investigated the influence of single and shared PTMs on differential regulation of protein–protein interactions. We provide evidence that molecular recognition features (MoRFs) show significant preferences for PTM sites, particularly shared PTM sites, implicating PTMs in the modulation of this specific type of macromolecular recognition. We conclude that intrinsic disorder is a strong structural prerequisite for complex PTM-based regulation, particularly in context-dependent protein–protein interactions related to transcriptional and developmental processes. Availability: http://www.modpred.org PMID:24888500

  8. The ‘Sticky Patch’ Model of Crystallization and Modification of Proteins for Enhanced Crystallizability

    Science.gov (United States)

    Derewenda, Zygmunt S.; Godzik, Adam

    2017-01-01

    Crystallization of macromolecules has long been perceived as a stochastic process, which cannot be predicted or controlled. This is consistent with another popular notion that the interactions of molecules within the crystal, i.e. crystal contacts, are essentially random and devoid of specific physicochemical features. In contrast, functionally relevant surfaces, such as oligomerization interfaces and specific protein-protein interaction sites, are under evolutionary pressures so their amino acid composition, structure and topology are distinct. However, current theoretical and experimental studies are significantly changing our understanding of the nature of crystallization. The increasingly popular ‘sticky patch’ model, derived from soft matter physics, describes crystallization as a process driven by interactions between select, specific surface patches, with properties thermodynamically favorable for cohesive interactions. Independent support for this model comes from various sources including structural studies and bioinformatics. Proteins that are recalcitrant to crystallization can be modified for enhanced crystallizability through chemical or mutational modification of their surface to effectively engineer ‘sticky patches’ which would drive crystallization. Here, we discuss the current state of knowledge of the relationship between the microscopic properties of the target macromolecule and its crystallizability, focusing on the ‘sticky patch’ model. We discuss state-of-art in silico methods that evaluate the propensity of a given target protein to form crystals based on these relationships, with the objective to design of variants with modified molecular surface properties and enhanced crystallization propensity. We illustrate this discussion with specific cases where these approaches allowed to generate crystals suitable for structural analysis. PMID:28573570

  9. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

    Directory of Open Access Journals (Sweden)

    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  10. Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.

    Science.gov (United States)

    Ehrenshaft, Marilyn; Deterding, Leesa J; Mason, Ronald P

    2015-12-01

    Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone and/or polymerization of monomers into dimers, multimers, and sometimes insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions that have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine, and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion-limited rate with hydroxyl radical and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral, whereas others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and the weaknesses of some of the techniques used to detect these oxidative interactions, and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants, and microbes. Published by Elsevier Inc.

  11. α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

    DEFF Research Database (Denmark)

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J

    2015-01-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites assoc...

  12. Acetylome of Acinetobacter baumannii SK17 Reveals a Highly-Conserved Modification of Histone-Like Protein HU

    Directory of Open Access Journals (Sweden)

    Jiahn-Haur Liao

    2017-11-01

    Full Text Available Lysine acetylation is a prevalent post-translational modification in both eukaryotes and prokaryotes. Whereas this modification is known to play pivotal roles in eukaryotes, the function and extent of this modification in prokaryotic cells remain largely unexplored. Here we report the acetylome of a pair of antibiotic-sensitive and -resistant nosocomial pathogen Acinetobacter baumannii SK17-S and SK17-R. A total of 145 lysine acetylation sites on 125 proteins was identified, and there are 23 acetylated proteins found in both strains, including histone-like protein HU which was found to be acetylated at Lys13. HU is a dimeric DNA-binding protein critical for maintaining chromosomal architecture and other DNA-dependent functions. To analyze the effects of site-specific acetylation, homogenously Lys13-acetylated HU protein, HU(K13ac was prepared by genetic code expansion. Whilst not exerting an obvious effect on the oligomeric state, Lys13 acetylation alters both the thermal stability and DNA binding kinetics of HU. Accordingly, this modification likely destabilizes the chromosome structure and regulates bacterial gene transcription. This work indicates that acetyllysine plays an important role in bacterial epigenetics.

  13. Prediction of protein modification sites of pyrrolidone carboxylic acid using mRMR feature selection and analysis.

    Directory of Open Access Journals (Sweden)

    Lu-Lu Zheng

    Full Text Available Pyrrolidone carboxylic acid (PCA is formed during a common post-translational modification (PTM of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR and incremental feature selection (IFS. We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations.

  14. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry

    Science.gov (United States)

    Engelen, Erik; Brandsma, Johannes H.; Moen, Maaike J.; Signorile, Luca; Dekkers, Dick H. W.; Demmers, Jeroen; Kockx, Christel E. M.; Ozgür, Zehila; van IJcken, Wilfred F. J.; van den Berg, Debbie L. C.; Poot, Raymond A.

    2015-01-01

    The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. PMID:25990348

  15. PHOXTRACK-a tool for interpreting comprehensive datasets of post-translational modifications of proteins.

    Science.gov (United States)

    Weidner, Christopher; Fischer, Cornelius; Sauer, Sascha

    2014-12-01

    We introduce PHOXTRACK (PHOsphosite-X-TRacing Analysis of Causal Kinases), a user-friendly freely available software tool for analyzing large datasets of post-translational modifications of proteins, such as phosphorylation, which are commonly gained by mass spectrometry detection. In contrast to other currently applied data analysis approaches, PHOXTRACK uses full sets of quantitative proteomics data and applies non-parametric statistics to calculate whether defined kinase-specific sets of phosphosite sequences indicate statistically significant concordant differences between various biological conditions. PHOXTRACK is an efficient tool for extracting post-translational information of comprehensive proteomics datasets to decipher key regulatory proteins and to infer biologically relevant molecular pathways. PHOXTRACK will be maintained over the next years and is freely available as an online tool for non-commercial use at http://phoxtrack.molgen.mpg.de. Users will also find a tutorial at this Web site and can additionally give feedback at https://groups.google.com/d/forum/phoxtrack-discuss. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders.

    Science.gov (United States)

    dos Santos-Pinto, José Roberto Aparecido; Lamprecht, Günther; Chen, Wei-Qiang; Heo, Seok; Hardy, John George; Priewalder, Helga; Scheibel, Thomas Rainer; Palma, Mario Sergio; Lubec, Gert

    2014-06-13

    Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk

  17. Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.; Donato, Dominique M.; Hanks, Steven K.; Zheng, Jie J.

    2017-08-31

    The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

  18. Molecular hydrogen modulates gene expression via histone modification and induces the mitochondrial unfolded protein response.

    Science.gov (United States)

    Sobue, Sayaka; Inoue, Chisato; Hori, Fumiko; Qiao, Shanlou; Murate, Takashi; Ichihara, Masatoshi

    2017-11-04

    Molecular hydrogen (H2) is a biologically active gas that is used medically to ameliorate various systemic pathological conditions. H2 also regulates gene expression involved in intracellular signaling and metabolic pathways. However, it is unclear whether H2 affects gene expression directly or through indirect effects as a consequence of health improvement. Therefore, we attempted to identify genes that exhibit similar changes in expression in response to H2 by employing DNA microarrays and gene set enrichment analysis to analyze RNA from liver and lung of rats and mice with or without dietary stress. We found that H2 activated the expression of sets of genes regulated by histone H3K27 methylation status. H2 also modified the expression of many genes regulated by a wide variety of signaling pathways. RT-qPCR showed that H2 up-regulated expression of Kcnc3, a H3K27-regulated gene, in organs such as liver, lung, kidney and brain. Furthermore, using immunohistochemistry and immunoblot analysis, we observed changes in H3K27 methylation status in the liver of mice and rats administered H2. Moreover, we showed that H2 simultaneously induced the H3K27 demethylase, Jmjd3, and mitochondrial unfolded protein response (mtUPR)-related genes. Recently, alteration of mitochondrial function was shown to cause induction of H3K27 demethylase or chromatin restructuring, followed by mtUPR activation through the alteration of H3K27 or H3K9 methylation states. Taken together, our study suggests that H2 can induce beneficial effects through mtUPR activation via epigenetic histone modification and by modification of gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

    Science.gov (United States)

    Flaherty, S P; Swann, N J; Primakoff, P; Myles, D G

    1993-03-01

    Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in

  20. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  1. Influence of nitriding atmosphere on the modification of surface titanium with focus on the behavior of blood platelets adhesion; Influencia da atmosfera nitretante na modificacao de superficies de titanio com enfase no comportamento de adesao de plaquetas sanguineas

    Energy Technology Data Exchange (ETDEWEB)

    Vitoriano, J.O.; Alves, C. [Universidade Federal Rural do Semi-Arido (UFERSA), RN (Brazil); Braz, D.C.; Camara, R.B.G.; Rocha, H.A.O., E-mail: clodomiro.jr@hotmail.com [Universidade Federal do Rio Grande do Norte (UFRN), RN (Brazil)

    2014-07-01

    The present study aimed to analyze the influence of surface modification of titanium on the adhesion of blood platelets, through techniques of adhesion and morphological analyzes. Discs of titanium grade II received different surface treatments with plasma of Ar + N{sub 2} + H{sub 2} and Ar + H{sub 2}, forming two experimental groups including only polished samples used as standard. Before and after treatment the samples were characterized according to topography, crystalline structure and wettability, using atomic force microscopy, X-ray diffraction, Raman spectroscopy and testing of sessile drop, respectively. Platelet rich plasma (PRP) was applied on the modified surfaces in a culture plates. Images obtained by electron microscopy of adhered platelets were analyzed to verify the behavior of platelets in the different experimental conditions. (author)

  2. Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system.

    Science.gov (United States)

    Gourbatsi, Evdoxia; Povey, Jane; Uddin, Shahid; Smales, C Mark

    2016-04-01

    The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.

  3. Vaspin inhibits cytokine-induced nuclear factor-kappa B activation and adhesion molecule expression via AMP-activated protein kinase activation in vascular endothelial cells.

    Science.gov (United States)

    Jung, Chang Hee; Lee, Min Jung; Kang, Yu Mi; Lee, Yoo La; Yoon, Hae Kyeong; Kang, Sang-Wook; Lee, Woo Je; Park, Joong-Yeol

    2014-02-12

    Vaspin is an adipocytokine that was recently identified in the visceral adipose tissue of diabetic rats and has anti-diabetic and anti-atherogenic effects. We hypothesized that vaspin prevents inflammatory cytokine-induced nuclear factor-kappa B (NF-κB) activation by activating AMP-activated protein kinase (AMPK) in vascular endothelial cells. We examined the effects of vaspin on NF-κB activation and the expression of the NF-κB-mediated genes intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1). Human aortic endothelial cells (HAECS) were used. Tumor necrosis factor alpha (TNFα) was used as a representative proinflammatory cytokine. Treatment with vaspin significantly increased the phosphorylation of AMPK and acetyl-CoA carboxylase, the down-stream target of AMPK. Furthermore, treatment with vaspin significantly decreased TNFα-induced activation of NF-κB, as well as the expression of the adhesion molecules ICAM-1, VCAM-1, E-selectin, and MCP-1. These effects were abolished following transfection of AMPKα1-specific small interfering RNA. In an adhesion assay using THP-1 cells, vaspin reduced TNFα-induced adhesion of monocytes to HAECS in an AMPK-dependent manner. Vaspin might attenuate the cytokine-induced expression of adhesion molecule genes by inhibiting NF-κB following AMPK activation.

  4. Improvement of Interfacial Adhesion by Bio-Inspired Catechol-Functionalized Soy Protein with Versatile Reactivity: Preparation of Fully Utilizable Soy-Based Film

    Directory of Open Access Journals (Sweden)

    Zhong Wang

    2017-03-01

    Full Text Available The development of materials based on renewable resources with enhanced mechanical and physicochemical properties is hampered by the abundance of hydrophilic groups because of their structural instability. Bio-inspired from the strong adhesion ability of mussel proteins, renewable and robust soy-based composite films were fabricated from two soybean-derived industrial materials: soluble soybean polysaccharide (SSPS and catechol-functionalized soy protein isolate (SPI-CH. The conjugation of SPI with multiple catechol moieties as a versatile adhesive component for SSPS matrix efficiently improved the interfacial adhesion between each segment of biopolymer. The biomimetic adherent catechol moieties were successfully bonded in the polymeric network based on catechol crosslinking chemistry through simple oxidative coupling and/or coordinative interaction. A combination of H-bonding, strong adhesion between the SPI-CH conjugation and SSPS matrix resulted in remarkable enhancements for mechanical properties. It was found that the tensile strength and Young’s modulus was improved from 2.80 and 17.24 MPa of unmodified SP film to 4.04 and 97.22 MPa of modified one, respectively. More importantly, the resultant films exhibited favorable water resistance and gas (water vapor barrier performances. The results suggested that the promising way improved the phase adhesion of graft copolymers using catechol-functionalized polymers as versatile adhesive components.

  5. Developmental expression patterns of Beta-ig (betaIG-H3) and its function as a cell adhesion protein.

    Science.gov (United States)

    Ferguson, Jill W; Mikesh, Michelle F; Wheeler, Esther F; LeBaron, Richard G

    2003-08-01

    Beta-ig is a secretory protein embodied by fasciclin I-like repeats containing sequences that might bind integrins and glycosaminoglycans in vivo. Expression of Beta-ig is responsive to Transforming Growth Factor-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating Beta-ig as an ECM adhesive protein of developmental processes. The spatiotemporal distribution of Beta-ig during various stages of murine development was examined and its ability to support adhesion of various cell types assessed. In situ hybridization of mouse embryos (E12.5-E18.5) indicated a prominent, distinct expression pattern for Beta-ig message in connective tissue. Beta-ig transcripts were abundantly expressed during mesenchymal cell condensation in areas of axial, craniofacial and appendicular primordial cartilage from E12.5-E14.5. Beginning at E15.5, Beta-ig transcripts appeared in collagen-rich tissues, including dura mater and corneal stroma. During E16.5-E18.5, Beta-ig transcripts were observed in proliferating chondrocytes and areas of endochondral ossification in joint and articular cartilage formation. Connective tissues expressed Beta-ig transcripts within the nasal septum and surrounding cartilage primordia, and in the pericardium, optic cup, kidney, ovary, esophagus, diaphragm, bronchi, trachea and corneal epithelium, and during cardiac valve formation. These patterns of expression indicate that Beta-ig may be involved in tissue morphogenesis. Cells derived from mesenchyme attached onto a substratum comprised of purified recombinant Beta-ig. Taken together, the results indicate that Beta-ig is expressed principally in collagen-rich tissues where it may interact with cells and ECM molecules, perhaps playing a role in tissue morphogenesis.

  6. Recycling of protein subunits during DNA translocation and cleavage by Type I restriction-modification enzymes.

    Science.gov (United States)

    Simons, Michelle; Szczelkun, Mark D

    2011-09-01

    The Type I restriction-modification enzymes comprise three protein subunits; HsdS and HsdM that form a methyltransferase (MTase) and HsdR that associates with the MTase and catalyses Adenosine-5'-triphosphate (ATP)-dependent DNA translocation and cleavage. Here, we examine whether the MTase and HsdR components can 'turnover' in vitro, i.e. whether they can catalyse translocation and cleavage events on one DNA molecule, dissociate and then re-bind a second DNA molecule. Translocation termination by both EcoKI and EcoR124I leads to HsdR dissociation from linear DNA but not from circular DNA. Following DNA cleavage, the HsdR subunits appear unable to dissociate even though the DNA is linear, suggesting a tight interaction with the cleaved product. The MTases of EcoKI and EcoAI can dissociate from DNA following either translocation or cleavage and can initiate reactions on new DNA molecules as long as free HsdR molecules are available. In contrast, the MTase of EcoR124I does not turnover and additional cleavage of circular DNA is not observed by inclusion of RecBCD, a helicase-nuclease that degrades the linear DNA product resulting from Type I cleavage. Roles for Type I restriction endonuclease subunit dynamics in restriction alleviation in the cell are discussed.

  7. IgA and IgM protein primarily drive plasma corona-induced adhesion reduction of PLGA nanoparticles in human blood flow.

    Science.gov (United States)

    Sobczynski, Daniel J; Eniola-Adefeso, Omolola

    2017-06-01

    The high abundance of immunoglobulins (Igs) in the plasma protein corona on poly(lactic-co-glycolic) acid (PLGA)-based vascular-targeted carriers (VTCs) has previously been shown to reduce their adhesion to activated endothelial cells (aECs) in human blood flow. However, the relative role of individual Ig classes (e.g., IgG, IgA, and IgM) in causing adhesion reduction remains largely unknown. Here, we characterized the influence of specific Ig classes in prescribing the binding efficiency of PLGA nano-sized VTCs in blood flow. Specifically, we evaluated the flow adhesion to aECs of PLGA VTCs with systematic depletion of various Igs in their corona. Adhesion reduction was largely eliminated for PLGA VTCs when all Igs were removed from the corona. Furthermore, re-addition of IgA or IgM to the Igs-depleted corona reinstated the low adhesion of PLGA VTCs, as evidenced by ∼40-70% reduction relative to particles with an Igs-deficient corona. However, re-addition of a high concentration of IgG to the Igs-depleted corona did not cause significant adhesion reduction. Overall, the presented results reveal that PLGA VTC adhesion reduction in blood flows is primarily driven by high adsorption of IgA and IgM in the particle corona. Pre-coating of albumin on PLGA VTCs mitigated the extent of adhesion reduction in plasma for some donors but was largely ineffective in general. Overall, this work may shed light into effective control of protein corona composition, thereby enhancing VTC functionality in vivo for eventual clinical use.

  8. Specific Degradation of the Mucus Adhesion-Promoting Protein (MapA) of Lactobacillus reuteri to an Antimicrobial Peptide ▿

    Science.gov (United States)

    Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B.; Holo, Helge; Nes, Ingolf F.

    2010-01-01

    The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota. PMID:20833791

  9. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    Science.gov (United States)

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

  10. Environment-Sensitive Turn-On Fluorescent Polyamino Acid: Fingerprinting Protein Populations with Post-Translational Modifications.

    Science.gov (United States)

    Tomita, Shunsuke; Ishihara, Sayaka; Kurita, Ryoji

    2017-07-12

    The identification of post-translational modifications (PTMs) in proteins has been of particular interest in the elucidation of human diseases and the improvement of therapeutic proteins. Herein, we report a novel strategy toward the construction of fingerprint-based PTM-sensing systems as an alternative to conventional specific recognition tools. Our strategy is based on poly-l-lysine (PLL) derivatives with two distinct properties suitable to fingerprint-based protein-sensing: (i) a turn-on fluorescent signal upon binding to proteins and (ii) condition-dependent cross-reactivity toward proteins and PTMs. One type of PLL derivative under varying solution properties (pH value and ionic strength) was sufficient to construct a sensing array that produces unique fluorescence fingerprints for structurally similar mammalian albumins with/without a wide variety of chemical modifications corresponding to PTMs. This approach was also applicable for the recognition of deviations in physicochemical properties of proteins as a result of realistic glycation and phosphorylation events. Multivariate analyses of the thus obtained fingerprints successfully identified analytes with 100% accuracy (qualitatively and quantitatively) in all cases. This study thus demonstrates for the first time a fingerprint-based sensing of proteins with/without PTMs using a single, highly accessible, and tunable synthetic polymer, and accordingly offers a powerful platform for simple high-throughput sensing of PTMs in proteins.

  11. Characterization of human GTPBP3, a GTP-binding protein involved in mitochondrial tRNA modification.

    Science.gov (United States)

    Villarroya, Magda; Prado, Silvia; Esteve, Juan M; Soriano, Miguel A; Aguado, Carmen; Pérez-Martínez, David; Martínez-Ferrandis, José I; Yim, Lucía; Victor, Victor M; Cebolla, Elvira; Montaner, Asunción; Knecht, Erwin; Armengod, M-Eugenia

    2008-12-01

    Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases.

  12. Protein and DNA modifications: evolutionary imprints of bacterial biochemical diversification and geochemistry on the provenance of eukaryotic epigenetics.

    Science.gov (United States)

    Aravind, L; Burroughs, A Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M

    2014-07-01

    Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth's history. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  13. Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

    DEFF Research Database (Denmark)

    Brockdorff, J; Kanner, S B; Nielsen, M

    1998-01-01

    beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors...... and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation...... experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fak...

  14. Molecular modification of Protein A to improve the elution pH and alkali resistance in affinity chromatography.

    Science.gov (United States)

    Xia, Hai-Feng; Liang, Zhen-Dong; Wang, Sha-Li; Wu, Pu-Qiang; Jin, Xiong-Hua

    2014-04-01

    Protein A of Staphylococcus aureus has been widely used as an affinity ligand for the purification of immunoglobulin. However, the low elution pH and the sensitivity to alkaline condition restricted the large-scale application of antibody purification. To overcome these disadvantages, the B domain was selected and mutated to Z domain and the recombinant Protein A was reconstructed by linking five Z domains. First, a section of six glycines was inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.0-5.0. Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work confirmed the modification of Protein A and exhibited the characteristics of recombinant Staphylococcal Protein A for antibody purification.

  15. Opsonic activity of cell adhesion proteins and beta-1,3-glucan binding proteins from two crustaceans.

    Science.gov (United States)

    Thörnqvist, P O; Johansson, M W; Söderhäll, K

    1994-01-01

    A beta-1,3-glucan binding protein (beta GBP) from the shore crab Carcinus maenas was purified from plasma by precipitation of the protein at low ionic strength. The protein had a molecular mass of 110 kDa, and was shown to affinity precipitate with laminarin, a soluble beta-1,3-glucan, and to cross-react with an antiserum directed toward beta GBP from the crayfish Pacifastacus leniusculus. Also, a protein from the haemocytes of C. maenas with a molecular mass of 80 kDa was found to mediate cell attachment and cause degranulation of crab cells, similar to the 76 kDa protein present in the haemocytes of P. leniusculus. Antibodies against the crayfish 76-kDa protein reacted with the crab 80-kDa protein present in the granular cells. No 80-kDa protein could be found in the hyaline cells. Using a method with FITC-conjugated yeast particles in a phagocytosis assay, both the beta GBP and the 80-kDa protein from C. maenas were shown to have opsonic activity as had beta GBP and 76-kDa protein from P. leniusculus, resulting in higher levels of phagocytosis by the crab hyaline cells. Treatment of the yeast particles with beta GBP previously reacted with laminarin (beta GBP-L) only resulted in a minor increase of phagocytosis. Moreover, if the phagocytic cells were preincubated with beta GBP-L or with the 80-kDa protein, the enhancement of the phagocytic activity by beta GBP or the 80-kDa protein were abolished, indicating that a saturable number of one kind of cell surface receptor seem to be involved in phagocytosis.

  16. IL-2 induces beta2-integrin adhesion via a wortmannin/LY294002-sensitive, rapamycin-resistant pathway. Phosphorylation of a 125-kilodalton protein correlates with induction of adhesion, but not mitogenesis

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S

    1996-01-01

    beta2-integrin-dependent, homotypic adhesion in Ag-specific, human T cell lines. The IL-2 adhesion response is blocked by wortmannin and LY294002, inhibitors of phosphatidylinositol-3 (PI-3) kinase activity. In contrast, rapamycin strongly inhibits IL-2-induced proliferation without inhibiting IL-2...... on mitogenesis. IL-2R ligation rapidly (PI-3 kinase, and an as yet unidentified 125-kDa protein (p125). Wortmannin, LY294002......, and cytochalasin E almost completely inhibit cytokine-induced tyrosine phosphorylation of p125, whereas tyrosine phosphorylation of PI-3 kinase, Janus kinases, Stat3, Stat5, and other proteins is unaffected. In contrast, rapamycin has little effect on IL-2-induced phosphorylation of p125. Taken together...

  17. Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins.

    Science.gov (United States)

    Kilpatrick, Lisa E; Kilpatrick, Eric L

    2017-09-01

    Intact protein analysis by liquid chromatography-mass spectrometry (LC-MS) is now possible due to the improved capabilities of mass spectrometers yielding greater resolution, mass accuracy, and extended mass ranges. Concurrent measurement of post-translational modifications (PTMs) during LC-MS of intact proteins is advantageous while monitoring critical proteoform status, such as for clinical samples or during production of reference materials. However, difficulties exist for PTM identification when the protein is large or contains multiple modification sites. In this work, analyses of low-abundance proteoforms of proteins of clinical or therapeutic interest, including C-reactive protein, vitamin D-binding protein, transferrin, and immunoglobulin G (NISTmAb), were performed on an Orbitrap Elite mass spectrometer. This work investigated the effect of various instrument parameters including source temperatures, in-source CID, microscan type and quantity, resolution, and automatic gain control on spectral quality. The signal-to-noise ratio was found to be a suitable spectral attribute which facilitated identification of low abundance PTMs. Source temperature and CID voltage were found to require specific optimization for each protein. This study identifies key instrumental parameters requiring optimization for improved detection of a variety of PTMs by LC-MS and establishes a methodological framework to ensure robust proteoform identifications, the first step in their ultimate quantification.

  18. Surface-tethered polymers to influence protein adsorption and microbial adhesion

    NARCIS (Netherlands)

    Norde, Willem

    2007-01-01

    In various applications it is desired that biological cells or protein molecules are immobilized at surfaces. Examples are enzymes or cells in bioreactors and biosensors, immuno-proteins in solid-state diagnostics and proteinaceous farmacons in drug delivery systems. In order to retain biological

  19. Molecular cloning and characterization of a surface-localized adhesion protein in Mycoplasma bovis Hubei-1 strain.

    Directory of Open Access Journals (Sweden)

    Xiaohui Zou

    Full Text Available Mycoplasma bovis (M. bovis is an important pathogen that causes various bovine diseases, such as mastitis in cows and pneumonia in calves. The surface proteins are generally thought to play a central role in the pathogenesis of this organism. We screened the entire genome of M. bovis Hubei-1 and discovered a gene named vpmaX that encodes the 25 kDa variable surface lipoprotein A (VpmaX. Sequence analysis revealed that VpmaX contains several repetitive units and a typical bacterial lipoprotein signal sequence. The vpmaX gene was cloned and expressed in E. coli to obtain recombinant VpmaX (rVpmaX. Western blot analysis using a rabbit antibody against rVpmaX demonstrated that VpmaX is a membrane protein. Immunostaining visualized via confocal laser scanning microscopy showed that rVpmaX was able to adhere to embryonic bovine lung cells (EBL, and this was also confirmed by a sandwich ELISA. In summary, a surface-localized adhesion protein was identified in M. bovis Hubei-1.

  20. Posttranslational modification of Birch and Ragweed allergen proteins by common gas phase pollutants, NO2 and O3

    Science.gov (United States)

    Mahmood, M. A.; Pope, F.; Bloss, W.

    2015-12-01

    The global incidence of hay fever has been rising for decades, however, the underlying reasons behind this rise remain unclear. It is hypothesized that exposure of pollen to common gas phase pollutants, such as nitrogen dioxide (NO2) and ozone (O3), increases the allergenicity of the pollen and thus increases hay fever incidence. Since atmospheric pollutants tend to have greater concentrations within urban areas (in particular NO2) the hypothesis suggests that greater allergenicity should occur in urban areas. Indeed, several studies do suggest higher hay fever incidence within urban areas compared to rural areas. Previous published work suggests a link between increased allergies with changes in the chemical composition of the pollen protein via posttranslational modification of the protein. This study investigates the posttranslational modification of two highly allergenic pollen species (Birch and Ragweed) that are common in Europe. Within the laboratory, we expose pollen grains to atmospherically relevant exposures of gas phase NO2, O3 and other common gas phase oxidants under a range of environmentally relevant conditions. The effects of the environmentally relevant exposures on the biochemistry of the pollen grains were probed using a proteomic approach (liquid chromatography coupled ultra-high resolution spectrometer). Our findings indicate the interaction between gas phase pollutants and pollen cause protein specific modifications; in particular, nitration occurs upon tyrosine residues and nitrosylation on cysteine residues. Possibly, these modifications may affect the immune response of the pollen protein, which may suggest a possible reason for increased allergies in reaction to such biologically altered protein. The laboratory-derived results will be supported with a time series analysis of asthma incidence rates for the London area, which take into account the pollen count, and pollutant concentrations. The implications of the results will be discussed

  1. Protein phosphatase 2A plays a critical role in interleukin-2-induced beta 2-integrin dependent homotypic adhesion in human CD4+ T cell lines

    DEFF Research Database (Denmark)

    Brockdorff, J; Nielsen, M; Svejgaard, A

    1997-01-01

    A, blocks PP1/PP2A activity and IL-2 induced adhesion, whereas cyclosporin A, an inhibitor of protein serine/threonine phosphatase 2B (PP2B), does not, suggesting that PP1 and/or PP2A are involved in IL-2 induced adhesion. Endothall, which preferentially inhibits PP2A, strongly inhibited cytokine...... modulates enzymatic activity and/or subcellular distribution of serine/threonine phosphatases 1 and 2A (PP1/PP2A) in T cells, we examined the role of these phosphatases in IL-2 induced homotypic adhesion in antigen specific human CD4+ T cell lines. We show that calyculin A, a potent inhibitor of PP1 and PP2...... induced adhesion, whereas the structurally related compound 1,4-dimethylendothall had no effect on either phosphatase activity or the adhesion response. Okadaic acid, which preferentially inhibits PP2A, almost completely blocked IL-2-induced adhesion, whereas tautomycin, a potent inhibitor of PP1, had...

  2. The yeast SR-like protein Npl3 links chromatin modification to mRNA processing.

    Directory of Open Access Journals (Sweden)

    Erica A Moehle

    Full Text Available Eukaryotic gene expression involves tight coordination between transcription and pre-mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA-independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre-mRNA splicing.

  3. Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community

    Science.gov (United States)

    Li, Zhou; Wang, Yingfeng; Yao, Qiuming; Justice, Nicholas B.; Ahn, Tae-Hyuk; Xu, Dong; Hettich, Robert L.; Banfield, Jillian F.; Pan, Chongle

    2014-01-01

    Detailed characterization of post-translational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution. PMID:25059763

  4. Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Wang, Yingfeng [ORNL; Yao, Qiuming [University of Missouri, Columbia; Justice, Nicholas B. [University of California, Berkeley; Ahn, Tae-Hyuk [ORNL; Xu, Dong [University of Missouri, Columbia; Hettich, Robert {Bob} L [ORNL; Banfield, Jillian F. [University of California, Berkeley; Pan, Chongle [ORNL

    2014-01-01

    Detailed characterization of posttranslational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.

  5. Most m6A RNA modifications in protein-coding regions are evolutionarily unconserved and likely nonfunctional.

    Science.gov (United States)

    Liu, Zhen; Zhang, Jianzhi

    2017-12-08

    Methylation of the adenosine base at the nitrogen-6 position (m6A) is the most prevalent internal posttranscriptional modification of mRNAs in many eukaryotes. Despite the rapid progress in the transcriptome-wide mapping of m6As, identification of proteins responsible for writing, reading, and erasing m6As, and elucidation of m6A functions in splicing, RNA stability, translation, and other processes, it is unknown whether most observed m6A modifications are functional. To address this question, we respectively analyze the evolutionary conservation of yeast and human m6As in protein-coding regions. Relative to comparable unmethylated As, m6As are overall no more conserved in yeasts and only slightly more conserved in mammals. Furthermore, yeast m6As and comparable unmethylated As have no significant difference in single nucleotide polymorphism (SNP) density or SNP site frequency spectrum. The same is true in human. The methylation status of a gene, not necessarily the specific sites methylated in the gene, is subject to purifying selection for no more than ∼20% of m6A-modified genes. These observations suggest that most m6A modifications in protein-coding regions are nonfunctional and nonadaptive, probably resulting from off-target activities of m6A methyltransferases. In addition, our reanalysis invalidates the recent claim of positive selection for newly acquired m6A modifications in human evolution. Regarding the small number of evolutionarily conserved m6As, evidence suggests that a large proportion of them are likely functional; they should be prioritized in future functional characterizations of m6As. Together, these findings have important implications for understanding the biological significance of m6A and other posttranscriptional modifications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Carbohydrate mediated bacterial adhesion.

    Science.gov (United States)

    Pieters, Roland J

    2011-01-01

    In the process of adhesion, bacteria often carry proteins on their surface, adhesins, that bind to specific components of tissue cells or the extracellular matrix. In many cases these components are carbohydrate structures. The carbohydrate binding specificities of many bacteria have been uncovered over the years. The design and synthesis of inhibitors of bacterial adhesion has the potential to create new therapeutics for the prevention and possibly treatment of bacterial infections. Unfortunately, the carbohydrate structures often bind only weakly to the adhesion proteins, although drug design approaches can improve the situation. Furthermore, in some cases linking carbohydrates covalently together, to create so-called multivalent systems, can also significantly enhance the inhibitory potency. Besides adhesion inhibition as a potential therapeutic strategy, the adhesion proteins can also be used for detection. Novel methods to do this are being developed. These include the use of microarrays and glyconanoparticles. New developments in these areas are discussed.

  7. 2-DE and MS analysis of key proteins in the adhesion of Lactobacillus plantarum, a first step toward early selection of probiotics based on bacterial biomarkers

    NARCIS (Netherlands)

    Izquierdo, Esther; Horvatovich, Peter; Marchioni, Eric; Aoude-Werner, Dalal; Sanz, Yolanda; Ennahar, Saied

    The identification of cell components involved in probiotic activities is a challenge in current probiotic research. In this work, a new approach based on proteomics as an analytical tool for the identification of characteristic protein profiles related to adhesion to mucin as a model probiotic

  8. Comparison of adhesive properties of water- and phosphate-buffer-washed cottonseed meals with cottonseed protein isolate on maple and poplar veneers

    Science.gov (United States)

    Water- and phosphate buffer (35 mM Na2HPO4/NaH2PO4, pH 7.5)-washed cottonseed meals (abbreviated as WCM and BCM, respectively) could be low-cost and environmentally friendly protein-based adhesives as their preparation does not involve corrosive alkali and acid solutions that are needed for cottonse...

  9. Extraction of Jatropha curcas proteins and application in polyketone-based wood adhesives

    NARCIS (Netherlands)

    Hamarneh, A. I.; Heeres, H. J.; Broekhuis, A. A.; Picchioni, F.

    2010-01-01

    Jatropha proteins were successfully extracted from the corresponding seeds using the principle of isoelectric precipitation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), elemental analysis and Fourier transform infrared spectroscopy (FTIR) were used to analyze the obtained

  10. [Influence of different adhesive composition on sporulation and protein synthesis by Bacillus thuringiensis collection strains].

    Science.gov (United States)

    Krut', V V; Dankevych, L A; Votselko, S K; Patyka, V P

    2014-01-01

    The influence of different sticky-gene composition on sporulation and protein synthesis by B. thuringiensis collection strains has been investigated. It has been detemined that the most effective according this characteristics were B. thuringiensis collection strains 0293 and 98. It has been shown that the best on protein synthesis processes and sporulation by investigated B. thuringiensis strains influences adding to the culture medium sticky-gene compositions A and E in a concentration of from 10 to 15%.

  11. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins.

    Science.gov (United States)

    Wilson, Rashaun S; Swatek, Kirby N; Thelen, Jay J

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants.

  12. Laminin-511: a multi-functional adhesion protein regulating cell migration, tumor invasion and metastasis.

    Science.gov (United States)

    Pouliot, Normand; Kusuma, Nicole

    2013-01-01

    Laminins are major constituents of basement membranes. At least 16 isoforms have now been described, each with distinct spatio-temporal expression patterns and functions. The laminin-511 heterotrimer (α5β1γ1) is one of the more recent isoforms to be identified and a potent adhesive and pro-migratory substrate for a variety of normal and tumor cell lines in vitro. As our understanding of its precise function in normal tissues and in pathologies is rapidly unraveling, current evidence suggests an important regulatory role in cancer. This review describes published data on laminin-511 expression in several malignancies and experimental evidence from both in vitro and in vivo studies supporting its functional role during tumor progression. A particular emphasis is put on more recent studies from our laboratory and that of others indicating that laminin-511 contributes to tumor dissemination and metastasis in advanced breast carcinomas and other tumor types. Collectively, the experimental evidence suggests that high expression of laminin-511 has prognostic significance and that targeting tumor-laminin-511 interactions may have therapeutic potential in advanced cancer patients.

  13. Amine-functionalized polypyrrole: Inherently cell adhesive conducting polymer.

    Science.gov (United States)

    Lee, Jae Y; Schmidt, Christine E

    2015-06-01

    Electrically conducting polymers (CPs) have been recognized as novel biomaterials that can electrically communicate with biological systems. For their tissue engineering applications, CPs have been modified to promote cell adhesion for improved interactions between biomaterials and cells/tissues. Conventional approaches to improve cell adhesion involve the surface modification of CPs with biomolecules, such as physical adsorption of cell adhesive proteins and polycationic polymers, or their chemical immobilization; however, these approaches require additional multiple modification steps with expensive biomolecules. In this study, as a simple and effective alternative to such additional biomolecule treatment, we synthesized amine-functionalized polypyrrole (APPy) that inherently presents cell adhesion-supporting positive charges under physiological conditions. The synthesized APPy provides electrical activity in a moderate range and a hydrophilic surface compared to regular polypyrrole (PPy) homopolymers. Under both serum and serum-free conditions, APPy exhibited superior attachment of human dermal fibroblasts and Schwann cells compared to PPy homopolymer controls. Moreover, Schwann cell adhesion onto the APPy copolymer was at least similar to that on poly-l-lysine treated PPy controls. Our results indicate that amine-functionalized CP substrates will be useful to achieve good cell adhesion and potentially electrically stimulate various cells. In addition, amine functionality present on CPs can further serve as a novel and flexible platform to chemically tether various bioactive molecules, such as growth factors, antibodies, and chemical drugs. © 2014 Wiley Periodicals, Inc.

  14. Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search.

    Science.gov (United States)

    Shortreed, Michael R; Wenger, Craig D; Frey, Brian L; Sheynkman, Gloria M; Scalf, Mark; Keller, Mark P; Attie, Alan D; Smith, Lloyd M

    2015-11-06

    Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify post-translational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified over 2200 unique, high-confidence modified peptides comprising 26 different PTM types in a single-pass database search.

  15. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    KAUST Repository

    An, Xiaomeng

    2017-06-21

    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

  16. Multiple {gamma}-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, Mai [Bioscience Course, Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Ikeda, Yuka [Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda 386-8567 (Japan); Kanzawa, Hideaki [Department of Biology, Faculty of Science, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Sakamoto, Mika [Bioscience Course, Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Goto, Mina [Department of Biology, Faculty of Science, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Tsunasawa, Susumu [Analytical and Measuring Instruments Division, Shimadzu Corporation, Nishinokyo Kuwabaracho 1, Nakagyo-Ku, Kyoto 604-8511 (Japan); Uchiumi, Toshio, E-mail: uchiumi@bio.sc.niigata-u.ac.jp [Bioscience Course, Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Department of Biology, Faculty of Science, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Odani, Shoji [Bioscience Course, Graduate School of Science and Technology, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan); Department of Biology, Faculty of Science, Niigata University, 2-8050 Ikarashi, Nishi-Ku, Niigata 950-2181 (Japan)

    2010-03-26

    A highly hydrophilic, glutamate-rich protein was identified in the aqueous phenol extract from the cytosolic fraction of brine shrimp (Artemia franciscana) diapausing cysts and termed Artemia phenol soluble protein (PSP). Mass spectrometric analysis revealed the presence of many protein peaks around m/z 11,000, separated by 129 atomic mass units; this value corresponds to that of glutamate, which is strongly suggestive of heterogeneous polyglutamylation. Polyglutamylation has long been known as the functionally important post-translational modification of tubulins, which carry poly(L-glutamic acid) chains of heterogeneous length branching off from the main chain at the {gamma}-carboxy groups of a few specific glutamate residues. In Artemia PSP, however, Edman degradation of enzymatic peptides revealed that at least 13, and presumably 16, glutamate residues were modified by the attachment of a single L-glutamate, representing a hitherto undescribed type of post-translational modification: namely, multiple {gamma}-glutamylation or the addition of a large number of glutamate residues along the polypeptide chain. Although biological significance of PSP and its modification is yet to be established, suppression of in vitro thermal aggregation of lactate dehydrogenase by glutamylated PSP was observed.

  17. topPTM: a new module of dbPTM for identifying functional post-translational modifications in transmembrane proteins.

    Science.gov (United States)

    Su, Min-Gang; Huang, Kai-Yao; Lu, Cheng-Tsung; Kao, Hui-Ju; Chang, Ya-Han; Lee, Tzong-Yi

    2014-01-01

    Transmembrane (TM) proteins have crucial roles in various cellular processes. The location of post-translational modifications (PTMs) on TM proteins is associated with their functional roles in various cellular processes. Given the importance of PTMs in the functioning of TM proteins, this study developed topPTM (available online at http://topPTM.cse.yzu.edu.tw), a new dbPTM module that provides a public resource for identifying the functional PTM sites on TM proteins with structural topology. Experimentally verified TM topology data were integrated from TMPad, TOPDB, PDBTM and OPM. In addition to the PTMs obtained from dbPTM, experimentally verified PTM sites were manually extracted from research articles by text mining. In an attempt to provide a full investigation of PTM sites on TM proteins, all UniProtKB protein entries containing annotations related to membrane localization and TM topology were considered potential TM proteins. Two effective tools were then used to annotate the structural topology of the potential TM proteins. The TM topology of TM proteins is represented by graphical visualization, as well as by the PTM sites. To delineate the structural correlation between the PTM sites and TM topologies, the tertiary structure of PTM sites on TM proteins was visualized by Jmol program. Given the support of research articles by manual curation and the investigation of domain-domain interactions in Protein Data Bank, 1347 PTM substrate sites are associated with protein-protein interactions for 773 TM proteins. The database content is regularly updated on publication of new data by continuous surveys of research articles and available resources.

  18. Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches.

    Science.gov (United States)

    Mast, Carole; Lyan, Bernard; Joly, Charlotte; Centeno, Delphine; Giacomoni, Franck; Martin, Jean-François; Mosoni, Laurent; Dardevet, Dominique; Pujos-Guillot, Estelle; Papet, Isabelle

    2015-04-29

    Liver protein can be altered under paracetamol (APAP) treatment. APAP-protein adducts and other protein modifications (oxidation/nitration, expression) play a role in hepatotoxicity induced by acute overdoses, but it is unknown whether liver protein modifications occur during long-term treatment with non-toxic doses of APAP. We quantified APAP-protein adducts and assessed other protein modifications in the liver from rats under chronic (17 days) treatment with two APAP doses (0.5% or 1% of APAP in the diet w/w). A targeted metabolomic method was validated and used to quantify APAP-protein adducts as APAP-cysteine adducts following proteolytic hydrolysis. The limit of detection was found to be 7ng APAP-cysteine/mL hydrolysate i.e. an APAP-Cys to tyrosine ratio of 0.016‰. Other protein modifications were assessed on the same protein hydrolysate by untargeted metabolomics including a new strategy to process the data and identify discriminant molecules. These two complementary mass spectrometry (MS)-based metabolic approaches enabled the assessment of a wide range of protein modifications induced by chronic treatment with APAP. APAP-protein adducts were detected even in the absence of glutathione depletion and hepatotoxicity, i.e. in the 0.5% APAP group, and increased by 218% in the 1% APAP group compared to the 0.5% APAP group. At the same time, the untargeted metabolomic method revealed a decrease in the binding of cysteine, cysteinyl-glycine and GSH to thiol groups of protein cysteine residues, an increase in the oxidation of tryptophan and proline residues and a modification in protein expression. This wide range of modifications in liver proteins occurred in rats under chronic treatment with APAP that did not induce hepatotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    Science.gov (United States)

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-01-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials. PMID:28378749

  20. Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

    Science.gov (United States)

    Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

    2017-04-01

    Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

  1. Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

    Science.gov (United States)

    Liu, Tong; Zhang, Wanjun; Zhang, Zheng; Chen, Mingli; Wang, Jianhua; Qian, Xiaohong; Qin, Weijie

    2018-02-06

    Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

  2. Systematic screening of protein modifications in four kinases using affinity enrichment and mass spectrometry analysis with unrestrictive sequence alignment.

    Science.gov (United States)

    Zhang, Kai; Zhu, Yixin; He, Xiwen; Zhang, Yukui

    2011-04-08

    Protein kinases transfer phosphate groups from ATP to substrate proteins, they are known to be involved in diverse cellular processes. They are also important therapeutic targets in pharmaceutical design. Previous studies indicated that multiple post-translational modifications (PTMs) exist in kinases in addition to phosphorylation, and these PTMs play an important role in regulating kinases activities. Nevertheless, a comprehensive analysis for PTMs of kinases is insufficient due to technical limitations, which prevent us from better understanding their functional regulation. Here, we have developed a novel strategy that combines glutathione S-transferase tag affinity enrichment with nano-liquid chromatography coupled with tandem mass spectrometry analysis and non-restrictive protein sequence alignment for identification of diverse PTMs in four yeast kinases. The method allows us to enrich and analyze the entire protein isomers and to minimize the loss of all isomers of protein sample during protein purification. In our study, nineteen phosphorylation sites and several other types of PTMs sites were localized in 4 protein kinases. In addition, we found that some interesting mass shifts can not match those of the known PTMs. It suggested the existence of some undescribed PTMs in the proteins. Accordingly, this study showed that the novel strategy holds a great potential for identification of full-spectrum PTMs in proteins. Our data serves as a stepping stone for future functional studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. S-(2-Succinyl)cysteine: a novel chemical modification of tissue proteins by a Krebs cycle intermediate.

    Science.gov (United States)

    Alderson, Nathan L; Wang, Yuping; Blatnik, Matthew; Frizzell, Norma; Walla, Michael D; Lyons, Timothy J; Alt, Nadja; Carson, James A; Nagai, Ryoji; Thorpe, Suzanne R; Baynes, John W

    2006-06-01

    S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

  4. Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: A cell-adhesive and plasmin-degradable biosynthetic material for tissue repair

    Science.gov (United States)

    Halstenberg, Sven

    2002-01-01

    The goal of the research presented in this dissertation was to create a biomimetic artificial material that exhibits functions of extracellular matrix relevant for improved nerve regeneration. Neural adhesion peptides were photoimmobilized on highly crosslinked poly(ethylene glycol)-based substrates that were otherwise non-adhesive. Neurons adhered in two-dimensional patterns for eleven hours, but no neurites extended. To enable neurite extension and nerve regeneration in three dimensions, and to address the need for specifically cell adhesive and cell degradable materials for clinical applications in tissue repair in general, an artificial protein was recombinantly expressed and purified that consisted of a repeating amino acid sequence based on fibrinogen and anti-thrombin III. The recombinant protein contained integrin-binding RGD sites, plasmin degradation sites, heparin binding sites, and six thiol-containing cysteine residues as grafting sites for poly(ethylene glycol) diacrylate via Michael-type conjugate addition. The resulting protein-graft-poly(ethylene glycol)acrylates were crosslinked by photopolymerization to form hydrogels. Although three-dimensional, RGD mediated and serine protease-dependent ingrowth of human fibroblasts into protein-graft-poly(ethylene glycol) hydrogels occurred, only surface neurite outgrowth was observed from chick dorsal root ganglia. Axonal outgrowth depended on the concentration of matrix-bound heparin, suggesting that improved mechanical strength of the hydrogels and possible immobilization of neuroactive factors due to the presence of heparin promoted neurite outgrowth. Together, the above results show that specific biological functions can be harnessed by protein-graft-poly(ethylene glycol) hydrogels to serve as matrices for tissue repair and regeneration. In particular, the two design objectives, specific cell adhesion and degradability by cell-associated proteases, were fulfilled by the material. In the future, this and

  5. Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

    Directory of Open Access Journals (Sweden)

    Alleaume-Butaux A

    2013-07-01

    Full Text Available Aurélie Alleaume-Butaux,1,2 Caroline Dakowski,1,2 Mathéa Pietri,1,2 Sophie Mouillet-Richard,1,2 Jean-Marie Launay,3,4 Odile Kellermann,1,2 Benoit Schneider1,2 1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris Cité, UMR-S 747, 3Public Hospital of Paris, Department of Biochemistry, INSERM UMR-S 942, Lariboisière Hospital, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland Abstract: Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1 neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2 neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3 the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC, a glycosylphosphatidylinositol (GPI-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that Pr

  6. Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Rossi, Barbara; Zenaro, Elena; Angiari, Stefano; Ottoboni, Linda; Bach, Simona; Piccio, Laura; Pietronigro, Enrica C; Scarpini, Elio; Fusco, Mariella; Leon, Alberta; Constantin, Gabriela

    2011-04-01

    It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show

  7. cAMP receptor protein regulates mouse colonization, motility, fimbria-mediated adhesion, and stress tolerance in uropathogenic Proteus mirabilis

    National Research Council Canada - National Science Library

    Yi-Lin Tsai; Hsiung-Fei Chien; Kuo-Tong Huang; Wen-Yuan Lin; Shwu-Jen Liaw

    2017-01-01

    .... This study demonstrated that Crp affects numerous virulence-related phenotypes, including colonization of mice, motility, fimbria-mediated adhesion, and glucose stress tolerance in uropathogenic Proteus mirabilis...

  8. A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    Rocha H.A.O.

    2001-01-01

    Full Text Available Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1 and the mutant type deficient in xylosyltransferase (CHO-745. The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5(and CHO-745 (2 x 10(5 and 5 x 10(5 cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.

  9. POTAMOS mass spectrometry calculator: computer aided mass spectrometry to the post-translational modifications of proteins. A focus on histones.

    Science.gov (United States)

    Vlachopanos, A; Soupsana, E; Politou, A S; Papamokos, G V

    2014-12-01

    Mass spectrometry is a widely used technique for protein identification and it has also become the method of choice in order to detect and characterize the post-translational modifications (PTMs) of proteins. Many software tools have been developed to deal with this complication. In this paper we introduce a new, free and user friendly online software tool, named POTAMOS Mass Spectrometry Calculator, which was developed in the open source application framework Ruby on Rails. It can provide calculated mass spectrometry data in a time saving manner, independently of instrumentation. In this web application we have focused on a well known protein family of histones whose PTMs are believed to play a crucial role in gene regulation, as suggested by the so called "histone code" hypothesis. The PTMs implemented in this software are: methylations of arginines and lysines, acetylations of lysines and phosphorylations of serines and threonines. The application is able to calculate the kind, the number and the combinations of the possible PTMs corresponding to a given peptide sequence and a given mass along with the full set of the unique primary structures produced by the possible distributions along the amino acid sequence. It can also calculate the masses and charges of a fragmented histone variant, which carries predefined modifications already implemented. Additional functionality is provided by the calculation of the masses of fragments produced upon protein cleavage by the proteolytic enzymes that are most widely used in proteomics studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. The Role of Histone Protein Modifications and Mutations in Histone Modifiers in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia

    Science.gov (United States)

    Janczar, Szymon; Janczar, Karolina; Pastorczak, Agata; Harb, Hani; Paige, Adam J. W.; Zalewska-Szewczyk, Beata; Danilewicz, Marian; Mlynarski, Wojciech

    2017-01-01

    While cancer has been long recognized as a disease of the genome, the importance of epigenetic mechanisms in neoplasia was acknowledged more recently. The most active epigenetic marks are DNA methylation and histone protein modifications and they are involved in basic biological phenomena in every cell. Their role in tumorigenesis is stressed by recent unbiased large-scale studies providing evidence that several epigenetic modifiers are recurrently mutated or frequently dysregulated in multiple cancers. The interest in epigenetic marks is especially due to the fact that they are potentially reversible and thus druggable. In B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) there is a relative paucity of reports on the role of histone protein modifications (acetylation, methylation, phosphorylation) as compared to acute myeloid leukemia, T-cell ALL, or other hematologic cancers, and in this setting chromatin modifications are relatively less well studied and reviewed than DNA methylation. In this paper, we discuss the biomarker associations and evidence for a driver role of dysregulated global and loci-specific histone marks, as well as mutations in epigenetic modifiers in BCP-ALL. Examples of chromatin modifiers recurrently mutated/disrupted in BCP-ALL and associated with disease outcomes include MLL1, CREBBP, NSD2, and SETD2. Altered histone marks and histone modifiers and readers may play a particular role in disease chemoresistance and relapse. We also suggest that epigenetic regulation of B-cell differentiation may have parallel roles in leukemogenesis. PMID:28054944

  11. The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases.

    Science.gov (United States)

    Voisin, Sébastien; Watson, David C; Tessier, Luc; Ding, Wen; Foote, Simon; Bhatia, Smita; Kelly, John F; Young, N Martin

    2007-12-01

    We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.

  12. O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection.

    Science.gov (United States)

    Pérez, José de Jesús; Udeshi, Namrata D; Shabanowitz, Jeffrey; Ciordia, Sergio; Juárez, Silvia; Scott, Cheryl L; Olszewski, Neil E; Hunt, Donald F; García, Juan Antonio

    2013-08-01

    O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Mussel adhesion - essential footwork.

    Science.gov (United States)

    Waite, J Herbert

    2017-02-15

    Robust adhesion to wet, salt-encrusted, corroded and slimy surfaces has been an essential adaptation in the life histories of sessile marine organisms for hundreds of millions of years, but it remains a major impasse for technology. Mussel adhesion has served as one of many model systems providing a fundamental understanding of what is required for attachment to wet surfaces. Most polymer engineers have focused on the use of 3,4-dihydroxyphenyl-l-alanine (Dopa), a peculiar but abundant catecholic amino acid in mussel adhesive proteins. The premise of this Review is that although Dopa does have the potential for diverse cohesive and adhesive interactions, these will be difficult to achieve in synthetic homologs without a deeper knowledge of mussel biology; that is, how, at different length and time scales, mussels regulate the reactivity of their adhesive proteins. To deposit adhesive proteins onto target surfaces, the mussel foot creates an insulated reaction chamber with extreme reaction conditions such as low pH, low ionic strength and high reducing poise. These conditions enable adhesive proteins to undergo controlled fluid-fluid phase separation, surface adsorption and spreading, microstructure formation and, finally, solidification. © 2017. Published by The Company of Biologists Ltd.

  14. Allozyme-specific modification of a maize seed chitinase by a protein secreted by the fungal pathogen Stenocarpella maydis.

    Science.gov (United States)

    Naumann, Todd A; Wicklow, Donald T

    2010-07-01

    Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

  15. Bacterial adhesion to orthopaedic implant materials and a novel oxygen plasma modified PEEK surface.

    Science.gov (United States)

    Rochford, E T J; Poulsson, A H C; Salavarrieta Varela, J; Lezuo, P; Richards, R G; Moriarty, T F

    2014-01-01

    Despite extensive use of polyetheretherketone (PEEK) in biomedical applications, information about bacterial adhesion to this biomaterial is limited. This study investigated Staphylococcus aureus and Staphylococcus epidermidis adhesion to injection moulded and machined PEEK OPTIMA(®) using a custom-built adhesion chamber with medical grade titanium and Thermanox for comparison. Additionally, bacterial adhesion to a novel oxygen plasma modified PEEK was also investigated in both a pre-operative model in physiological saline, and additionally in a post-operative model in human blood plasma. In the pre-operative model, the rougher machined PEEK had a significantly greater number of adherent bacteria compared to injection moulded PEEK. Bacterial adhesion to titanium and Thermanox was similar. Oxygen plasma surface modification of PEEK did not lead to a significant change in bacterial adhesion in the pre-operative contamination model, despite observed changes in surface characteristics. In the post-operative contamination model, S. aureus adhesion was increased from 5×10(5) CFU cm(-2) to approximately 1.3×10(7) CFU cm(-2) on the modified surfaces due to differential protein adhesion during the conditioning period. However, S. epidermidis adhesion to modified PEEK was less than to unmodified PEEK in the post-operative model. These results illustrate the importance of testing bacterial adhesion of several strains in both a pre-operative and post-operative, clinically relevant bacterial contamination model. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Fluorescent protein-imprinted polymers capable of signal transduction of specific binding events prepared by a site-directed two-step post-imprinting modification.

    Science.gov (United States)

    Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2014-02-09

    Protein recognition polymers capable of highly specific transduction of protein binding events into fluorescence change were prepared by molecular imprinting in conjunction with a newly developed two-step post-imprinting chemical modification of functional groups located within the protein recognition cavity.

  17. Increase of the adhesion ability and display of a rumen fungal xylanase on the cell surface of Lactobacillus casei by using a listerial cell-wall-anchoring protein.

    Science.gov (United States)

    Hsueh, Hsiang-Yun; Yu, Bi; Liu, Chi-Te; Liu, Je-Ruei

    2014-02-01

    Lactobacillus, which has great adhesion ability to intestinal mucosa and is able to hydrolyse plant cell walls, can be used more efficiently as a feed additive. To increase the adhesion ability and display a fungal xylanase on the cell surface of Lactobacillus casei, the Listeria monocytogenes cell-wall-anchoring protein gene, mub, was introduced into L. casei ATCC 393 cells and used as a fusion partner to display the rumen fungal xylanase XynCDBFV on the cell surface of the transformed strains. The transformed strain L. casei pNZ-mub, which harboured mub gene, displayed recombinant Mub on its cell surface and showed greater adhesion ability to Caco-2 cells than the parental strain. The transformed strain L. casei pNZ-mub/xyn, which harboured mub-xynCDBFV fusion gene, acquired the capacity to break down oat spelt xylan and exhibited greater competition ability against the adhesion of L. monocytogenes to Caco-2 cells, in comparison with the parental strain. Mub has a potential to be used as a fusion partner to display heterologous proteins on the cell surface of Lactobacillus. Moreover, this is the first report of the successful display of xylanase on the cell surface of Lactobacillus. © 2013 Society of Chemical Industry.

  18. Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Doug A Medvetz

    Full Text Available Birt-Hogg-Dube (BHD is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN, the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1 activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

  19. Using ProteomeScout: A Resource of Post-Translational Modifications, Their Experiments, and the Proteins That They Annotate.

    Science.gov (United States)

    Mooradian, Arshag D; Held, Jason M; Naegle, Kristen M

    2017-09-13

    Post-translational modifications (PTMs) of protein amino acids are ubiquitous and important to protein function, localization, degradation, and more. In recent years, there has been an explosion in the discovery of PTMs as a result of improvements in PTM measurement techniques, including quantitative measurements of PTMs across multiple conditions. ProteomeScout is a repository for such discovery and quantitative experiments and provides tools for visualizing PTMs within proteins, including where they are relative to other PTMS, domains, mutations, and structure. ProteomeScout additionally provides analysis tools for identifying statistically significant relationships in experimental datasets. This unit describes four basic protocols for working with the ProteomeScout Web interface or programmatically with the database download. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  20. In vitro studies of platelet adhesion, activation, and protein adsorption on curcumin-eluting biodegradable stent materials.

    Science.gov (United States)

    Pan, C J; Shao, Z Y; Tang, J J; Wang, J; Huang, N

    2007-09-01

    A major complication of coronary stenting is in-stent restenosis (ISR) due to thrombus formation. We hypothesized that locally released curcumin from coronary stent surface would inhibit ISR due to thrombus formation because of antithrombosis of curcumin. In the present work, curcumin-eluting polylactic acid-co-glycolic acid (PLGA) films were fabricated and their properties in vitro were investigated. The in vitro platelet adhesion and activation, as well as protein adsorption on curcumin-loading PLGA films were investigated to evaluate the blood compatibility of curcumin-eluting films. The structure of curcumin-eluting PLGA film and control was examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicating that the peaks of curcumin did not shift in curcumin-eluting films. The results of contact angle and surface free energy indicated that loading curcumin in PLGA would make PLGA become more hydrophilic, which contributed to the increase of polar fraction of surface free energy. With the increase of curcumin in films, platelets adhering to the curcumin-eluting films decreased significantly. The number of activation platelets decreased after incorporating curcumin in PLGA films. Loading curcumin in PLGA film can markedly reduce the fibrinogen adsorption. All results indicated that incorporating curcumin in PLGA film can improve the blood compatibility of PLGA films. It can be used to fabricate drug-eluting stent to prevent thrombosis formation.

  1. Heat shock protein 90β stabilizes focal adhesion kinase and enhances cell migration and invasion in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiangyang [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi 330006 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Wang, Yao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Liu, Chengmei [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047 (China); Lu, Quqin [Department of Biostatistics and Epidemiology, School of Public Health, Nanchang University, Nanchang, Jiangxi 330006 (China); Liu, Tao [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006 (China); Chen, Guoan [Department of Hematology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006 (China); Rao, Hai [Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78229 (United States); Luo, Shiwen, E-mail: shiwenluo@ncu.edu.cn [Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, 17 Yongwai Street, Donghu District, Nanchang, Jiangxi 330006