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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

    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

  2. Laser surface modification and adhesion

    CERN Document Server

    Mittal, K L

    2014-01-01

    The book provides a unique overview on laser techniques and applications for the purpose of improving adhesion by altering surface chemistry and topography/morphology of the substrate. It details laser surface modification techniques for a wide range of industrially relevant materials (plastics, metals, ceramics, composites) with the aim to improve and enhance their adhesion to other materials. The joining of different materials is of critical importance in the fabrication of many and varied products.

  3. Laser- and UV-assisted modification of polystyrene surfaces for control of protein adsorption and cell adhesion

    Science.gov (United States)

    Pfleging, Wilhelm; Torge, Maika; Bruns, Michael; Trouillet, Vanessa; Welle, Alexander; Wilson, Sandra

    2009-03-01

    An appropriate choice of laser and process parameters enables new approaches for the fabrication of polymeric lab-on-chip devices with integrated functionalities. We will present our current research results in laser-assisted modification of polystyrene (PS) with respect to the fabrication of polymer devices for cell culture applications. For this purpose laser micro-patterning of PS and subsequent surface functionalization was investigated as function of laser and process parameters. A high power ArF-excimer laser radiation source with a pulse length of 19 ns as well as a high repetition ArF-excimer laser source with a pulse length of 5 ns were used in order to study the influence of laser pulse length on laser-induced surface oxidation. The change in surface chemistry was characterized by X-ray photoelectron spectroscopy and contact angle measurements. The difference between laser-assisted modification versus UV-lamp assisted modification was investigated. A photolytic activation of specific areas of the polymer surface and subsequent oxidization in oxygen or ambient air leads to a chemically modified polymer surface bearing carboxylic acid groups well-suited for controlled competitive protein adsorption or protein immobilization. Finally, distinct areas for cell growth and adhesion are obtained.

  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. 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

  6. Modification on epoxy-based adhesive

    Institute of Scientific and Technical Information of China (English)

    ZhengXiaoxia; QianChunxiang

    2003-01-01

    This research adopted four methods to toughen epoxy adhesives. They were liquid hydroxyl group terminated polybutadiene (HTPB) rubber modification, silicon rubber modification, polyacrylate multiplicity elastomer particulates emulsion modification and chemical grafting modification. After modification, the shearing strength and the rapture elongation were tested. The interface and the chemical reaction between the modifiers and the epoxy were analyzed by scanning electron microscope (SEM) and infrared optical spectrum. The results show that the elastomer particulates modification and the chemical grafting modification can reach the better toughening effects.

  7. 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...

  8. Modification of gold surface by grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion.

    Science.gov (United States)

    Zhang, F; Kang, E T; Neoh, K G; Huang, W

    2001-01-01

    Gold surfaces were first treated in an alkanethiol solution to form self-assembled monolayers (SAMs). The thiolated Au surface was then subjected to Ar plasma pretreatment, followed by air exposure and UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer. In comparison with the 3-mercaptopropionic acid-2-ethylhexyl ester (MPAEE) SAM, the (3-mercaptoproply)trimethoxysilane (MPTMS) SAM on Au exhibited higher stability under the conditions of Ar plasma pretreatment. The graft concentration of the PEGMA polymer on SAM-modified Au surface increased with increasing PEGMA macromonomer concentration and UV-graft polymerization time. The modified-Au surfaces were characterized by X-ray spectroscopy (XPS), atomic force microscopy (AFM), and water contact angle measurement. The Au surface with a high concentration of grafted PEGMA polymer could completely repel protein adsorption and platelet adhesion.

  9. Investigation of modified cottonseed protein adhesives for wood composites

    Science.gov (United States)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  10. Modification of Si(100) surface by the grafting of poly(ethylene glycol) for reduction in protein adsorption and platelet adhesion.

    Science.gov (United States)

    Zhang, F; Kang, E T; Neoh, K G; Wang, P; Tan, K L

    2001-09-05

    The modification of argon plasma-pretreated single-crystal Si(100) wafer surfaces via the UV-induced graft polymerization of poly(ethylene glycol) methacrylate (PEGMA) macromonomer (molecular weight approximately 340) for biomaterials applications was explored. The modified Si(100) surfaces were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Surface peroxide concentrations resulting from the argon plasma treatment and subsequent atmospheric exposure were determined by a coupling reaction with diphenylpicrylhydrazyl. The results suggested that a short plasma treatment time of 10 s and brief air exposure were sufficient for generating an optimum amount of peroxides and hydroperoxides for the subsequent UV-induced graft polymerization. The graft concentration of the PEGMA polymer increased with increasing PEGMA macromonomer concentration for the graft polymerization and with increasing UV graft polymerization time. The PEGMA graft-polymerized silicon surface with a high poly(ethylene glycol) graft concentration was very effective in preventing protein adsorption and platelet adhesion. The grafted PEGMA polymer layer on the Si(100) surface exhibited fairly good stability during storage in a buffer solution.

  11. 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...

  12. 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.

  13. Impact of Acquired Pellicle Modification on Adhesion of Early Colonizers.

    Science.gov (United States)

    Cheaib, Zeinab; Rakmathulina, Ekaterina; Lussi, Adrian; Eick, Sigrun

    2015-01-01

    New preventive approaches against dental erosion caused by acidic drinks and beverages include fortification of beverages with natural polymers. We have shown that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. This study aimed to investigate the effect of pellicle modification by casein, mucin and a casein-mucin mixture on the adhesion of early bacterial colonizers. Test specimens of human tooth enamel were prepared, covered with saliva and coated with 0.5% aqueous (aq.) casein, 0.27% aq. mucin or with 0.5% aq. casein-0.27% aq. mucin, after which the adhesion of Streptococcus gordonii, Streptococcus oralis, and Actinomyces odontolyticus was measured after incubation for 30 min and 2 h. log10 colony-forming units were compared by nonparametric tests. All three bacterial strains adhered in higher number to pellicle-coated enamel than to native enamel. The protein modifications of pellicle all decreased the counts of adhering bacteria up to 0.34 log10/mm2, the most efficient being the casein-mucin mixture. In addition to the recently shown erosion-reducing effect by casein-mucin, modification of the pellicle may inhibit bacterial adherence compared to untreated human pellicle.

  14. Surface modification of ultrahigh molecular weight polyethylene by the poly(ethylene glycol)-grafted method and its effect on the adsorption of proteins and the adhesion of blood platelets.

    Science.gov (United States)

    Xia, Bing; Xie, Meiju; Yang, Bangcheng

    2013-01-01

    With the help of a silane coupling agent, poly(ethylene glycol) (PEG), a well-biocompatable agent, was grafted onto the surface of ultrahigh molecular weight polyethylene (UHMWPE) by ultraviolet initiation. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analysis proved the success of PEG grafting. Water contact angle measurement showed that the modified UHMWPE was obviously improved in surface hydrophilicity and thermogravimetric analysis result showed that its thermostability did not decline even it was pretreated by strong acids. Then, the protein adsorption of the modified UHMWPE was investigated using three model proteins including bovine serum albumin, lysozyme, and fibrinogen. Rabbit blood was used to study the platelet adhesion on the surface of modified UHMWPE. The results indicated that the quantity of protein adsorption on the modified UHMWPE grafted PEG reduced apparently for all the model proteins while there was some specific differences or exceptions among them. It was ascribed to the changed surface chemical composition, surface hydrophilicity and surface topography after modification. The adhesive ability of blood platelets on the modified surface of UHMWPE decreased after PEG grafting. Owing to the improved resistance to fibrinogen adsorption and platelet adhesion, the surface modification might endow the UHWMPE surface better anticoagulation ability according to clotting mechanism.

  15. Posttranslational protein modification in Archaea.

    Science.gov (United States)

    Eichler, Jerry; Adams, Michael W W

    2005-09-01

    One of the first hurdles to be negotiated in the postgenomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various posttranslational modifications that proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation, and proteolytic cleavage. Whereas these and other posttranslational protein modifications have been well characterized in Eucarya and Bacteria, posttranslational modification in Archaea has received far less attention. Although archaeal proteins can undergo posttranslational modifications reminiscent of what their eucaryal and bacterial counterparts experience, examination of archaeal posttranslational modification often reveals aspects not previously observed in the other two domains of life. In some cases, posttranslational modification allows a protein to survive the extreme conditions often encountered by Archaea. The various posttranslational modifications experienced by archaeal proteins, the molecular steps leading to these modifications, and the role played by posttranslational modification in Archaea form the focus of this review.

  16. Chemical Protein Modification through Cysteine.

    Science.gov (United States)

    Gunnoo, Smita B; Madder, Annemieke

    2016-04-01

    The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance--both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.

  17. Posttranslational Protein Modification in Archaea

    OpenAIRE

    Eichler, Jerry; Adams, Michael W. W.

    2005-01-01

    One of the first hurdles to be negotiated in the postgenomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various posttranslational modifications that proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation, and proteolytic cleavage. Whereas these and other posttranslational protein modifications have been well characterized in Eucarya and B...

  18. Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification.

    Science.gov (United States)

    Tang, Chad; Kligman, Faina; Larsen, Coby C; Kottke-Marchant, Kandice; Marchant, Roger E

    2009-02-01

    A prominent failure mechanism of small diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts is platelet-mediated thrombosis. We have designed a surface modification for ePTFE consisting of a self-assembling fluorosurfactant polymer (FSP) bearing biologically active ligands, including adhesive peptides and polysaccharide moieties. The goal of this biomimetic construct is to improve graft hemocompatibility by promoting rapid surface endothelialization, whereas minimizing platelet adhesion. Here we present a direct comparison of platelet and endothelial cell (EC) adhesion to FSPs containing one of three cell-adhesion peptides: cyclic Arg-Gly-Asp-D-Phe-Glu (cRGD), cyclic *Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys* (cRRE, *denotes disulfide bond cyclization), linear Gly-Arg-Gly-Asp-Ser-Pro-Ala (RGD), or a polysaccharide moiety: oligomaltose (M-7), later designed to prevent nonspecific protein adhesion. Measurements of soluble peptide-integrin binding indicated that cRRE exhibits very low affinity for the alpha(IIb)beta(3) platelet fibrinogen receptor. Static and dynamic adhesion of washed, activated platelets on FSP-modified surfaces revealed that M-7 and cRRE promote significantly less platelet adhesion compared to RGD and cRGD FSPs, whereas EC adhesion was similar on all peptide FSPs and minimal on M-7 FSP. These results illustrate the potential for ligands presented in a FSP surface modification to selectively adhere ECs with limited platelet attachment.

  19. 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...

  20. Chemical Modification of Food Proteins

    Institute of Scientific and Technical Information of China (English)

    Allaoua Achouri; Wang Zhang; Xu Shiying

    1999-01-01

    Acylation has been shown to be an effective tool for improving surface functional properties of plant proteins.Soy bean protein has been extensively modified through chemical and enzymatic treatments. Their effectiveness lies in their high nutritional value and low cost, which promote their use as ingredients for the formulation of food products.This paper reports a complete review of chemical modification of various proteins from plant and animal sources. The nutritive and toxicological aspects through in vitro and in vivo tests are also described.

  1. 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.

  2. 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.

  3. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    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. The MRL proteins: adapting cell adhesion, migration and growth.

    Science.gov (United States)

    Coló, Georgina P; Lafuente, Esther M; Teixidó, Joaquin

    2012-01-01

    MIG-10, RIAM and Lamellipodin (Lpd) are the founding members of the MRL family of multi-adaptor molecules. These proteins have common domain structures but display distinct functions in cell migration and adhesion, signaling, and in cell growth. The binding of RIAM with active Rap1 and with talin provides these MRL molecules with important regulatory roles on integrin-mediated cell adhesion and migration. Furthermore, RIAM and Lpd can regulate actin dynamics through their binding to actin regulatory Ena/VASP proteins. Recent data generated with the Drosophila MRL ortholog called Pico and with RIAM in melanoma cells indicate that these proteins can also regulate cell growth. As MRL proteins represent a relatively new family, many questions on their structure-function relationships remain unanswered, including regulation of their expression, post-translational modifications, new interactions, involvement in signaling and their knockout mice phenotype.

  5. 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-...

  6. Glycosylated hydroxytryptophan in a mussel adhesive protein from Perna viridis.

    Science.gov (United States)

    Zhao, Hua; Sagert, Jason; Hwang, Dong Soo; Waite, J Herbert

    2009-08-28

    The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW(1)TAW(2)K and APPPAW(1)TAW(2)K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C(2)-hexosylated tryptophan (W(1)) and C(2)-hexosylated hydroxytryptophan (W(2)), the latter of which is redox active. The UV absorbance spectrum of W(2) is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.

  7. Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis*

    Science.gov (United States)

    Zhao, Hua; Sagert, Jason; Hwang, Dong Soo; Waite, J. Herbert

    2009-01-01

    The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW1TAW2K and APPPAW1TAW2K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C2-hexosylated tryptophan (W1) and C2-hexosylated hydroxytryptophan (W2), the latter of which is redox active. The UV absorbance spectrum of W2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion. PMID:19584055

  8. Purification of adhesive proteins from mussels.

    Science.gov (United States)

    Pardo, J; Gutierrez, E; Sáez, C; Brito, M; Burzio, L O

    1990-11-01

    The adhesive polyphenolic proteins from the mussels Mytilus chilensis and Choromytilus chorus have been purified based on their solubility in dilute perchloric acid and on differential precipitation with acetone containing about 0.3 N HCl. The specific activity of the proteins obtained was 0.16 mg of 3,4-dihydroxyphenylalanine per milligram of protein, or higher. The proteins have an apparent molecular weight of about 100,000 and they contain a high proportion of 3,4-dihydroxyphenylalanine, lysine, and proline.

  9. Study on Modification of Octyl-α-Cyanoacrylate Medical Adhesive

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective:In order that the adhesive character could be improved to modify the octyl-α-cyanoacrylate(OCA) medical adhesive.Methods:Suitable modifiers involving polycaprolactone(PCL),dibutyl phthalate (DBP),dioctyl phthalate(DOP) and poly octyl methacrylat(POMA) have been chosen to modify the OCA adhesive,then tensile shear strength and adhesive strength are tested to evaluate the bond character of adhesives.Results:The PCL group's tensile shear strength and adhesive strength in normal temperature are descen...

  10. Assessing the impact of modifications neoprene adhesives amine-containing compounds the mechanisms to improve adhesion

    OpenAIRE

    KABLOV V.F.; KEYBAL N.A.; S. N. Bondarenko; RUDENKO K.U.; Zaikov, G. E.

    2015-01-01

    Possible mechanisms for an increase in the adhesion parameters of neopren-based adhesive compositions modified with adhesion promoters on the basis of epoxy compounds and aniline derivatives are studied.

  11. Soy and cottonseed protein blends as wood adhesives

    Science.gov (United States)

    As an environmentally friendlier alternative to adhesives from petroleum feedstock, soy proteins are currently being formulated as wood adhesives. Cottonseed proteins have also been found to provide good adhesive properties. In at least some cases, cottonseed proteins appear to form greater shear ...

  12. Studies on polyurethane adhesives and surface modification of hydrophobic substrates

    Science.gov (United States)

    Krishnamoorthy, Jayaraman

    This thesis work deals with (a) Curing of reactive, hot-melt polyurethane adhesives and (b) Adsorption studies using different interactions. Research on polyurethanes involves characterization of polyurethane prepolymers and a novel mechanism to cure isocyanate-terminated polyurethane prepolymer by a "trigger" mechanism. Curing of isocyanate-terminated polyurethane prepolymers has been shown to be influenced by morphology and environmental conditions such as temperature and relative humidity. Although the initial composition, final morphology and curing kinetics are known, information regarding the intermediate prepolymer mixture is yet to be established. Polyurethane prepolymers prepared by the reaction of diisocyanates with the primary hydroxyls of polyester diol (PHMA) and secondary hydroxyls of polyether diol (PPG) were characterized. The morphology and crystallization kinetics of a polyurethane prepolymer was compared with a blend of PPG prepolymer (the product obtained by the reaction of PPG with diisocyanate) and a PHMA prepolymer (the product obtained by the reaction of PHMA with diisocyanate) to study the effect of copolymer formed in the polyurethane prepolymer on the above-mentioned properties. Although the morphology of the polyurethane prepolymer is determined in the first few minutes of application, the chemical curing of isocyanate-terminated prepolymer occurs over hours to days. In the literature, different techniques are described to follow the curing kinetics. But there is no established technique to control the curing of polyurethane prepolymer. To make the curing process independent of environmental factors, a novel approach using a trigger mechanism was designed and implemented by using ammonium salts as curing agents. Ammonium salts that are stable at room temperature but decompose on heating to yield active hydrogen-containing compounds, NH3 and H2O, were used as 'Trojan horses' to cure the prepolymer chemically. Research on adsorption

  13. Assessment of posttranslational modification of mitochondrial proteins.

    Science.gov (United States)

    Ande, Sudharsana R; Padilla-Meier, G Pauline; Mishra, Suresh

    2015-01-01

    Mitochondria play vital roles in the maintenance of cellular homeostasis. They are a storehouse of cellular energy and antioxidative enzymes. Because of its immense role and function in the development of an organism, this organelle is required for the survival. Defects in mitochondrial proteins lead to complex mitochondrial disorders and heterogeneous diseases such as cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases. It is widely known in the literature that some of the mitochondrial proteins are regulated by posttranslational modifications. Hence, designing methods to assess these modifications in mitochondria will be an important way to study the regulatory roles of mitochondrial proteins in greater detail. In this chapter, we outlined procedures to isolate mitochondria from cells and separate the mitochondrial proteins by two-dimensional gel electrophoresis and identify the different posttranslational modifications in them by using antibodies specific to each posttranslational modification.

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

    Science.gov (United States)

    Schaubroeck, David; Mader, Lothar; Dubruel, Peter; Vanfleteren, Jan

    2015-10-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Ming; Liu, Jianguo, E-mail: liujg@mail.hust.edu.cn; Zeng, Xiaoyan; Du, Qifeng; Ai, Jun

    2015-10-30

    Highlights: • High-adhesion copper patterns on alumina ceramic were obtained conveniently. • Effects of processing parameters on adhesion were investigated. • The adhesion of copper–ceramic was higher than the tensile strength of tin-lead solder. • Failure mechanism was studied by the analysis of fracture surfaces. - Abstract: 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:YVO{sub 4} 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.

  16. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    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...... 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...

  17. 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

  18. 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.

  19. 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

    2015-10-01

    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.

  20. 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...

  1. Integrated Microfluidics for Protein Modification Discovery*

    Science.gov (United States)

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-01-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research. PMID:26276765

  2. Integrated Microfluidics for Protein Modification Discovery.

    Science.gov (United States)

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-10-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.

  3. Diagonal chromatography to study plant protein modifications.

    Science.gov (United States)

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

    2016-08-01

    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 thereby allows for the enrichment of specific, though different types of peptides. Here, we focus on the application of diagonal chromatography for the study of modifications of plant proteins. In particular, we show how diagonal chromatography allows for studying proteins processed by proteases, protein ubiquitination, and the oxidation of protein-bound methionines. We discuss the actual sorting steps needed for each of these applications and the obtained results. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

  4. Posttranslational Modification Assays on Functional Protein Microarrays.

    Science.gov (United States)

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng

    2016-10-03

    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  5. 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.

  6. Characterization of canine platelet adhesion to extracellular matrix proteins.

    Science.gov (United States)

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  7. 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.

  8. Soy protein isolate molecular level contributions to bulk adhesive properties

    Science.gov (United States)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  9. 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 rep

  10. Spatial distribution of proteins in the quagga mussel adhesive apparatus.

    Science.gov (United States)

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

    2016-01-01

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

  11. Impact of various modifications of biodegradable membranous scaffolds surface on multipotent mesenchimal stromal cells adhesion and viability

    Directory of Open Access Journals (Sweden)

    L. V. Antonova

    2012-01-01

    Full Text Available Designing a hybrid vascular graft using biocompatible and biodegradable polymers is one of the ways to fill the market with vascular grafts of a small diameter necessary for coronary artery bypass surgery. Good adhesive properties of cultivated cells and low cytotoxicity of biopolymers can improve biocompatibility of polymer-based vascular grafts. The impact of protein modification of membranous polyhydroxyalkanoate and polycaprolactone scaffolds surface on their adhesive properties and cytotoxicity to multipotent mesenchimal stromal cells was evaluated. We used the following compositions of membranous scaffolds: № 1 polyhydroxybutyrate valerate ММ 2 307 kDa with polycaprolactone ММ 80 000 kDa, № 2 had the same composition but was made using a magnetic stirrer, №3 polyhydroxybutyrate ММ 541 kDa with polycaprolactone ММ 80 000 kDa and № 4 polycaprolactone ММ 80 000 kDa only. Premodification of scaffolds surfaces with 75, 50 and 25% fetal calf serum was found to significantly reduce cytotoxicity of scaffold № 4 and improve the adhesive properties of scaffolds № 1, 2 and 3. Premodification with fibronectin (10 µg/ml mostly improved the adhesive properties of scaffolds № 1—4.

  12. Mussel-mimetic protein-based adhesive hydrogel.

    Science.gov (United States)

    Kim, Bum Jin; Oh, Dongyeop X; Kim, Sangsik; Seo, Jeong Hyun; Hwang, Dong Soo; Masic, Admir; Han, Dong Keun; Cha, Hyung Joon

    2014-05-12

    Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications.

  13. 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

  14. 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.

  15. Platelet and endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification

    OpenAIRE

    Tang, Chad; Kligman, Faina; Larsen, Coby C.; KOTTKE-MARCHANT, KANDICE; Marchant, Roger E.

    2009-01-01

    A prominent failure mechanism of small-diameter expanded polytetrafluoroethylene (ePTFE) vascular grafts is platelet-mediated thrombosis. We have designed surface modification for ePTFE consisting of a self-assembling fluorosurfactant polymer (FSP) bearing biologically active ligands, including adhesive peptides and polysaccharide moieties. The goal of this biomimetic construct is to improve graft hemocompatibility by promoting rapid surface endothelialization, while minimizing platelet adhes...

  16. Cell adhesive and antifouling polyvinyl chloride surfaces via wet chemical modification.

    Science.gov (United States)

    Gabriel, Matthias; Strand, Dennis; Vahl, Christian-Friedrich

    2012-09-01

    Polyvinyl chloride (PVC) is one of the most frequently used polymers for the manufacturing of medical devices. Limitations for its usage are based upon unfavorable surface properties of the polymer including its hydrophobicity and lack of functionalities in order to increase its versatility. To address this issue, wet chemical modification of PVC was performed through surface amination using the bifunctional compound ethylene diamine. The reaction was conducted in order to achieve maximum surface amination while leaving the bulk material unaffected. The initial activation step was characterized by means of various methods including contact angle measurements, colorimetric amine quantification, infrared spectroscopy, and gel permeation chromatography. Depth profiles were obtained by a confocal microscopic method using fluorescence labeling. Exclusive surface modification was thus confirmed. To demonstrate biological applications of the presented technique, two examples were chosen: The covalent immobilization of the cell adhesive Asp-Gly-Asp-Ser-peptide (RGD) onto PVC samples yielded a surface that strongly supported cellular adhesion and proliferation of fibroblasts. In contrast, the decoration of PVC with the hydrophilic polymer polyethylene glycol prevented cellular adhesion to a large extent. The impact of these modifications was demonstrated by cell culture experiments.

  17. Nanoscale adhesion, friction and wear of proteins on polystyrene.

    Science.gov (United States)

    Bhushan, Bharat; Utter, Jason

    2013-02-01

    Protein layers are routinely deployed on biomaterials and biological micro/nanoelectromechanical systems (bioMEMS/NEMS) as a functional layer allowing for specific molecular recognition, binding properties or to facilitate biocompatibility. In addition, uncoated biomaterial surfaces will have uncontrolled protein layers adsorbing to the surface within seconds of implantation, so a pre-defined protein layer will improve the host response. Implanted biomaterials also experience micromotion over time which may degrade any surface protein layers. Degradation of these protein layers may lead to system failure or an unwanted immune response. Therefore, it is important to characterize the interfacial properties of proteins on biomaterial surfaces. In this study, the nanoscale adhesion, friction and wear properties of proteins adsorbed to a spin coated polystyrene surface were measured using atomic force microscopy (AFM) in deionized (DI) water and phosphate buffered saline. Adhesion, friction and wear have been measured for bovine serum albumin (BSA), collagen, fibronectin and streptavidin (STA) in DI water and PBS as a function of protein concentration. These proteins were chosen due to their importance and widespread application in the biotechnology field. Adhesion and friction were also measured for BSA and STA at two different temperatures and different pH values to simulate a biological environment. Based on this study, adhesion, friction and wear mechanisms of the different proteins are discussed.

  18. Cell Adhesion Selectivity of Stent Material to improve Bio-functionality by Ion Beam Modification

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jaesang; Park, JUngchan; Jung, Myunghwan; Kim, Yongki [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Park, Junkyu [Bio alpha., Co. Ltd., Gimhae (Korea, Republic of)

    2014-05-15

    In this study, ion implantation into collagen coated Co-Cr alloy, which is a cheaper material of the artificial stent product comparing with Ti alloy, has been studied to develop small diameter artificial stent by the cell adhesion control. The size of stent was 1.6mm of the diameter and 18mm of the length. The life-time of artificial stent depends on adhesion property of endothelial-cells. We successfully controlled cell adhesion selectivity between endothelial cell and muscle cell by using collagen coated and He{sup +} ion beam irradiated Co-Cr-alloy to apply to artificial stent. But, we did not achieve the inhibition of platelet adhesion, yet by using collagen coating and He{sup +} ion beam irradiation. Based on this study, we have plan to research about separation between collagen coating effect and ion beam effect. Also, we will have more detail analysis of the mechanism of cell attachment. In recent years, ion implantation has been applied to the surface modification of prosthesis to improve blood compatibility and tissue compatibility in field of biomedical application. As well known, bio compatibility was concerned with the cell adhesion selectivity for bio-functionality. The biomedical application of ion beam technology would be used more widely in the future such as catheter and artificial graft.

  19. Influence of surface modifications to titanium on oral bacterial adhesion in vitro.

    Science.gov (United States)

    Yoshinari, M; Oda, Y; Kato, T; Okuda, K; Hirayama, A

    2000-11-01

    The influence of surface modifications to titanium on the initial adherence of Porphyromonas gingivalis ATCC33277 and Actinobacillus actinomycetemcomitans ATCC43718 was evaluated. Surface modifications were performed with dry processes including ion implantation (Ca(+), N(+), F(+)), oxidation (anode oxidation, titania spraying), ion plating (TiN, alumina), and ion beam mixing (Ag, Sn, Zn, Pt) with Ar(+) on polished pure titanium plates. Comparatively large amounts of P. gingivalis and A. actinomycetemcomitans adhered to polished titanium plates. The degree of P. gingivalis adhesion showed a positive correlation with surface energy and the amount of calcium-ion adsorption. Adherence of both P. gingivalis and A. actinomycetemcomitans increased on calcium-implanted surfaces compared with polished titanium surfaces, whereas adherence of P. gingivalis was remarkably decreased on alumina-coated surfaces. These findings indicate that titanium implants exposed to the oral cavity require surface modification to inhibit the adherence of oral bacteria, and that surface modification with a dry process is useful in controlling the adhesion of oral bacteria as well as ensuring resistance against wear. Copyright 2000 John Wiley & Sons, Inc.

  20. Posttranslational Protein Modifications in Plant Metabolism.

    Science.gov (United States)

    Friso, Giulia; van Wijk, Klaas J

    2015-11-01

    Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cai Ning; Gong Yingxue; Chan, Vincent; Liao Kin [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Chian, Kerm Sin [School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 639798 (Singapore)], E-mail: askliao@ntu.edu.sg

    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{sup -7} J m{sup -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.

  2. Pursuing DNA catalysts for protein modification.

    Science.gov (United States)

    Silverman, Scott K

    2015-05-19

    Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening have provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead surprisingly led to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 10(5)-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, we were able to identify deoxyribozymes for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion of chemically modified DNA

  3. Nanoscale adhesion forces between enamel pellicle proteins and hydroxyapatite.

    Science.gov (United States)

    Vukosavljevic, D; Hutter, J L; Helmerhorst, E J; Xiao, Y; Custodio, W; Zaidan, F C; Oppenheim, F G; Siqueira, W L

    2014-05-01

    The acquired enamel pellicle (AEP) is important for minimizing the abrasion caused by parafunctional conditions as they occur, for instance, during bruxism. It is a remarkable feature of the AEP that a protein/peptide film can provide enough protection in normofunction to prevent teeth from abrasion and wear. Despite its obvious critical role in the protection of tooth surfaces, the essential adhesion features of AEP proteins on the enamel surface are poorly characterized. The objective of this study was to measure the adhesion force between histatin 5, a primary AEP component, and hydroxyapatite (HA) surfaces. Both biotinylated histatin 5 and biotinylated human serum albumin were allowed to adsorb to streptavidin-coated silica microspheres attached to atomic force microscope (AFM) cantilevers. A multimode AFM with a Nanoscope IIIa controller was used to measure the adhesion force between protein-functionalized silica microspheres attached to cantilever tips and the HA surface. The imaging was performed in tapping mode with a Si3N4 AFM cantilever, while the adhesion forces were measured in AFM contact mode. A collection of force-distance curves (~3,000/replicate) was obtained to generate histograms from which the adhesion forces between histatin 5 or albumin and the HA surface were measured. We found that histatin 5 exhibited stronger adhesion forces (90% >1.830 nN) to the HA surface than did albumin (90% > 0.282 nN). This study presents an objective approach to adhesion force measurements between histatin 5 and HA, and provides the experimental basis for measuring the same parameters for other AEP constituents. Such knowledge will help in the design of synthetic proteins and peptides with preventive and therapeutic benefits for tooth enamel.

  4. 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

    Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial...

  5. Regulation of embryonic cell adhesion by the prion protein.

    Directory of Open Access Journals (Sweden)

    Edward Málaga-Trillo

    2009-03-01

    Full Text Available Prion proteins (PrPs are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1 mediates Ca(+2-independent homophilic cell adhesion and signaling; and (2 modulates Ca(+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin-based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development.

  6. 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.

  7. 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.

  8. Posttranslational Modifications of Ribosomal Proteins in Escherichia coli.

    Science.gov (United States)

    Nesterchuk, M V; Sergiev, P V; Dontsova, O A

    2011-04-01

    А number of ribosomal proteins inEscherichia coliundergo posttranslational modifications. Six ribosomal proteins are methylated (S11, L3, L11, L7/L12, L16, and L33), three proteins are acetylated (S5, S18, and L7), and protein S12 is methylthiolated. Extra amino acid residues are added to protein S6. С-terminal amino acid residues are partially removed from protein L31. The functional significance of these modifications has remained unclear. These modifications are not vital to the cells, and it is likely that they have regulatory functions. This paper reviews all the known posttranslational modifications of ribosomal proteins inEscherichia coli. Certain enzymes responsible for the modifications and mechanisms of enzymatic reactions are also discussed.

  9. 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...

  10. The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function

    OpenAIRE

    Larsen, Coby C.; Kligman, Faina; KOTTKE-MARCHANT, KANDICE; Marchant, Roger E.

    2006-01-01

    We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on ePTFE vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluen...

  11. 水性聚氨酯的合成及改性%Synthesis and modification of waterborne polyurethane adhesive

    Institute of Scientific and Technical Information of China (English)

    王洪祚; 王颖

    2012-01-01

    The preparation principles,methods and developments of waterborne polyurethancs adhesive were introduced.The major modification methods of waterborne polyurethane adhesive were also reviewed briefly.%对水性聚氨酯胶粘剂的制备原理、方法及研拓进行了扼要介绍,并对其主要改性途径作了简要综.

  12. Site specific protein labeling by enzymatic posttranslational modification.

    Science.gov (United States)

    Sunbul, Murat; Yin, Jun

    2009-09-07

    Site specific protein labeling plays a key role in elucidating the function of the proteins at the molecular level by revealing their locations in the cell, their interaction networks with other cellular components and the dynamic mechanisms of their bio-generation, trafficking and degradation in response to regulatory signals in a biological system. Site specific protein labeling is, in essence, artificial modification of proteins with new chemical entities at the posttranslational stage. Based on the analogy between protein labeling and protein posttranslational modification, enzymatic tools have been developed for site specific and efficient labeling of target proteins with chemical probes of diverse structures and functionalities. This perspective surveys a number of protein labeling methods based on the application of protein posttranslational modification enzymes.

  13. 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

    2016-01-01

    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

  14. Chemical modification of polyvinyl chloride and silicone elastomer in inhibiting adhesion of Aeromonas hydrophila.

    Science.gov (United States)

    Kregiel, Dorota; Berlowska, Joanna; Mizerska, Urszula; Fortuniak, Witold; Chojnowski, Julian; Ambroziak, Wojciech

    2013-07-01

    Disease-causing bacteria of the genus Aeromonas are able to adhere to pipe materials, colonizing the surfaces and forming biofilms in water distribution systems. The aim of our research was to study how the modification of materials used commonly in the water industry can reduce bacterial cell attachment. Polyvinyl chloride and silicone elastomer surfaces were activated and modified with reactive organo-silanes by coupling or co-crosslinking silanes with the native material. Both the native and modified surfaces were tested using the bacterial strain Aeromonas hydrophila, which was isolated from the Polish water distribution system. The surface tension of both the native and modified surfaces was measured. To determine cell viability and bacterial adhesion two methods were used, namely plate count and luminometry. Results were expressed in colony-forming units (c.f.u.) and in relative light units (RLU) per cm(2). Almost all the chemically modified surfaces exhibited higher anti-adhesive and anti-microbial properties in comparison to the native surfaces. Among the modifying agents examined, poly[dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammoniopropyl chloride) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer gave the most desirable results. The surface tension of this modifier, was comparable to the non-polar native surface. However, almost half of this value was due to the result of polar forces. In this case, in an adhesion analysis, only 1 RLU cm(-2) and less than 1 c.f.u. cm(-2) were noted. For the native gumosil, the results were 9,375 RLU cm(-2) and 2.5 × 10(8) c.f.u. cm(-2), respectively. The antibacterial activity of active organo-silanes was associated only with the carrier surface because no antibacterial compounds were detected in liquid culture media, in concentrations that were able to inhibit cell growth.

  15. Cell adhesive ability of a biological foam ceramic with surface modification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yong; Li Xiaoyu; Feng Fan; Lin Yunfeng [West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Liao Yunmao [State Key Laboratory of Oral Diseases, Chengdu 610044 (China); Tian, Weidong [State Key Laboratory of Oral Diseases, Chengdu 610044 (China)], E-mail: drtwd@sina.com; Liu Lei [West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)], E-mail: drliulei@163.com

    2008-11-15

    Biological foam ceramic is a promising material for tissue engineering scaffold because of its biocompatibility, biodegradation and adequate pores measured from micrometer to nanometers. The aim of this study was to evaluate the adhesion and proliferation of adipose-derived stromal cells (ADSCs) on the biological foam ceramic coated with fibronectin. ADSCs were harvested from SD rats and passaged three times prior to seeding onto biological foam surface modified with fibronectin (50 {mu}g/ml). Scaffold without surface modification served as control. To characterize cellular attachment, cells were incubated on the scaffold for 1 h and 3 h and then the cells attached onto the scaffold were counted. The difference of proliferation was appraised using MTT assay at day 1, 3, 5 and 7 before the cells reached confluence. After 7 days of culture, scanning electron microscope (SEM) was chosen to assess cell morphology and attachment of ADSCs on the biological foam ceramic. Attachment of ADSCs on the biological foam ceramic surface modified with fibronectin at 1 h or 3 h was substantially greater than that in control. MTT assay revealed that ADSCs proliferation tendency of the experimental group was nearly parallel to that of control. SEM view showed that ADSCs in the experimental groups connected more tightly and excreted more collagen than that in control. The coating of fibronectin could improve the cell adhesive ability of biological foam ceramics without evident effect on proliferation.

  16. The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

    Science.gov (United States)

    Larsen, Coby C; Kligman, Faina; Kottke-Marchant, Kandice; Marchant, Roger E

    2006-10-01

    We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.

  17. Rho family proteins in cell adhesion and cell migration.

    Science.gov (United States)

    Evers, E E; Zondag, G C; Malliri, A; Price, L S; ten Klooster, J P; van der Kammen, R A; Collard, J G

    2000-06-01

    Cell migration and the regulation of cadherin-mediated homotypic cell-cell interactions are critical events during development, morphogenesis and wound healing. Aberrations in signalling pathways involved in the regulation of cell migration and cadherin-mediated cell-cell adhesion contribute to tumour invasion and metastasis. The rho family proteins, including cdc42, rac1 and rhoA, regulate signalling pathways that mediate the distinct actin cytoskeleton changes required for both cellular motility and cell-cell adhesion. Recent studies indicate that rac directly influences rho activity at the GTPase level and that the reciprocal balance between rac and rho activity can determine epithelial or mesenchymal cell morphology and migratory behaviour of epithelial (tumour) cells.

  18. Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton

    NARCIS (Netherlands)

    Tudor, C.; Riet, J. te; Eich, C.; Harkes, R.; Smisdom, N.; Bouhuijzen-Wenger, J.; Ameloot, M.; Holt, M.; Kanger, J.S.; Figdor, C.G.; Cambi, A.; Subramaniam, V.

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both

  19. Principles of protein group SUMO modification substantiated in DNA repair

    OpenAIRE

    Psakhye, Ivan

    2013-01-01

    Posttranslational modifications (PTMs) of proteins by covalent attachment of functional groups (like phosphorylation, acetylation, methylation, glycosylation, etc.) are of key importance for the cell as they regulate various aspects of protein behavior after its synthesis, e.g., dictate protein interaction properties, change catalytic activity of enzymes, induce conformational changes, guide subcellular localization and determine protein stability. A special class of protein PTMs is the conju...

  20. High Mobility Group Proteins and Their Post-Translational Modifications

    OpenAIRE

    Zhang, Qingchun; Wang, Yinsheng

    2008-01-01

    The high mobility group (HMG) proteins, including HMGA, HMGB and HMGN, are abundant and ubiquitous nuclear proteins that bind to DNA, nucleosome and other multi-protein complexes in a dynamic and reversible fashion to regulate DNA processing in the context of chromatin. All HMG proteins, like histone proteins, are subjected to extensive post-translational modifications (PTMs), such as lysine acetylation, arginine/lysine methylation and serine/threonine phosphorylation, to modulate their inter...

  1. Homocysteine and its thiolactone-mediated modification of fibrinogen affect blood platelet adhesion.

    Science.gov (United States)

    Malinowska, Joanna; Olas, Beata

    2012-01-01

    Homocysteine (Hcys) and homocysteine thiolactone (HTL) concentrations in organism are correlated with a number of serious pathologies. In the literature, there are few papers describing studies on the effects of homocysteine on proteins that participate in blood coagulation and fibrinolysis in human. However, mechanisms involved in the relationship between hyperhomocysteinemia and hemostatic process are still unclear. The role of N- or S-homocysteinylation (induced by Hcys and its derivatives) of different hemostatic proteins, including fibrinogen is also still poorly known. The aim of this study was to establish the functional changes of the fibrinogen molecule induced by Hcys (at final doses of 10-100 µM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (0.1-1 µM), and to examine the effects of these changes on the capability of fibrinogen to interact with human blood platelets (by measuring the platelet adhesion). Our present results demonstrated that Hcys-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion. Both, unstimulated and thrombin-activated platelets showed a reduced ability to adhere to Hcys-mediated fibrinogen. HTL (at all tested concentrations) had similar properties when we used thrombin-activated platelets. In conclusion, the results reported in this study could be useful for a better understanding of changes in hemostasis during hyperhomocysteinemia.

  2. Post-translational modification of PII signal transduction proteins

    Directory of Open Access Journals (Sweden)

    Mike eMerrick

    2015-01-01

    Full Text Available The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target. PII proteins in the Proteobacteria and the Actinobacteria are subject to post-translational modification by uridylylation or adenylylation respectively, whilst in some Cyanobacteria they can be modified by phosphorylation. In all these cases the protein’s modification state is influenced by the cellular nitrogen status and is thought to regulate its activity. However in many organisms there is no evidence for modification of PII proteins and indeed the ability of these proteins to respond to the cellular nitrogen status is fundamentally independent of post-translational modification. In this review we explore the role of post-translational modification in PII proteins in the light of recent studies.

  3. Sumoylation: a regulatory protein modification in health and disease.

    Science.gov (United States)

    Flotho, Annette; Melchior, Frauke

    2013-01-01

    Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

  4. 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.

  5. 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.

  6. 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.

  7. Significant Enhancement of the Adhesion between Metal Films and Polymer Substrates by UV-Ozone Surface Modification in Nanoscale.

    Science.gov (United States)

    Liu, Junshan; He, Licheng; Wang, Liang; Man, Yuncheng; Huang, Luyi; Xu, Zheng; Ge, Dan; Li, Jingmin; Liu, Chong; Wang, Liding

    2016-11-09

    Polymer metallization is extensively used in a variety of micro- and nanosystem technologies. However, the deposited metal film exhibits poor adhesion to polymer substrates, which may cause difficulties in many applications. In this work, ultraviolet (UV)-ozone surface modification is for the first time put forward to enhance the adhesion between metal films and polymer substrates. The adhesion of sputtered Cu films on UV-ozone modified poly(methyl methacrylate) (PMMA) substrates is enhanced by a factor of 6, and that of Au films is improved by a factor of 10. Moreover, metal films on the modified PMMA substrates can withstand a long-time liquid immersion. To understand the mechanism for the adhesion enhancement, the surface modification is studied with contact angle measurements, attenuated total reflection Fourier-transform infrared spectrometry (ATR-FTIR) and atomic force microscopy (AFM). Detailed characterization results indicate that the significant adhesion enhancement is attributed to the increases of both the surface wettability by generating some polar functional groups and the roughness of the surface in nanoscale. To demonstrate this novel polymer metallization method, a 6-in. PMMA chip with arrays of three-electrode electrochemical microsensors is designed and fabricated, and the microsensor exhibits excellent reproducibility, uniformity, and long-term stability.

  8. Anti-proteolytic property and bonding durability of mussel adhesive protein-modified dentin adhesive interface.

    Science.gov (United States)

    Fang, Hui; Li, Quan-Li; Han, Min; Mei, May Lei; Chu, Chun Hung

    2017-10-01

    To evaluate the effect of mussel adhesive protein (MAP) on collagenase activity, dentin collagen degradation and microtensile dentin bond strength (μTBS). Three groups were designed: 1. experimental group: treated with MAP; 2. positive control: treated with GM6001 (collagenase-inhibitor); 3. negative control: treated with distilled water (DW). For collagenase activity, Clostridiopeptidase-A was added to each group (n=5), and collagenase activity was assessed by colorimetric assay. For dentin collagen degradation, thirty dentin slabs were allocated to the three above groups (n=10). Dentin collagen degradation was evaluated by measuring released hydroxyproline by colorimetric assay after being incubated in Clostridiopeptidase-A for 7 days. For microtensile bond strength, sixty human third molars with flat dentin surfaces were etched by phosphoric acid and then assigned to the three above groups (n=20). An etch-and-rinse adhesive system was applied to all three groups as stated in standard clinic protocol. The test of μTBS was performed before and after thermocycling and collagenase challenge. The collagenase activities (nmol/min/mg) in the group of MAP was significantly less inactive compared to the group of GM6001 and DW (MAP0.06), the value of μTBSs after thermocycling and collagenase challenge was significantly greater in the group of MAP and GM6001 compared to the group of DW (MAP, GM6000>DW, pcomposite restoration over time. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  9. Research progress in protein post-translational modification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Protein post-translational modification plays an important role in organism. It makes the protein obtain more complicated structures, perfect functions, more accurate regulations and more specific operations. The most common protein post- translational modifications include ubiquitylation, phosphorylation, glycosylation, lipodation, methylation, and acetylation and so on. Ubiquitylation plays an essential role in cellular functions such as cellular differentiation, apoptosis, DNA repair, antigen processing, and stress response. Phosphorylation is related to physiological and pathological processes including cellular signal conduction, nervous activity, muscle contraction and proliferation, development and differentiation of cells. Protein glycosylation is of great importance for many cell processes like immunoprotection, virus replication, cell growth, and occurrence of inflammation and so on. Lipodation is vital to signal conduction. Histone methylation and acetylation are responsible for transcription regulation. In vivo, different post-translational modifications do not occur isolatedly, but influence each other's function and cooperate with each other. Understanding what influences the post-translational modifications will help to uncover cellular processes and protein network in molecular level and finally direct more precise drug design targeting molecules. Post-translational modification mimics are set to dominate the next wave of protein therapeutics and become powerful medicinal tools in the 21st century.

  10. Surface modification with fibronectin or collagen to improve the cell adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Li Xiaoyu [West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Yao Jinfeng [State Key Laboratory of Oral Diseases, Chengdu 610044 (China); Yang Xiaojuan [West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Tian Weidong [State Key Laboratory of Oral Diseases, Chengdu 610044 (China)], E-mail: drtwd@sina.com; Liu Lei [West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)], E-mail: drliulei@163.com

    2008-11-15

    Objective: The surface of biomaterials plays a critical role in determining bioactivity. The aim of this study was to evaluate the cell adhesion and proliferation of ADSCs on the surface of biomaterial which is modified with fibronectin or collagen. Materials and methods: Adipose-derived stromal cells (ADSCs) were obtained from SD rats, expanded in culture, and seeded onto scaffold surface-modified with fibronectin or collagen. To characterize cellular attachment, cells were incubated on scaffold for 1 and 2 h and then counted the cells attached onto the scaffold. The MTT assay was chosen to evaluate the proliferation at days 1, 4, 7 and 14. After 7 d of culture, scanning electron microscope was chosen to observe cell morphology and attachment of ADSCs on the scaffolds. Results: Attachment at 1 and 2 h of cells on scaffold modified with fibronectin was significantly greater than in control, but not with collagen. The MTT assay revealed that ADSCs proliferation tendency was nearly parallel to that in control. The scanning electron microscope (SEM) showed that ADSCs in experiment expanded thoroughly and excreted much extracellular materials. Conclusions: Surface modification with fibronectin or collagen can enhance the attachment of cultured ADSCs on the scaffold, but it had not evident effect to proliferation.

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

    Indian Academy of Sciences (India)

    USHA P KAR; HIMANI DEY; ABDUR RAHAMAN

    2017-06-01

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agentsinvolved in several biological processes such as endocytosis, maintenance of organelle morphology and viralresistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion ortubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulatedby 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 themechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In thisreview, we discuss major post-translational modifications along with their roles in the mechanism of dynaminfunctions and implications in various cellular processes.

  12. Chemical modification of muscle protein in diabetes.

    Science.gov (United States)

    Alt, Nadja; Carson, James A; Alderson, Nathan L; Wang, Yuping; Nagai, Ryoji; Henle, Thomas; Thorpe, Suzanne R; Baynes, John W

    2004-05-15

    Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.

  13. Use of additives to enhance the properties of cottonseed protein as wood adhesives

    Science.gov (United States)

    Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed...

  14. 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.

  15. Affixing plant sections without protein based adhesives for protease histochemistry.

    Science.gov (United States)

    Jona, R; Griglione, R

    1999-01-01

    To submit a section of plant tissue to histochemical analysis using protease, the protein based adhesives which keep the slices attached to the slides must be replaced because they are attacked by the enzyme and the slices are washed off the slides. We devised a method to keep the slices attached to the slides during histochemical extractions and subsequent staining. Slides are frosted on two lateral zones by spreading on them a fluoride paste composed of 15 g barium sulfate, 15 g ammonium difluoride, 8 g oxalic acid, 40 ml glycerine and 12 ml deionized water using a thin paint brush. After removing the paste with tap water and drying the slides, the sections are placed on the central clear zone of the slide and covered with an ethyl-cellulose film that keeps the slices in place and allows the reagents to act through it. To do this, the slides are dipped into 0.5% ethyl cellulose (ETC) prepared in a 4:1 mixture of toluene and absolute ethanol. The ETC coating is layered three times to improve its firmness and its ability to retain the slices on the slides. To obtain perfect adhesion, the slide should be oven dried (40-50 C for 10-15 min) to remove any trace of humidity before applying each layer of ETC. Subsequently the sections can be extracted and stained without undue loss of material.

  16. Generation of protein-derived redox cofactors by posttranslational modification.

    Science.gov (United States)

    Davidson, Victor L

    2011-01-01

    Redox enzymes which catalyze the oxidation and reduction of substrates are ubiquitous in nature. These enzymes typically possess exogenous cofactors to allow them to perform catalytic functions which cannot be accomplished using only amino acid residues. It is now evident that nature also employs an alternative strategy of generating catalytic and redox-active sites in proteins by posttranslational modification of amino acid residues. This review describes the structures and functions of several of these protein-derived cofactors and the diverse mechanisms of posttranslational modification through which they are generated.

  17. Modifications of blood platelet proteins of patients with schizophrenia.

    Science.gov (United States)

    Dietrich-Muszalska, Anna; Olas, Beata

    2009-03-01

    Oxidative damage to lipids in plasma, blood platelets and neurons in patients with schizophrenia was described. The aim of our present study was to evaluate oxidative/nitrative modifications of blood platelets proteins by measurement the level of biomarkers of oxidative stress such as carbonyl groups, thiol groups and 3-nitrotyrosine in proteins in patients with schizophrenia and compare with a control group. Levels of carbonyl groups and 3-nitrotyrosine residues in platelet proteins were measured by ELISA and competition ELISA, respectively. The method with 5,5'-dithio-bis(2-nitro-benzoic acid) has been used to analyse thiol groups in platelet proteins. We demonstrated for the first time in platelet proteins from patients with schizophrenia a statistically significant increase of the level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine; in schizophrenic patients the amount of thiol groups in platelet proteins was lower than in platelets from healthy subjects. Our results strongly indicate that in patients with schizophrenia reactive oxygen species and reactive nitrogen species induce not only peroxidation of lipids, but also may stimulate oxidative/nitrative modifications of platelet proteins. The consequence of these modifications may be the alteration of platelet protein structure and function.

  18. Modification of protein synthetic components by aflatoxin B1.

    Science.gov (United States)

    Lyman, B A; Erki, L; Biedrzycka, D W; Devlin, T M; Ch'ih, J J

    1988-04-15

    Molecular sites of perturbation by the hepatocarcinogen aflatoxin B1 (AFB1) in the protein synthesis initiation complex were assessed using isolated hepatocytes and a cell-free activating system containing microsomes and cytoplasmic ribonucleoprotein complexes (cRPC). Ribosomal proteins showed no detectable modification by the toxin in either system. With hepatocytes, initiation factors demonstrated only slight modification by AFB1. RNAs from both hepatocytes and the cell-free system with microsomes and cRPC were modified, with poly(A)-containing RNA exhibiting at least a 5-fold higher modification than poly(A)-lacking RNA. The poly(A)-lacking RNAs were modified in the order 28S rRNA greater than 18S rRNA greater than 5-6S rRNA greater than 4S tRNA. Guanine was the target base of AFB1, but only 10% of the AFB1-GMP adducts were on guanines located in a poly(G) region. These results suggest that guanine modification in RNAs may be responsible for the observed inhibition of translational initiation by AFB1 to a greater extent than modification of either ribosomal intrinsic or associated proteins.

  19. Synthesis, modification and turnover of proteins during aging.

    Science.gov (United States)

    Rattan, Suresh I S

    2010-01-01

    Iterations in the rate and extent of protein synthesis, accuracy, post-translational modifications and turnover are among the main molecular characteristics of aging. A decline in the cellular capacity through proteasomal and lysosomal pathways to recognize and preferentially degrade damaged proteins leads to the accumulation of abnormal proteins during aging. The consequent increase in molecular heterogeneity and impaired functioning of proteins is the basis of several age-related pathologies, such as cataracts, sarcopenia and neurodegerative diseases. Understanding the proteomic spectrum and its functional implications during aging can facilitate developing effective means of intervention, prevention and therapy of aging and age-related diseases.

  20. 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...

  1. Monocotyledons in Particleboard Production: Adhesives, Additives, and Surface Modification of Reed Canary Grass

    Directory of Open Access Journals (Sweden)

    Johann Trischler

    2014-05-01

    Full Text Available As a supplier to the furniture industry, the particleboard industry is searching for opportunities to reduce costs, weight, and formaldehyde emissions. One such opportunity is to use monocotyledons such as straw and hemp, as well as grasses like reed canary grass. A major problem when using reed canary grass or other monocotyledons in combination with wood is the difference in their surface properties, leading to poor reactivity and wettability with adhesives such as melamine urea formaldehyde. To this end, either the surface of the particles must be modified in some way, or different adhesives must be used. The purpose of this paper is to present adhesives, surfactants, coupling agents, and pre-treatment methods that can be used in combination with monocotyledons to improve compatibility with wood. Some of the methods have been tested on reed canary grass. The results show a wide range of strength values for the joint between wood and untreated or pre-treated reed canary grass glued with different adhesives, with and without a surfactant and a coupling agent. Isocyanate-based adhesives provided relatively strong bonds, and polyvinyl acetate, acryl, and epoxy adhesives were also effective. The most effective method was pre-treatment followed by adhesives in combination with a coupling agent.

  2. 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.

  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......-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. 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.

  5. Chemical modification of proteins by lipids in diabetes.

    Science.gov (United States)

    Baynes, John W

    2003-09-01

    Advanced glycation and lipoxidation end-products (AGE/ALE) increase in tissue proteins with age and at an accelerated rate in diabetes. This Review focuses on the nature and source of AGEs/ALEs and the factors affecting their formation in tissue and plasma proteins. Lipids are identified as an important source of chemical modification of proteins in diabetes, and the role of diabetes, dyslipidemia and renal disease in formation of AGEs/ALEs is reviewed. The article concludes with a discussion of ELISA assays for AGEs/ALEs and the merits of measuring AGEs/ALEs in the clinical laboratory.

  6. Recent approaches in physical modification of protein functionality.

    Science.gov (United States)

    Mirmoghtadaie, Leila; Shojaee Aliabadi, Saeedeh; Hosseini, Seyede Marzieh

    2016-05-15

    Today, there is a growing demand for novel technologies, such as high hydrostatic pressure, irradiation, ultrasound, filtration, supercritical carbon dioxide, plasma technology, and electrical methods, which are not based on chemicals or heat treatment for modifying ingredient functionality and extending product shelf life. Proteins are essential components in many food processes, and provide various functions in food quality and stability. They can create interfacial films that stabilize emulsions and foams as well as interact to make networks that play key roles in gel and edible film production. These properties of protein are referred to as 'protein functionality', because they can be modified by different processing. The common protein modification (chemical, enzymatic and physical) methods have strong effects on the structure and functionality of food proteins. Furthermore, novel technologies can modify protein structure and functional properties that will be reviewed in this study.

  7. Adhesion protein networks reveal functions proximal and distal to cell-matrix contacts.

    Science.gov (United States)

    Byron, Adam; Frame, Margaret C

    2016-04-01

    Cell adhesion to the extracellular matrix is generally mediated by integrin receptors, which bind to intracellular adhesion proteins that form multi-molecular scaffolding and signalling complexes. The networks of proteins, and their interactions, are dynamic, mechanosensitive and extremely complex. Recent efforts to characterise adhesions using a variety of technologies, including imaging, proteomics and bioinformatics, have provided new insights into their composition, organisation and how they are regulated, and have also begun to reveal unexpected roles for so-called adhesion proteins in other cellular compartments (for example, the nucleus or centrosomes) in diseases such as cancer. We believe this is opening a new chapter on understanding the wider functions of adhesion proteins, both proximal and distal to cell-matrix contacts.

  8. Aberrant Glycosylation of Plasma Proteins in Severe Preeclampsia Promotes Monocyte Adhesion

    Science.gov (United States)

    Kazanjian, Avedis A.; Tinnemore, Deborah; Gafken, Philip R.; Ogata, Yuko; Napolitano, Peter G.; Stallings, Jonathan D.; Ippolito, Danielle L.

    2014-01-01

    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte–endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte–endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia. PMID:23757314

  9. Protein adhesion on silicon-supported hyperbranched poly(ethylene glycol) and poly(allylamine) thin films.

    Science.gov (United States)

    Dyer, Maureen A; Ainslie, Kristy M; Pishko, Michael V

    2007-06-19

    Hyperbranching poly(allylamine) (PAAm) and poly(ethylene glycol) (PEG) on silicon and its effect on protein adhesion was investigated. Hyperbranching involves sequential grafting of polymers on a surface with one of the components having multiple reactive sites. In this research, PAAm provided multiple amines for grafting PEG diacrylate. Current methodologies for generating PEG surfaces include PEG-silane monolayers or polymerized PEG networks. Hyperbranching combines the nanoscale thickness of monolayers with the surface coverage afforded by polymerization. A multistep approach was used to generate the silicon-supported hyperbranched polymers. The silicon wafer surface was initially modified with a vinyl silane followed by oxidation of the terminal vinyl group to present an acid function. Carbodiimide activation of the surface carboxyl group allowed for coupling to PAAm amines to form the first polymer layer. The polymers were hyperbranched by grafting alternating PEG and PAAm layers to the surface using Michael addition chemistry. The alternating polymers were grafted up to six total layers. The substrates remained hydrophilic after each modification. Static contact angles for PAAm (32-44 degrees) and PEG (33-37 degrees) were characteristic of the corresponding individual polymer (30-50 degrees for allylamine, 34-42 degrees for PEG). Roughness values varied from approximately 1 to 8 nm, but had no apparent affect on protein adhesion. Modifications terminating with a PEG layer reduced bovine serum albumin adhesion to the surface by approximately 80% as determined by ELISA and radiolabel binding studies. The hyperbranched PAAm and PEG surfaces described in this paper are nanometer-scale, multilayer films capable of reducing protein adhesion.

  10. 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 PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from......, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLß domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1...

  11. A poly(acrylic acid)-block-poly(L-glutamic acid) diblock copolymer with improved cell adhesion for surface modification.

    Science.gov (United States)

    Cao, Bin; Yan, Shifeng; Zhang, Kunxi; Song, Zhijiang; Cao, Tian; Chen, Xuesi; Cui, Lei; Yin, Jingbo

    2011-07-07

    A novel PAA-b-PLGA diblock copolymer is synthesized and characterized that has excellent cell adhesion and biocompatibility. Fluorescent DiO labeling is used to monitor the attachment and growth of hASCs on the film surface, and cell proliferation over time is studied. Results show that PLLA modified by a CS/PAA-b-PLGA multilayer film can promote the attachment of human hASCs and provide an advantageous environment for their proliferation. The multilayer film presents excellent biocompatibility and cell adhesive properties, which will provide a new choice for improving the cell attachment in surface modification for tissue engineering. Hydroxyl, carboxyl and amine groups in the CS/PAA-b-PLGA multilayer film may be combined with drugs and growth factors for therapy and differentiation.

  12. 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

  13. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.

    Science.gov (United States)

    George, Margaret D; Wine, Robert N; Lackford, Brad; Kissling, Grace E; Akiyama, Steven K; Olden, Kenneth; Roberts, John D

    2013-12-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

  14. Medium-density particleboards from modified rice husks and soybean protein concentrate-based adhesives.

    Science.gov (United States)

    Ciannamea, Emiliano M; Stefani, Pablo M; Ruseckaite, Roxana A

    2010-01-01

    The main goal of this work was to evaluate the technical feasibility of using rice husk (RH) as wood substitute in the production of environmentally sound medium-density particleboards using adhesives from soybean protein concentrate (SPC). Chemical modification of rice husk with sodium hydroxide and sodium hydroxide followed by hydrogen peroxide (bleaching) were undertaken to evaluate the effect of such treatments on the composition and topology of rice husk and the performance of produced panels. Both treatments were efficient in partially eliminating hemicelluloses, lignin and silica from RH, as evidenced by thermo-gravimetric analysis (TGA). Scanning electron microscopy observations suggested that alkaline treatment resulted in a more damaged RH substrate than bleaching. The dependence of mechanical properties (modulus of rupture, modulus of elasticity, and internal bond) and the physical properties (water absorption and thickness swelling) on chemical treatments performed on both, rice husk and SPC was studied. Bleached-rice husk particleboards bonded with alkaline-treated soybean protein concentrate displayed the best set of final properties. Particleboards with this formulation met the minimum requirements of internal bond, modulus of elasticity and modulus of rupture recommended by the US Standard ANSI/A208.1 specifications for M1, MS and M2-grade medium-density particleboards, but failed to achieve the thickness swelling value recommended for general use panels. This limitation of soybean protein concentrate-bonded rice husk particleboards was counterbalanced by the advantage of being formaldehyde-free which makes them a suitable alternative for indoor applications.

  15. Oxidative modification of proteins: age-related changes.

    Science.gov (United States)

    Chakravarti, Bulbul; Chakravarti, Deb N

    2007-01-01

    Aging is a complex biological phenomenon which involves progressive loss of different physiological functions of various tissues of living organisms. It is the inevitable fate of life and is a major risk factor for death and different pathological disorders. Based on a wide variety of studies performed in humans as well as in various animal models and microbial systems, reactive oxygen species (ROS) are believed to play a key role in the aging process. The production of ROS is influenced by cellular metabolic activities as well as environmental factors. ROS can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since, in general, proteins are the key molecules that play the ultimate role in various structural and functional aspects of living organisms, this review will focus on the age-related oxidative modifications of proteins as well as on mechanism for removal or repair of the oxidized proteins. The topics covered include protein oxidation as a marker of oxidative stress, experimental evidence indicating the role of ROS in protein oxidation, protein carbonyl content, enzymatic degradation of oxidized proteins, and effects of caloric restriction on protein oxidation in the context of aging. Finally, we will discuss different strategies which have been or can be undertaken to slow down the oxidative damage of proteins and the aging process.

  16. Surface modification of CoCr alloy using varying concentrations of phosphoric and phosphonoacetic acids: albumin and fibrinogen adsorption, platelet adhesion, activation, and aggregation studies.

    Science.gov (United States)

    Thiruppathi, Eagappanath; Larson, Mark K; Mani, Gopinath

    2015-01-01

    CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.

  17. 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

  18. [Processing and Modification of Recombinant Spider Silk Proteins].

    Science.gov (United States)

    Liu, Bin; Wang, Tao; Liu, Xiaobing; Luo, Yongen

    2015-08-01

    Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein.

  19. 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 metho...

  20. 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.

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

    Science.gov (United States)

    Zhang, Chunming; Zhao, Meihua; Wang, Libing; Qu, Lijun; Men, Yajing

    2017-04-01

    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(Cdbnd O, Csbnd 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.

  2. Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading.

    Science.gov (United States)

    Norman, Leann L; Oetama, Ratna J; Dembo, Micah; Byfield, F; Hammer, Daniel A; Levitan, Irena; Aranda-Espinoza, Helim

    2010-06-01

    Cellular cholesterol is a critical component of the plasma membrane, and plays a key role in determining the physical properties of the lipid bilayer, such as elasticity, viscosity, and permeability. Surprisingly, it has been shown that cholesterol depletion increases cell stiffness, not due to plasma membrane stiffening, but rather, due to the interaction between the actin cytoskeleton and the plasma membrane. This indicates that traction stresses of the acto-myosin complex likely increase during cholesterol depletion. Here we use force traction microscopy to quantify the forces individual cells are exerting on the substrate, and total internal reflection fluorescence microscopy as well as interference reflection microscopy to observe cell-substrate adhesion and spreading. We show that single cells depleted of cholesterol produce larger traction forces and have large focal adhesions compared to untreated or cholesterol-enriched cells. Cholesterol depletion also causes a decrease in adhesion area for both single cells and monolayers. Spreading experiments illustrate a decrease in spreading area for cholesterol-depleted cells, and no effect on cholesterol-enriched cells. These results demonstrate that cholesterol plays an important role in controlling and regulating the cell-substrate interactions through the actin-plasma membrane complex, cell-cell adhesion, and spreading.

  3. 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 contac

  4. 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...

  5. Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene

    NARCIS (Netherlands)

    Dekker, A.; Reitsma, K.; Beugeling, T.; Bantjes, A.; Feijen, J.; Aken, van 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 modif

  6. PROTEIN EXTRACTION FROM SECONDARY SLUDGE OF PAPER MILL WASTEWATER AND ITS UTILIZATION AS A WOOD ADHESIVE

    Directory of Open Access Journals (Sweden)

    Muhammad Pervaiz

    2011-04-01

    Full Text Available In this study, secondary sludge (SS from a kraft paper mill was used as a source of biomass to recover protein and investigate its potential use as a wood adhesive. The process of protein recovery involved disruption of the floc structure in alkaline medium to disintegrate and release intercellular contents into the aqueous phase followed by separation of soluble protein. Finally, the soluble protein was subjected to low pH precipitation and the pelletized sludge protein, referred to as recovered sludge protein (RSP was tested for crude protein, moisture, and other contents. A significant process yield of 90% in terms of precipitation of soluble protein from disintegrated sludge was estimated through calorimetric studies, whereas an overall material balance confirmed a RSP yield of up to 23% based on total suspended solids of raw sludge. The RSP containing 30% crude protein was used as a wood adhesive and its adhesion performance was compared with soy protein isolate (SPI and phenol formaldehyde (PF resin. The testing of plywood lap joints has shown up to 41% shear strength level of RSP adhesive compared to PF. This work demonstrates the technical feasibility and potential of SS as a biomass resource to develop eco-friendly adhesives for wood composite applications.

  7. Modification of proteins by norepinephrine is important for vascular contraction

    Directory of Open Access Journals (Sweden)

    Kyle B Johnson

    2010-10-01

    Full Text Available Norepinephrine (NE is thought to mediate its effects through G-protein coupled receptors. However, previous studies have shown that norepinephrine and another primary amine, serotonin, also have the ability to exert effects in a receptor-independent manner. We hypothesized that the enzyme transglutaminase II (TG II has the ability modify proteins with NE and that this modification is physiologically relevant. As our model we used rat aortic and vena cava tissues, two tissues that depend on NE to modulate vascular tone. Immunohistochemical and immunocytochemical staining showed that NE and TG II are present in smooth muscle cells of these tissues. Western analysis shows aorta and vena cava homogenate proteins are recognized by an anti-NE antibody. NE and α-actin colocalize in cultured aorta and vena cava smooth muscle cells. Freshly dissociated smooth muscle cells from these vessels were able to take up NE-biotin. In isolated tissue baths, inhibition of TG II with cystamine (0.5 mM completely abolished NE-induced contraction in the aorta but only attenuated the receptor-independent contractant KCl (max contraction to 100 mM KCl in cystamine treated = 88.8 ± 7.5% of vehicle treated, p<0.05. In the vena cava, contraction to NE was abolished with 0.1 mM cystamine and KCl contraction was attenuated (max contraction to 100 mM KCl in cystamine treated = 54.8 ± 21.2% of vehicle treated, p<0.05. Taken together, these results show that vascular smooth muscle cells take up and utilize NE for the modification of proteins, and that this modification may play an important role in vascular contraction.

  8. 改性水性聚氨酯胶黏剂研究进展%Progress of modification of waterborne polyurethane adhesive

    Institute of Scientific and Technical Information of China (English)

    邓威; 黄洪; 傅和青

    2011-01-01

    The classification and preparation of waterborne polyurethane adhesive are introduced. The modification methods of waterborne polyurethane, such as acrylate modification, epoxy resin modification, organic fluorine modification, silicone, nanomaterials modification, multi-modification and hyperbranched prepolymer modification are summarized. The advantages and disadvantages of these modification methods are compared, and the application of the modified waterborne polyurethane adhesive is proposed. The development of waterborne polyurethane adhesive is discussed.%介绍了水性聚氨酯胶黏剂的分类和合成方法.综述了水性聚氨酯的改性方法,包括丙烯酸酯改性、环氧树脂改性、有机氟改性、有机硅改性、纳米材料改性、复合改性和超支化预聚体改性.比较了各种改性方法的优势和缺陷,提出了每种方法改性的胶黏剂的适用领域,指出了水性聚氨酯胶黏剂的发展趋势.

  9. Surface Modification of a PCB Substrate for Better Adhesion of Inkjet Printed Circuit Structures

    OpenAIRE

    Sridhar, A.; Dijk, van, JMF Jan; Akkerman, R.

    2009-01-01

    The robustness and service life of inkjet printed electronic circuit structures are highly influenced by the state of the interface between these structures and the substrate. In the case of polymeric substrate materials, surface modification is necessary to realise a favourable interface, as these materials are generally not very receptive to chemical bond formation with the deposited ink. This paper deals with the surface modification of a high frequency laminate (substrate) using two diffe...

  10. Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion.

    Science.gov (United States)

    Milani, Renato; Ferreira, Carmen V; Granjeiro, José M; Paredes-Gamero, Edgar J; Silva, Rodrigo A; Justo, Giselle Z; Nader, Helena B; Galembeck, Eduardo; Peppelenbosch, Maikel P; Aoyama, Hiroshi; Zambuzzi, Willian F

    2010-04-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.

  11. 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

  12. Recent Progress in Predicting Posttranslational Modification Sites in Proteins.

    Science.gov (United States)

    Xu, Yan; Chou, Kuo-Chen

    2016-01-01

    The posttranslational modification or PTM is a later but subtle step in protein biosynthesis via which to change the properties of a protein by adding a modified group to its one or more amino acid residues. PTMs are responsible for many significant biological processes, and meanwhile for many major diseases as well, such as cancer. Facing the avalanche of biological sequences generated in the post-genomic age, it is important for both basic research and drug development to timely identify the PTM sites in proteins. This Review is devoted to summarize the recent progresses in this area, with a focus on those predictors, which were developed based on the pseudo amino acid composition or PseAAC approach, and for which a publicly accessible web-server has been established. Meanwhile, the future challenge in this area has also been briefly addressed.

  13. 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.

  14. Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

    Science.gov (United States)

    Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

    2013-12-14

    Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored.

  15. 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...

  16. Effects of surface modification on placement and adhesion of NG108-15 cells

    Science.gov (United States)

    Schneider, Thomas W.; Schessler, Heather M.; Sheffer, Kara M.; Dumm, Judy; Aloi, L. E.

    2000-12-01

    A graded height flow cell was used to measure the adhesion properties of the neural cell line of neuroblastoma X glioma (NG108-15) cells cultured on substrates of organosilane self-assembled monolayers (SAMs). The SAMs tested in this study were 13F, 15F, PEG 550, OTS, DETA and APTS. Utilizing deep UV lithography, patterning of the SAMs create three regions for cell attachment; the original SAM, the backfilled SAM, and the interface between the two. Upon plating, the cell soma show no preference for any of the three regions. One exception was on PEG 550, which was found to resist cell adhesion upon normal plating conditions. The cell processes of the NG108-15 cells show a preference for growth at the interface between two patterned surfaces. A factor of three increase in adhesion properties was found for the patterned surface over an uncoated glass surface. Design rules of a single whole cell biosensor using theNG108-15 cells can be developed based on these findings.

  17. Excimer laser surface modification of coated steel for enhancement of adhesive bonding

    Science.gov (United States)

    Jahani, Hamid R.; Moffat, B.; Mueller, R. E.; Fumo, D.; Duley, W.; North, T.; Gu, Bo

    1998-05-01

    Zinc coated sheet steel in the form of temper rolled galvanize and galvanneal are used extensively in the automotive industry. Through a process of excimer laser surface treatment, we have developed a procedure to significantly enhance the adhesion characteristics of these coated steels. We report here results of processing trials using both XeCl (308 nm) and KrF (248 nm) excimer lasers and a two-part epoxy adhesive (3M DP-460) with a range of processing conditions. Bond strengths are measured by T-peel and shear test methods. Using T-peel tests, bond strength improvements greater than five times than for untreated surfaces have been observed. With the improved surface condition, the bond strength becomes limited by the cohesive strength of the adhesive. Detailed measurements of the physical structure and chemical composition of the excimer laser processed surfaces are presented. The enhancement in bond strength is correlated with the observed changes in physical and chemical structure of the laser processed surfaces. Surface structure is observed using SEM and physical characteristics are quantified using a Talysurf profilometer. The chemical composition of the treated surface has been analysed using XPS and time-of-flight mass spectroscopy.

  18. Adhesion modification of neural stem cells induced by nanoscale ripple patterns

    Science.gov (United States)

    Pedraz, P.; Casado, S.; Rodriguez, V.; Giordano, M. C.; Buatier de Mongeot, F.; Ayuso-Sacido, A.; Gnecco, E.

    2016-03-01

    We have studied the influence of anisotropic nanopatterns (ripples) on the adhesion and morphology of mouse neural stem cells (C17.2) on glass substrates using cell viability assay, optical microscopy and atomic force microscopy. The ripples were produced by defocused ion beam sputtering with inert Ar ions, which physically remove atoms from the surface at the energy of 800 eV. The ripple periodicity (∼200 nm) is comparable to the thickness of the cytoplasmatic microspikes (filopodia) which link the stem cells to the substrate. All methods show that the cell adhesion is significantly lowered compared to the same type of cells on flat glass surfaces. Furthermore, the AFM analysis reveals that the filopodia tend to be trapped parallel or perpendicular to the ripples, which limits the spreading of the stem cell on the rippled substrate. This opens the perspective of controlling the micro-adhesion of stem cells and the orientation of their filopodia by tuning the anisotropic substrate morphology without chemical reactions occurring at the surface.

  19. Cell Adhesion Modification of Streptococcus viridians in the Presence of Xylitol

    Science.gov (United States)

    Esmacher, Jason; Vidakovich, Blair; Giangrande, Michael; Hoffmann, Peter

    2012-10-01

    There is scientific documentation that those who chew gum sweetened by the sugar alcohol xylitol report a dramatically lower incident of both dental caries and otitis media compared to those who chew conventional gum sweetened by sucrose. An explanation contends that xylitol interferes with the ability of Streptococcus viridian (SV) to form biofilms which is a necessary precursor to the bacteria's ability to damage human tissues. We have used atomic force microscopy to study the cell wall/fimbria properties at the nanonewton level in both the presence and absence of xylitol. The first set of measurements used varying concentrations of xylitol incorporated within the incubation medium. The second used non-xylitol grown bacteria, the xylitol was added externally at various concentrations. Our study suggests that growing SV with xylitol reduces their ability to adhere together. Additionally, externally added xylitol showed grouping of cell adhesion to a relatively narrow nanonewton spread that is concentration dependent. Measurement of the adhesion properties of the bacterial cell wall have found that there is a dramatic increase in the cell wall's firmness which simultaneously accompanied a decrease in its ability to support adhesion, even at very low concentrations of xylitol.

  20. A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

    Science.gov (United States)

    Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

    2006-07-01

    Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

  1. 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.

  2. Reversible lysine modification on proteins by using functionalized boronic acids.

    Science.gov (United States)

    Cal, Pedro M S D; Frade, Raquel F M; Cordeiro, Carlos; Gois, Pedro M P

    2015-05-26

    Iminoboronates have been utilized to successfully install azide and alkyne bioorthogonal functions on proteins, which may then be further reacted with their bioorthogonal counterparts. These constructs were also used to add polyethylene glycol (PEG) to insulin, a modification which has been shown to be reversible in the presence of fructose. Finally, iminoboronates were used to assemble a folic acid/paclitaxel small-molecule/drug conjugate in situ with an IC50  value of 20.7 nM against NCI-H460 cancer cells and negligible cytotoxicity against the CRL-1502 noncancer cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions.

    OpenAIRE

    1995-01-01

    Focal adhesions are sites of cell-extracellular matrix interactions that function in anchoring stress fibers to the plasma membrane and in adhesion-mediated signal transduction. Both focal adhesion structure and signaling ability involve protein tyrosine phosphorylation. LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion-like ectodomain and two intracellular protein tyrosine phosphatase domains. We have identified a novel cytoplasmic 160 kDa pho...

  4. Strong underwater adhesives made by self-assembling multi-protein nanofibres.

    Science.gov (United States)

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M; Lu, Timothy K

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m(-2), which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  5. Strong underwater adhesives made by self-assembling multi-protein nanofibres

    Science.gov (United States)

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A.; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M.; Lu, Timothy K.

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m-2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  6. Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

    Directory of Open Access Journals (Sweden)

    Jaimie-Leigh Jonker

    Full Text Available 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.

  7. Investigating the BSA protein adsorption and bacterial adhesion of Al-alloy surfaces after creating a hierarchical (micro/nano) superhydrophobic structure.

    Science.gov (United States)

    Moazzam, Parisa; Razmjou, Amir; Golabi, Mohsen; Shokri, Dariush; Landarani-Isfahani, Amir

    2016-09-01

    Bacterial adhesion and subsequent biofilm formation on metals such as aluminum (Al) alloys lead to serious issues in biomedical and industrial fields from both an economical and health perspective. Here, we showed that a careful manipulation of Al surface characteristics via a facile two-steps superhydrophobic modification can provide not only biocompatibility and an ability to control protein adsorption and bacterial adhesion, but also address the issue of apparent long-term toxicity of Al-alloys. To find out the roles of surface characteristics, surface modification and protein adsorption on microbial adhesion and biofilm formation, the surfaces were systematically characterized by SEM, EDX, XPS, AFM, FTIR, water contact angle (WCA) goniometry, surface free energy (SFE) measurement, MTT, Bradford, Lowry and microtiter plate assays and also flow-cytometry and potentiostat analyses. Results showed that WCA and SFE changed from 70° to 163° and 36.3 to 0.13 mN m(-1) , respectively. The stable and durable modification led to a substantial reduction in static/dynamic BSA adsorption. The effect of such a treatment on the biofilm formation was analyzed by using three different bacteria of Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus. The microtiter plate assay and flow cytometry analysis showed that the modification not only could substantially reduce the bacterial adhesion but this biofouling resistance is independent of bacterium type. An excellent cell viability after exposure of HeLa cells to waters incubated with the modified samples was observed. Finally, the corrosion rate reduced sharply from 856.6 to 0.119 MPY after superhydrophobic modifications, which is an excellent stable corrosion inhibition property. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2220-2233, 2016.

  8. Surface Modification of a PCB Substrate for Better Adhesion of Inkjet Printed Circuit Structures

    NARCIS (Netherlands)

    Sridhar, A.; Dijk, van D.J.; Akkerman, R.

    2009-01-01

    The robustness and service life of inkjet printed electronic circuit structures are highly influenced by the state of the interface between these structures and the substrate. In the case of polymeric substrate materials, surface modification is necessary to realise a favourable interface, as these

  9. 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......-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p2+/-3.2 vs 16.9+/-2.6 vs......-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans...

  10. 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.

  11. Protein pI shifts due to posttranslational modifications in the separation and characterization of proteins.

    Science.gov (United States)

    Zhu, Kan; Zhao, Jia; Lubman, David M; Miller, Fred R; Barder, Timothy J

    2005-05-01

    Proteins from breast cancer cell lines are characterized using a 2-D liquid separation technique in which protein pI is used as the first-dimension separation parameter. To effect this protein pI separation, chromatofocusing(CF) is employed whereby a pH gradient is generated on-column using a weak anion exchange medium with the intact proteins fractionated and collected every 0.2 pH unit. It is demonstrated that the pI for expressed intact proteins as generated by CF is an important parameter for identification and characterization of the actual protein modifications occurring in the cancer cell. For most proteins, the experimentally determined pI is very close to that predicted by the databases. In other cases, however, where the pI is observed to be shifted from the expected value, it is shown that this shift is often correlated to protein modifications. The modifications that cause such shifts include truncations and deletions often observed in cancer cells or phosphorylations that can shift the pI by several pH units. It is also shown that the effects of phosphorylation on the observed shift can vary depending upon the protein and the amount of phosphorylation. Moreover, large changes in the pI are often observed for proteins with a pI above 7.0 upon phosphorylation, whereas little change is observed for proteins with a pI of approximately 5.0. The expressed protein's pI value thus becomes an important parameter together with the intact MW value, peptide map, and MS/MS results for identification of the presence and type of posttranslational modifications occurring in the cancer cell.

  12. Surface engineering and adhesion modification of SAM surfaces of 1-hexanethiol and 1-decanethiol: confining Staphylococcus aureus

    Science.gov (United States)

    Olsen, Morgan; Amroski, Alicia; Calabrese, Joseph; Senevirathne, Reshani; Senevirathne, Indrajith

    2012-02-01

    Engineering surfaces for adhesion and confinement of bacteria is interesting towards development of respective biosensors, and bio machine interfacing. Investigation was focused towards modification of surfaces towards confinement and entrapment of the nonpathogenic strain Staphylococcus aureus or similar pathogenic strains and to study surface engineering. Clean, flat Au(111) on mica surfaces were used for self assembly for Self Assembled Monolayers (SAM). 1-hexanethiol, and 1-decanethiol were used at total 5 mM solutions in varying ratios, in 200 proof Ethanol solution. Resulting SAM layers were investigated for surface corrugation, morphology and structure variation at different thiol ratios. Observations will be discussed, quantitatively and qualitatively. Eventual mixture ratios were so selected towards optimum conditions for confining Staphylococcus aureus as a model system. SAM surfaces were investigated using intermittent contact, noncontact, lateral force and contact modes of Atomic Force Microscopy (AFM).

  13. Polyimide surface modification by using microwave plasma for adhesion enhancement of Cu electroless plating.

    Science.gov (United States)

    Cho, Sang-Jin; Nguyen, Trieu; Boo, Jin-Hyo

    2011-06-01

    Microwave (MW) plasma was applied to the surface of polyimide (PI) films as a treatment to enhance the adhesion between copper deposition layer and PI surface for electroless plating. The influences of nitrogen MW plasma treatment on chemical composition of the PI surface were investigated by using X-Ray photoelectron spectroscopy (XPS). The wettability was also investigated by water contact angle measurement. The surface morphologies of PI films before and after treatment were characterized with atomic force microscopy (AFM). The contact angle results show that was dramatically decreased to 16.1 degrees at the optimal treatment condition from 72.1 degrees (untreated PI). However, the root mean square (RMS) roughness of treated PI film was almost unchanged. The AFM roughness was stayed from 1.0 to 1.2 with/without plasma treatment. XPS data show a nitrogen increase when PI films exposed to N2 MW plasma. Electroless copper depositions were carried out with the free-formaldehyde method using glyoxylic acid as the reducing reagent and mixture palladium chloride, tin chloride as activation solution. Adhesion property between polyimide surface and copper layer was investigated by tape test.

  14. Protein-protein-interaction Network Organization of the Hypusine Modification System*

    Science.gov (United States)

    Sievert, Henning; Venz, Simone; Platas-Barradas, Oscar; Dhople, Vishnu M.; Schaletzky, Martin; Nagel, Claus-Henning; Braig, Melanie; Preukschas, Michael; Pällmann, Nora; Bokemeyer, Carsten; Brümmendorf, Tim H.; Pörtner, Ralf; Walther, Reinhard; Duncan, Kent E.; Hauber, Joachim; Balabanov, Stefan

    2012-01-01

    Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: “bioreactor-TAP-MS/MS.” By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions. PMID:22888148

  15. Protein-protein-interaction network organization of the hypusine modification system.

    Science.gov (United States)

    Sievert, Henning; Venz, Simone; Platas-Barradas, Oscar; Dhople, Vishnu M; Schaletzky, Martin; Nagel, Claus-Henning; Braig, Melanie; Preukschas, Michael; Pällmann, Nora; Bokemeyer, Carsten; Brümmendorf, Tim H; Pörtner, Ralf; Walther, Reinhard; Duncan, Kent E; Hauber, Joachim; Balabanov, Stefan

    2012-11-01

    Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.

  16. Adhesion mechanism in a DOPA-deficient foot protein from green mussels†

    Science.gov (United States)

    Hwang, Dong Soo; Zeng, Hongbo; Lu, Qingye; Israelachvili, Jacob; Waite, J. Herbert

    2012-01-01

    The holdfast or byssus of Asian green mussels, Perna viridis, contains a foot protein, pvfp-1, that differs in two respects from all other known adhesive mussel foot proteins (mfp): (1) instead of the hallmark L-3,4-dihydroxyphenylalanine (DOPA) residues in mfp-1, for example, pvfp-1 contains C2-mannosyl-7-hydroxytryptophan (Man7OHTrp). (2) In addition, pvfp-1 chains are not monomeric like mfp-1 but trimerized by collagen and coiled-coil domains near the carboxy terminus after a typical domain of tandemly repeated decapeptides. Here, the contribution of these peculiarities to adhesion was examined using a surface forces apparatus (SFA). Unlike previously studied mfp-1s, pvfp-1 showed significant adhesion to mica and, in symmetric pvfp-1 films, substantial cohesive interactions were present at pH 5.5. The role of Man7OHTrp in adhesion is not clear, and a DOPA-like role for Man7OHTrp in metal complexation (e.g., Cu2+, Fe3+) was not observed. Instead, cation–π interactions with low desolvation penalty between Man7OHTrp and lysyl side chains and conformational changes (raveling and unraveling of collagen helix and coiled-coil domains) are the best explanations for the strong adhesion between pvfp-1 monomolecular films. The strong adhesion mechanism induced by cation–π interactions and conformational changes in pvfp-1 provides new insights for the development of biomimetic underwater adhesives. PMID:23105946

  17. 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.

  18. 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.

  19. Protein adhesion on dental surfaces-a combined surface analytical approach.

    Science.gov (United States)

    Müller, Christine; Wald, Johanna; Hoth-Hannig, Wiebke; Umanskaya, Natalia; Scholz, Daniel; Hannig, Matthias; Ziegler, Christiane

    2011-05-01

    Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-07-12

    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.

  1. 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...

  2. Bacterial binding to extracellular proteins - in vitro adhesion

    DEFF Research Database (Denmark)

    Schou, C.; Fiehn, N.-E.

    1999-01-01

    Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis......Viridans streptococci, bacterial adherence, extracellular matrix proteins, surface receptors, endocarditis...

  3. Effect of Monocyte Chemotactic Protein-1 on the Intraperitoneal Adhesion Formation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to study the role of monocyte chemotactic protein-1 (MCP-1) in the intra-peritoneal adhesion formation, 23 infertile patients undergoing laparoscopic operation were divided into two groups: experimental group including 12 patients with intra-peritoneal adhesion and control group including 11 patients without intra-peritoneal adhesion. Peritoneal fluid (PF) and peritoneum were collected from these patients during laparoscopic examination. The expression levels of MCP-l protein and MCP-1 mRNA were detected by using enzyme-linked immunosorbent assay (ELISA) and dot blot analysis method respectively. It was found that the levels of MCP-1 protein in PF of the patients with peritoneal adhesion were significantly higher than in the control group (0. 44±0.11 ng/ml vs 0. 19+0. 09 ng/ml respectively, P<0. 01 ). The level of MCP-1 mRNA in the peritoneum of the patients with peritoneal adhesion was significantly higher than in the control group (48.61±3.72 vs 19. 87±2.54 respectively, P<0. 01). It was suggested that MCP-1 might play a role in the adhesion formation, and chemotactic cytokines expressing in the peritoneal mesothelial cells might be take part in the process.

  4. Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins.

    Science.gov (United States)

    Lee, J H; Lee, H B

    1998-08-01

    A wettability gradient was prepared on lowdensity polyethylene (PE) sheets by treating them in air with a corona from a knife-type electrode the power of which increased gradually along the sample length. The PE surfaces oxidized gradually with the increasing corona power and a wettability gradient was created on the surfaces, as evidenced by the measurement of water contact angles, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. The wettability gradient surfaces prepared were used to investigate the adhesion behavior of platelets in the absence and presence of plasma proteins in terms of the surface hydrophilicity/hydrophobicity of polymeric materials. The platelets adhered to the wettability gradient surfaces along the sample length were counted and examined by scanning electron microscopy (SEM). It was observed that the platelet adhesion in the absence of plasma proteins increased gradually as the surface wettability increased along the sample length. The platelets adhered to the hydrophilic positions of the gradient surface also were more activated (possessed more pseudo pods as examined by SEM) than on the more hydrophobic ones. However, platelet adhesion in the presence of plasma proteins decreased gradually with the increasing surface wettability; the platelets adhered to the surface also were more activated on the hydrophobic positions of the gradient surface. This result is closely related to plasma protein adsorption on the surface. Plasma protein adsorption on the wettability gradient surface increased with the increasing surface wettability. More plasma protein adsorption on the hydrophilic positions of the gradient surface caused less platelet adhesion, probably due to platelet adhesion inhibiting proteins, such as high-molecular-weight kininogen, which preferably adsorbs onto the surface by the so-called Vroman effect. It seems that both the presence of plasma proteins

  5. Adhesive performance of sorghum protein extracted from sorghum DDGS and flour

    Science.gov (United States)

    Distillers dried grains with solubles (DDGS) is the main co-product from grain-based ethanol production. The objective of this research was to compare the adhesive performance of three types of sorghum proteins: acetic acid-extracted sorghum protein from DDGS (PI), aqueous ethanol-extracted sorghum ...

  6. Regulation by S-Nitrosylation of Protein Post-translational Modification*

    OpenAIRE

    Hess, Douglas T.; Stamler, Jonathan S.

    2011-01-01

    Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this mi...

  7. 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.

  8. 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...

  9. 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.

  10. Posttranslational modification of autophagy-related proteins in macroautophagy.

    Science.gov (United States)

    Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J; Tang, Daolin

    2015-01-01

    Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.

  11. Assays for Posttranslational Modifications of Intermediate Filament Proteins.

    Science.gov (United States)

    Snider, Natasha T; Omary, M Bishr

    2016-01-01

    Intermediate filament (IF) proteins are known to be regulated by a number of posttranslational 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.

  12. 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

  13. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    Science.gov (United States)

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  14. 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

  15. 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.

  16. 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

  17. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    Science.gov (United States)

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  18. 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.

  19. Identification of macrophage external membrane proteins and their possible role in cell adhesion.

    Science.gov (United States)

    Pearlstein, E; Dienstman, S R; Defendi, V

    1978-10-01

    Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl-sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.

  20. [Study on FAK regulation of migration of vascular endothelial cells depending upon focal adhesion proteins].

    Science.gov (United States)

    Gao, Min; Liu, Xiaoheng; Sun, Heng; Ren, Hongyi; Wang, Lijuan; Shen, Yang

    2013-06-01

    Tumor angiogenesis induced by vascular endothelial cells (VECs) migration is a necessary condition for tumor growth and metastasis. The purpose of this study is to investigate the effect of focal adhesion kinase (FAK) inhibitor (50nmol/mL) on the adhesion and migration of endothelial cells(ECs) and the expression of focal adhesion proteins vinculin, talin and paxillin. Scratch wound migration assay was performed to examine the effect of FAK inhibitor with 50nmol/mL on ECs migration at 0, 5, 10, 30, 60 and 120min, respectively. And immunofluorescence analysis was performed to detect the expression of F-actin in ECs treated with FAK inhibitor within 2h. Western blot was carried out to determine the effect of FAK inhibitor on expression of vinculin, talin and paxillin proteins. The results showed that the migration distance and the expression of F-actin in ECs treated with FAK inhibitor decreased significantly compared with that of the controls, and the level of vinculin showed no significant difference with increasing of treated time of FAK inhibitor. However, the talin and paxillin showed an identical decreasing tendency in 5-10min, but slowly going up in 30min and then after subsequently decreasing. The results of this study proved that blocking phosphorylation of FAK could inhibit VECs adhesion and migration by downregulating focal adhesion proteins so that it may inhibit tumor angiogenesis. This may provide a new approach for tumor therapy.

  1. Hydrophobic enhancement of Dopa-mediated adhesion in a mussel foot protein.

    Science.gov (United States)

    Wei, Wei; Yu, Jing; Broomell, Christopher; Israelachvili, Jacob N; Waite, J Herbert

    2013-01-09

    Dopa (3,4-dihydroxyphenylalanine) is recognized as a key chemical signature of mussel adhesion and has been adopted into diverse synthetic polymer systems. Dopa's notorious susceptibility to oxidation, however, poses significant challenges to the practical translation of mussel adhesion. Using a surface forces apparatus to investigate the adhesion of mussel foot protein 3 (Mfp3) "slow", a hydrophobic protein variant of the Mfp3 family in the plaque, we have discovered a subtle molecular strategy correlated with hydrophobicity that appears to compensate for Dopa instability. At pH 3, where Dopa is stable, Mfp3 slow, like Mfp3 "fast" adhesion to mica, is directly proportional to the mol % of Dopa present in the protein. At pH of 5.5 and 7.5, however, loss of adhesion in Mfp3 slow was less than half that occurring in Mfp3 fast, purportedly because Dopa in Mfp3 slow is less prone to oxidation. Indeed, cyclic voltammetry showed that the oxidation potential of Dopa in Mfp3 slow is significantly higher than in Mfp3 fast at pH of 7.5. A much greater difference between the two variants was revealed in the interaction energy of two symmetric Mfp3 slow films (E(ad) = -3 mJ/m(2)). This energy corresponds to the energy of protein cohesion which is notable for its reversibility and pH independence. Exploitation of aromatic hydrophobic sequences to protect Dopa against oxidation as well as to mediate hydrophobic and H-bonding interactions between proteins provides new insights for developing effective artificial underwater adhesives.

  2. Considering protonation as a posttranslational modification regulating protein structure and function.

    Science.gov (United States)

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

    2013-01-01

    Posttranslational modification is an evolutionarily conserved mechanism for regulating protein activity, binding affinity, and stability. Compared with established posttranslational modifications such as phosphorylation or ubiquitination, posttranslational 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, posttranslational modification by protons can drive dynamic changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and, in contrast to most other posttranslational 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 posttranslational modification of pH-sensing proteins regulating different cellular processes.

  3. 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.

  4. Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis.

    Science.gov (United States)

    Wu, Xia; Oh, Man-Ho; Schwarz, Eliezer M; Larue, Clayton T; Sivaguru, Mayandi; Imai, Brian S; Yau, Peter M; Ort, Donald R; Huber, Steven C

    2011-04-01

    Lysine acetylation (LysAc), a form of reversible protein posttranslational modification previously known only for histone regulation in plants, is shown to be widespread in Arabidopsis (Arabidopsis thaliana). Sixty-four Lys modification sites were identified on 57 proteins, which operate in a wide variety of pathways/processes and are located in various cellular compartments. A number of photosynthesis-related proteins are among this group of LysAc proteins, including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins (LHCb), Rubisco large and small subunits, and chloroplastic ATP synthase (β-subunit). Using two-dimensional native green/sodium dodecyl sulfate gels, the loosely PSII-bound LHCb was separated from the LHCb that is tightly bound to PSII and shown to have substantially higher level of LysAc, implying that LysAc may play a role in distributing the LHCb complexes. Several potential LysAc sites were identified on eukaryotic elongation factor-1A (eEF-1A) by liquid chromatography/mass spectrometry and using sequence- and modification-specific antibodies the acetylation of Lys-227 and Lys-306 was established. Lys-306 is contained within a predicted calmodulin-binding sequence and acetylation of Lys-306 strongly inhibited the interactions of eEF-1A synthetic peptides with calmodulin recombinant proteins in vitro. These results suggest that LysAc of eEF-1A may directly affect regulatory properties and localization of the protein within the cell. Overall, these findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism of a large number of nonhistone proteins affecting a wide range of pathways and processes in Arabidopsis and likely in all plants.

  5. 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.

  6. The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces.

    Science.gov (United States)

    Schuster, Swen; Yu, Wenqi; Nega, Mulugeta; Chu, Ya-Yun; Zorn, Stefan; Zhang, Fajun; Götz, Friedrich; Schreiber, Frank

    2014-11-01

    Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins

    Science.gov (United States)

    Kirchenbüchler, David; Born, Simone; Kirchgeßner, Norbert; Houben, Sebastian; Hoffmann, Bernd; Merkel, Rudolf

    2010-05-01

    Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile

  8. The human and mouse repertoire of the adhesion family of G-protein-coupled receptors.

    Science.gov (United States)

    Bjarnadóttir, Thóra K; Fredriksson, Robert; Höglund, Pär J; Gloriam, David E; Lagerström, Malin C; Schiöth, Helgi B

    2004-07-01

    The adhesion G-protein-coupled receptors (GPCRs) (also termed LN-7TM or EGF-7TM receptors) are membrane-bound proteins with long N-termini containing multiple domains. Here, 2 new human adhesion-GPCRs, termed GPR133 and GPR144, have been found by searches done in the human genome databases. Both GPR133 and GPR144 have a GPS domain in their N-termini, while GPR144 also has a pentraxin domain. The phylogenetic analyses of the 2 new human receptors show that they group together without close relationship to the other adhesion-GPCRs. In addition to the human genes, mouse orthologues to those 2 and 15 other mouse orthologues to human were identified (GPR110, GPR111, GPR112, GPR113, GPR114, GPR115, GPR116, GPR123, GPR124, GPR125, GPR126, GPR128, LEC1, LEC2, and LEC3). Currently the total number of human adhesion-GPCRs is 33. The mouse and human sequences show a clear one-to-one relationship, with the exception of EMR2 and EMR3, which do not seem to have orthologues in mouse. EST expression charts for the entire repertoire of adhesion-GPCRs in human and mouse were established. Over 1600 ESTs were found for these receptors, showing widespread distribution in both central and peripheral tissues. The expression patterns are highly variable between different receptors, indicating that they participate in a number of physiological processes. Copyright 2003 Elsevier Inc.

  9. 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...

  10. 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...

  11. Catalytic protein modification with dirhodium metallopeptides: specificity in designed and natural systems.

    Science.gov (United States)

    Chen, Zhen; Vohidov, Farrukh; Coughlin, Jane M; Stagg, Loren J; Arold, Stefan T; Ladbury, John E; Ball, Zachary T

    2012-06-20

    In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.

  12. Protein cysteine modifications: (2) reactivity specificity and topics of medicinal chemistry and protein engineering.

    Science.gov (United States)

    Nagahara, Noriyuki; Matsumura, Tomohiro; Okamoto, Ryo; Kajihara, Yasuhiro

    2009-01-01

    Cysteine (cysteinyl residue) modifications in proteins result in diversity in protein functions. The reaction specificity of a protein with a modified cysteine residue is determined by the overall conditions of the protein, including the spatial position of the cysteine residue, electrostatic interactions between cysteine residue and other charged residues, spatial interactions between the cysteine residue and a chemical compound, electrophilicity of the chemical compound, and the pH of the solution. In cysteine-dependant enzymes, each specific type of cysteine modification characterizes the catalytic mechanism of the enzyme. Recently, the catalytic mechanisms of peroxiredoxins and cysteine proteases, which contain a cysteine residue(s) in their catalytic sites, have been elucidated. In the catalytic process of peroxiredoxins, a sulfenyl intermediate is formed by oxidation of the catalytic cysteine residue. On the other hand, in cysteine proteases, the catalytic cysteine residue reacts with the carboxyl carbon of a peptide substrate to form an intermediate complex via S-alkylation. In this review, we introduce the most current information on the applications of cysteine thiol chemistry for in vitro glycoprotein synthesis. Recently, a glycoprotein (monocyte chemotactic protein-3), containing an intact human complex-type sialyloligosaccharide has been chemically synthesized. The procedure used for this could have applications in the development of new protein-based drugs, including antineoplastic drugs and antibiotics. It can also potentially be applied for improving the half-life and reducing the toxicity of these drugs, and for preventing the development of multidrug resistance.

  13. Polylysine modification of adenoviral fiber protein enhances muscle cell transduction.

    Science.gov (United States)

    Bouri, K; Feero, W G; Myerburg, M M; Wickham, T J; Kovesdi, I; Hoffman, E P; Clemens, P R

    1999-07-01

    Adenoviral vectors (ADVs) are used widely for gene delivery to different tissues including muscle. One particularly promising use for ADVs is in the transfer of the dystrophin gene to the muscle of patients with Duchenne muscular dystrophy (DMD). However, studies in different animal models of DMD suggest that ADVs inefficiently transduce mature skeletal muscle. In this article we test whether AdZ.F(pK7), a genetically modified ADV that expresses a polylysine moiety on the end of the fiber protein, could enhance transduction of muscle cells and circumvent the maturation-dependent loss of muscle infectivity by ADVs. The efficiency of transduction was tested at different levels of muscle maturation. In vitro, AdZ.F(pK7) showed a higher level of transduction at all stages of differentiation including myoblasts, myotubes, and single muscle fibers. In vivo, mature skeletal muscle was transduced fourfold better by AdZ.F(pK7) than by the unmodifled vector (AdZ.F). Together, these observations demonstrate improved ADV transduction of skeletal muscle by modifying ADV tropism, and provide a proof-of-principle that modification of ADVs to target muscle-specific molecules could result in tissue-specific transfer of skeletal muscle tissue as well.

  14. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  15. Adhesive ability means inhibition activities for lactobacillus against pathogens and S-layer protein plays an important role in adhesion.

    Science.gov (United States)

    Zhang, Wenming; Wang, Haifeng; Liu, Jianxin; Zhao, Yunhao; Gao, Kan; Zhang, Juan

    2013-08-01

    Eighty-five strains of lactobacillus were isolated from the pig intestine and identified by sequencing analysis based on 16S rRNA gene, from which five lactobacillus strains with high adhesive ability were selected. The inhibition ability of the five lactobacillus strains with or without S-layer proteins against adherence of Escherichia coli K88 and Salmonella enteritidis 50335 to Caco-2 was evaluated in vitro with Lactobacillus rhamnosus GG strain (LGG) as a positive control. In addition, tolerance of lactobacilli to heat, acid, bile, Zn(2+) and Cu(2+) were assessed. All five selected strains, Lactobacillus salivarius ZJ614 (JN981856), Lactobacillus reuteri ZJ616 (JN981858), L. reuteri ZJ617 (JN981859), L. reuteri ZJ621 (JN981863) and L. reuteri ZJ623 (JN981865), showed inhibition against the two pathogens, E. coli K88 and S. enteritidis 50335. L. reuteri ZJ621 showed higher inhibition ability than the others to S. enteritidis 50335 (P S-layer protein, the inhibition activities of the lactobacilli against pathogens decreased significantly (P S-layer proteins plays an important role.

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  17. 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.

  18. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-05

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Use of soy proteins in polyketone-based wood adhesives

    NARCIS (Netherlands)

    Hamarneh, A.; Heeres, H. J.; Broekhuis, A. A.; Sjollema, K. A.; Zhang, Y.; Picchioni, F.

    2010-01-01

    This paper describes the preparation of aqueous emulsions consisting of soy proteins and chemically modified thermosetting aliphatic polyketones. Emulsions were prepared in a range of total solids contents and different addition protocols were tested. Room temperature stability and structure of the

  20. 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.

  1. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  2. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling.

    Science.gov (United States)

    Vowinckel, Jakob; Stahlberg, Silke; Paulmann, Nils; Bluemlein, Katharina; Grohmann, Maik; Ralser, Markus; Walther, Diego J

    2012-11-02

    Posttranslational modifications (PTM) have been shown to be essential for protein function and signaling. Here we report the identification of a novel modification, protein transfer of histamine, and provide evidence for its function in G protein signaling. Histamine, known as neurotransmitter and mediator of the inflammatory response, was found incorporated into mastocytoma proteins. Histaminylation was dependent on transglutaminase II. Mass spectrometry confirmed histamine modification of the small and heterotrimeric G proteins Cdc42, Gαo1 and Gαq. The modification was specific for glutamine residues in the catalytic core, and triggered their constitutive activation. TGM2-mediated histaminylation is thus a novel PTM that functions in G protein signaling. Protein αmonoaminylations, thus including histaminylation, serotonylation, dopaminylation and norepinephrinylation, hence emerge as a novel class of regulatory PTMs.

  3. 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.

  4. Streptococcal collagen-like surface protein 1 promotes adhesion to the respiratory epithelial cell

    Directory of Open Access Journals (Sweden)

    Chang Cherng-Shyang

    2010-12-01

    Full Text Available Abstract Background Collagen-like surface proteins Scl1 and Scl2 on Streptococcus pyogenes contain contiguous Gly-X-X triplet amino acid motifs, the characteristic structure of human collagen. Although the potential role of Scl1 in adhesion has been studied, the conclusions may be affected by the use of different S. pyogenes strains and their carriages of various adhesins. To explore the bona fide nature of Scl1 in adherence to human epithelial cells without the potential interference of other streptococcal surface factors, we constructed a scl1 isogenic mutant from the Scl2-defective S. pyogenes strain and a Scl1-expressed Escherichia coli. Results Loss of Scl1 in a Scl2-defective S. pyogenes strain dramatically decreased the adhesion of bacteria to HEp-2 human epithelial cells. Expression of Scl1 on the surface of the heterologous bacteria E. coli significantly increased adhesion to HEp-2. The increase in adhesion was nullified when Scl1-expressed E. coli was pre-incubated with proteases or antibodies against recombinant Scl1 (rScl1 protein. Treatment of HEp-2 cells with rScl protein or pronase drastically reduced the binding capability of Scl1-expressed E. coli. These findings suggest that the adhesion is mediated through Scl1 on bacterial surface and protein receptor(s on epithelial cells. Further blocking of potential integrins revealed significant contributions of α2 and β1 integrins in Scl1-mediated binding to epithelial cells. Conclusions Together, these results underscore the importance of Scl1 in the virulence of S. pyogenes and implicate Scl1 as an adhesin during pathogenesis of streptococcal infection.

  5. 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...

  6. Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion

    DEFF Research Database (Denmark)

    Köwitsch, Alexander; Yang, Yuan; Ma, Ning

    2011-01-01

    Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification...... affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated...

  7. Mechanical and water soaking properties of medium density fiberboard with wood fiber and soybean protein adhesive.

    Science.gov (United States)

    Li, Xin; Li, Yonghui; Zhong, Zhikai; Wang, Donghai; Ratto, Jo A; Sheng, Kuichuan; Sun, Xiuzhi Susan

    2009-07-01

    Soybean protein is a renewable and abundant material that offers an alternative to formaldehyde-based resins. In this study, soybean protein was modified with sodium dodecyl sulfate (SDS) as an adhesive for wood fiber medium density fiberboard (MDF) preparation. Second-order response surface regression models were used to study the effects and interactions of initial moisture content (IMC) of coated wood fiber, press time (PT) and temperature on mechanical and water soaking properties of MDF. Results showed that IMC of coated fiber was the dominant influencing factor. Mechanical and soaking properties improved as IMC increased and reached their highest point at an IMC of 35%. Press time and temperature also had a significant effect on mechanical and water soaking properties of MDF. Second-order regression results showed that there were strong relationships between mechanical and soaking properties of MDF and processing parameters. Properties of MDF made using soybean protein adhesive are similar to those of commercial board.

  8. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    Science.gov (United States)

    Seong, Jihye; Tajik, Arash; Sun, Jie; Guan, Jun-Lin; Humphries, Martin J; Craig, Susan E; Shekaran, Asha; García, Andrés J; Lu, Shaoying; Lin, Michael Z; Wang, Ning; Wang, Yingxiao

    2013-11-26

    Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

  9. Post-translation modification of proteins; methodologies and applications in plant sciences.

    Science.gov (United States)

    Bond, A E; Row, P E; Dudley, E

    2011-07-01

    Proteins have the potential to undergo a variety of post-translational modifications and the different methods available to study these cellular processes has advanced rapidly with the continuing development of proteomic technologies. In this review we aim to detail five major post-translational modifications (phosphorylation, glycosylaion, lipid modification, ubiquitination and redox-related modifications), elaborate on the techniques that have been developed for their analysis and briefly discuss the study of these modifications in selected areas of plant science. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Research Progress and Modification of Polyurethane Adhesive%聚氨酯胶粘剂的研究及改性现状

    Institute of Scientific and Technical Information of China (English)

    韩红青

    2012-01-01

    简述了聚氨酯胶粘剂的分类及特点、常见的改性方法,对国内外聚氨酯胶粘剂改性研究的现状做了分析.从研究现状来看,国内对高性能溶剂型聚氨酯胶粘剂的研究较多,其生产技术日趋成熟;国内外对水性聚氨酯胶粘剂的研究做了大量的工作,但其在国内大规模生产的技术还不具备,特种功能性的改性聚氨酯胶粘剂和环境友好型的水性聚氨酯胶粘剂成为将来的发展趋势.%The classification and characteristics of polyurethane adhesive, the modification methods and the recent research and development in home and abroad were summarized. Viewed from present research current situation , the excellent properties of solvent base polyurethane adhesive was mainly studied in China whose technology was coming to maturity. A lot of researches on waterborne polyurethane adhesive had been done at home and a-broad, but China didn' t have the capability of mass production technology. The special function of the modified polyurethane adhesive and environment friendly waterborne polyurethane adhesive were the trends in the future.

  11. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology.

  12. The Effect of Primer Application Modifications on the Bond Strength of 4th Generation Adhesive Bonding Agents

    Science.gov (United States)

    2012-03-30

    The influence of deviations from the manufacturer’s instructions for the use of six adhesive systems on the bond strengths to enamel and dentin...application of two layers of the self- etching adhesive systems produced significant improvement in bond strength to enamel compared to the passive...2010) evaluated the relationship between the number of adhesive layers and internal adaptation on the microtensile bond strength to enamel and dentin

  13. Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

    Directory of Open Access Journals (Sweden)

    O.C. Lima

    1999-05-01

    Full Text Available The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate (poly-HEMA was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent. The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin was statistically significant (P<0.05 when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

  14. Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

    Science.gov (United States)

    Linder, Markus; Szilvay, Geza R.; Nakari-Setälä, Tiina; Söderlund, Hans; Penttilä, Merja

    2002-01-01

    Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C-terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface-bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins. PMID:12192081

  15. OmpA-like protein influences cell shape and adhesive activity of Tannerella forsythia.

    Science.gov (United States)

    Abe, T; Murakami, Y; Nagano, K; Hasegawa, Y; Moriguchi, K; Ohno, N; Shimozato, K; Yoshimura, F

    2011-12-01

    Tannerella forsythia, a gram-negative fusiform rod, is implicated in several types of oral anaerobic infections. Most gram-negative bacteria have OmpA-like proteins that are homologous to the OmpA protein in Escherichia coli. We identified an OmpA-like protein in T. forsythia encoded by the tf1331 gene as one of the major proteins by mass spectrometric analysis. Two-dimensional, diagonal electrophoresis showed that the OmpA-like protein formed a dimeric or trimeric structure via intermolecular disulfide bonds. A biotin labeling experiment revealed that a portion of the protein was exposed on the cell surface, even though T. forsythia possesses an S-layer at the outermost cell surface. Using a tf1331-deletion mutant, we showed that the OmpA-like protein affected cell morphology. The length of the mutant cell was reduced almost by half. Cell swelling was observed in more than 40% of the mutant cells. Moreover, the mutant exhibited decreased adhesion to fibronectin, retarded autoaggregation, and reduced cell surface hydrophobicity. These results suggest that the OmpA-like protein in T. forsythia plays an important role in cellular integrity and adhesive function.

  16. Glycopolymer functionalization of engineered spider silk protein-based materials for improved cell adhesion.

    Science.gov (United States)

    Hardy, John G; Pfaff, André; Leal-Egaña, Aldo; Müller, Axel H E; Scheibel, Thomas R

    2014-07-01

    Silk protein-based materials are promising biomaterials for application as tissue scaffolds, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) and their functionalization with glycopolymers are described. The glycopolymers bind proteins found in the extracellular matrix, providing a biomimetic coating on the films that improves cell adhesion to the surfaces of engineered spider silk films. Such silk-based materials have potential as coatings for degradable implantable devices. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    Science.gov (United States)

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-03-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments.

  18. High-Throughput Liquid-Liquid Fractionation of Multiple Protein Post-Translational Modifications*

    Science.gov (United States)

    DeFord, James H.; Nuss, Jonathan E.; Amaning, James; English, Robert D.; Tjernlund, Don; Papaconstantinou, John

    2009-01-01

    Post-translational protein modifications have contributed significantly to the identification of macromolecular biomarkers of biological processes. We have modified a 2-dimensional HPLC system (Beckman Coulter PF2D ProteomeLab) to create proteome maps of post-translational protein modifications. This system resolves complex protein mixtures by anion exchange chromatofocusing in the first dimension and hydrophobicity (reverse phase chromatography) in the second dimension. The simultaneous identification of multiple protein modifications, accomplished by incorporating a photo diode array (PDA) detector into the PF2D system, facilitates the simultaneous production of three dimensional proteome maps and visualization of both unmodified and post-translationally modified (PTM) proteins at their signature wavelengths within the proteome. We describe procedures for the simultaneous resolution of proteome maps, the identification of proteins modified by nitration, carbonylation, and phosphorylation, and proteins with unique spectra such as the heme containing proteins. PMID:19099502

  19. Pseudouridine and N(6)-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 N(6)-methyladenosine (m(6)A)-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 m(6)A 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.

  20. Salivary protein adsorption and Streptococccus gordonii adhesion to dental material surfaces.

    Science.gov (United States)

    Schweikl, Helmut; Hiller, Karl-Anton; Carl, Ulrich; Schweiger, Rainer; Eidt, Andreas; Ruhl, Stefan; Müller, Rainer; Schmalz, Gottfried

    2013-10-01

    The initial adhesion of microorganisms to clinically used dental biomaterials is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polytetrafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer cement as well as cobalt-chromium-molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1. Wettability of material surfaces precoated with salivary proteins or left in phosphate-buffered saline was determined by the measurement of water contact angles. Amounts of adsorbed proteins were determined directly on material surfaces after biotinylation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The same technique was used to analyze for the binding of biotinylated bacteria to material surfaces. The highest amount of proteins (0.18μg/cm(2)) adsorbed to hydrophobic PTFE samples, and the lowest amount (0.025μg/cm(2)) was detected on silicone. The highest number of S. gordonii (3.2×10(4)CFU/mm(2)) adhered to the hydrophilic glassionomer cement surface coated with salivary proteins, and the lowest number (4×10(3)CFU/mm(2)) was found on the hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the number of attached microorganisms were weakly negatively correlated. No such correlation between hydrophobicity and the number of bacteria was detected when surfaces were coated with salivary proteins. Functional groups added by the adsorption of specific salivary proteins to material surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a physical property. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  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. 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.

  3. 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.

  4. Endocytosis regulates cell soma translocation and the distribution of adhesion proteins in migrating neurons.

    Directory of Open Access Journals (Sweden)

    Jennifer C Shieh

    Full Text Available Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.

  5. Enhancement of CNT/PET film adhesion by nano-scale modification for flexible all-solid-state supercapacitors

    Science.gov (United States)

    Kang, Yu Jin; Chung, Haegeun; Kim, Min-Seop; Kim, Woong

    2015-11-01

    We demonstrate the fabrication of high-integrity flexible supercapacitors using carbon nanotubes (CNTs), polyethylene terephthalate (PET) films, and ion gels. Although both CNTs and PET films are attractive materials for flexible electronics, they have poor adhesion properties. In this work, we significantly improve interfacial adhesion by introducing nanostructures at the interface of the CNT and PET layers. Simple reactive ion etching (RIE) of the PET substrates generates nano-scale roughness on the PET surface. RIE also induces hydrophilicity on the PET surface, which further enhances adhesive strength. The improved adhesion enables high integrity and excellent flexibility of the fabricated supercapacitors, demonstrated over hundreds of bending cycles. Furthermore, the supercapacitors show good cyclability with specific capacitance retention of 87.5% after 10,000 galvanostatic charge-discharge (GCD) cycles. Our demonstration may be important for understanding interfacial adhesion properties in nanoscale and for producing flexible, high-integrity, high-performance energy storage systems.

  6. 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.

  7. Progress and application of modification starch on wood adhesive%改性淀粉在木材胶粘剂中的应用研究进展

    Institute of Scientific and Technical Information of China (English)

    葛金龙; 孟庆佑; 邹宜哲

    2012-01-01

    淀粉在胶粘剂领域占有重要地位.经过氧化改性后,在淀粉分子链上引入大量分布均匀的化学键与氢键结合,改善耐水性,更能满足木材胶粘剂行业要求.文章综述了淀粉改性后在水性高分子-异氰酸酯(API)胶粘剂、聚乙酸乙烯酯胶粘剂和三聚氰胺-甲醛胶粘剂中作为木材胶粘剂的应用,分析了研究现状,探讨了发展趋势和研究方向.%The starch plays an important role in the field of wood adhesive.Most of the modification of starch are trying to make the full use of macromolecular chains of the starch and to connect chemical bonds evenly on the chain in order to enhance the water resistance and to meet the professional requirements in the wood adhesive industry.It is mainly summed that the modified starch is widely used in wood adhesive and the progress of application in polymer-isocyanate adhesive for wood splice,polyvinyl acetate emulsion,melamine-formaldehyde wood adhesive.Then,the present progress of the research,the tendency and prospect of modified starch on wood adhesive have been discussed in the paper.

  8. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  9. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  10. 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.

  11. Protein microarray applications: Autoantibody detection and posttranslational modification.

    Science.gov (United States)

    Atak, Apurva; Mukherjee, Shuvolina; Jain, Rekha; Gupta, Shabarni; Singh, Vedita Anand; Gahoi, Nikita; K P, Manubhai; Srivastava, Sanjeeva

    2016-10-01

    The discovery of DNA microarrays was a major milestone in genomics; however, it could not adequately predict the structure or dynamics of underlying protein entities, which are the ultimate effector molecules in a cell. Protein microarrays allow simultaneous study of thousands of proteins/peptides, and various advancements in array technologies have made this platform suitable for several diagnostic and functional studies. Antibody arrays enable researchers to quantify the abundance of target proteins in biological fluids and assess PTMs by using the antibodies. Protein microarrays have been used to assess protein-protein interactions, protein-ligand interactions, and autoantibody profiling in various disease conditions. Here, we summarize different microarray platforms with focus on its biological and clinical applications in autoantibody profiling and PTM studies. We also enumerate the potential of tissue microarrays to validate findings from protein arrays as well as other approaches, highlighting their significance in proteomics.

  12. 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.

  13. Modulating cell adhesion dynamics on carbon nanotube monolayer engineered with extracellular matrix proteins.

    Science.gov (United States)

    Cai, Ning; Wong, Chee C; Gong, Ying X; Tan, Samuel C W; Chan, Vincent; Liao, Kin

    2010-04-01

    Although it has been demonstrated that carbon nanotubes (CNTs) may have potentials for tissue engineering applications because of their unparalleled physical properties, little has been known on the cell adhesion mechanisms on model CNT monolayer pertaining to the design of novel cell therapeutics device. In this study, the adhesion dynamics of primary porcine esophageal fibroblasts (PEFs) on CNT monolayer were elucidated with confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy. Moreover, CNT monolayer (CNT-ML) was functionalized with two typical extracellular matrix (ECM) proteins including collagen type I (COL) and fibronectin (FN) in order to promote its biocompatibility. First, it is shown by atomic force microscopy that the topographical features of CNT-ML were dependent on the types of immobilized ECM protein. Second, significant time lag in adhesion contact evolution (around 10 min) for PEFs was found on both CNT-ML and CNT-COL compared to the negligible time lag on CNT-FN. It was found that adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces reached steady state at 60 and 30 min after cell seeding compared to 70 min on CNT-ML surface. At steady state, the adhesion energy of PEFs on the CNT-COL and CNT-FN surfaces was about twice as much than that on the CNT-ML surface. Moreover, immobilization of collagen or fibronectin on CNT monolayer led to an increase in seeding efficiency and proliferation rate of PEFs. Scanning electron microscopy and immunostaining together demonstrated that PEFs displayed an elongated morphology and highly polarized actin network on both CNT-COL and CNT-FN surfaces, whereas PEFs displayed nonuniform cell morphology and actin organization on the CNT-ML surface. Overall, our results demonstrated that the biophysical responses and biological behavior of PEFs on unmodified or functionalized CNT monolayer were different. Functionalization of CNT through extracellular matrix

  14. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    Science.gov (United States)

    Himmel, Mirko; Ritter, Anett; Rothemund, Sven; Pauling, Björg V; Rottner, Klemens; Gingras, Alexandre R; Ziegler, Wolfgang H

    2009-05-15

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and there are contradictory models with respect to the determinants of adhesion site localization. Here, we report turnover rates and protein-protein interactions in a range of talin rod domain constructs varying in helix bundle structure. We conclude that several bundles of the C terminus cooperate to regulate targeting and concomitantly tailor high affinity interactions of the talin rod in cell adhesions. Intrinsic control of ligand binding activities is essential for the coordination of adhesion site function of talin.

  15. 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......H 3.5 ( pKa) incubated fruits (~2 N). The protein bands at ~29 kDa, ~75 kDa, ~32 kDa and 87 kDa were exclusively or prominently found in ICA strengthened fruits (pH 3.5

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

    OpenAIRE

    2013-01-01

    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 t...

  17. Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence.

    Science.gov (United States)

    Avilés-Reyes, Alejandro; Miller, James H; Simpson-Haidaris, Patricia J; Hagen, Fred K; Abranches, Jacqueline; Lemos, José A

    2014-08-01

    Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.

  18. Post-translational modification of PII signal transduction proteins

    OpenAIRE

    Mike eMerrick

    2015-01-01

    The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target. PII prot...

  19. Glucose Autoxidation Induces Functional Damage to Proteins via Modification of Critical Arginine Residues†

    Science.gov (United States)

    Chetyrkin, Sergei; Mathis, Missy; Pedchenko, Vadim; Sanchez, Otto A.; McDonald, W. Hayes; Hachey, David L.; Madu, Hartman; Stec, Donald; Hudson, Billy; Voziyan, Paul

    2011-01-01

    Non-enzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to αVβ3 integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while non-oxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation. PMID:21661747

  20. Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues.

    Science.gov (United States)

    Chetyrkin, Sergei; Mathis, Missy; Pedchenko, Vadim; Sanchez, Otto A; McDonald, W Hayes; Hachey, David L; Madu, Hartman; Stec, Donald; Hudson, Billy; Voziyan, Paul

    2011-07-12

    Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.

  1. 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/.

  2. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

    Science.gov (United States)

    Chamma, Ingrid; Letellier, Mathieu; Butler, Corey; Tessier, Béatrice; Lim, Kok-Hong; Gauthereau, Isabel; Choquet, Daniel; Sibarita, Jean-Baptiste; Park, Sheldon; Sainlos, Matthieu; Thoumine, Olivier

    2016-01-01

    The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures. PMID:26979420

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

    Science.gov (United States)

    Hamann, Jörg; Aust, Gabriela; Araç, Demet; Engel, Felix B; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A; Harty, Breanne L; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C; Monk, Kelly R; Peeters, Miriam C; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schiöth, Helgi B

    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 names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-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. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  4. 淀粉胶黏剂的应用及改性研究进展%Application of Starch Adhesive and Research Progress in its Modification

    Institute of Scientific and Technical Information of China (English)

    杜郢; 王政; 董全江; 罗莉娟

    2013-01-01

    淀粉是常见的天然高分子材料,具有来源广泛、价格低廉、可再生、可降解等优点,作为胶黏剂在各领域的应用逐渐受到重视,但是淀粉胶黏剂在使用过程中存在很多不足,例如耐水性差、流动性不好、易霉变、储存稳定性差等,需要对其进行物理或化学改性,才能满足各行业使用要求.简要介绍了淀粉胶黏剂在各行业的应用情况,针对主要缺陷,详细阐述了近年来国内外研究者对淀粉胶黏剂的改性研究进展,并提出了改性淀粉胶黏剂未来的发展方向.%Starch is a common natural polymer material,owing to its advantages,such as abundant resource,low cost,biodegradable and renewable,it is becoming more and more attractive in the field of adhesive; however,starch has some defects such as poor water-resistance,poor mobility,easy to mold and poor storage stability when it is used as adhesive,so physical or chemical modification is necessary in order to make it meet the needs of industrial requirements.The application progresses in starch adhesives are summarized briefly.Considering of the major defects,its recent research situation on modification at home and abroad are detailed,and the development trends of modified starch adhesives are proposed.

  5. 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

    Cell adhesion molecules (CAMs) mediate cell-to-cell interactions and interactions between cells and the extracellular matrix (ECM). The neural cell adhesion molecule (NCAM), a prototypic member of the immunoglobulin (Ig) superfamily of CAMs, mediates adhesion through homophilic and heterophilic i...

  6. Mechanical Activation of a Multimeric Adhesive Protein through Domain Conformational Change

    Science.gov (United States)

    Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Patel, Jay M.; Moake, Joel; Dong, Jing-fei; Kiang, Ching-Hwa

    2013-01-01

    The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers. PMID:23521301

  7. In Vivo Detection of Vascular Adhesion Protein-1 in Experimental Inflammation

    Science.gov (United States)

    Jaakkola, Kimmo; Nikula, Tuomo; Holopainen, Riikka; Vähäsilta, Tommi; Matikainen, Marja-Terttu; Laukkanen, Marja-Leena; Huupponen, Risto; Halkola, Lauri; Nieminen, Lauri; Hiltunen, Jukka; Parviainen, Sakari; Clark, Michael R.; Knuuti, Juhani; Savunen, Timo; Kääpä, Pekka; Voipio-Pulkki, Liisa Maria; Jalkanen, Sirpa

    2000-01-01

    Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation. PMID:10934150

  8. Flocculation protein structure and cell-cell adhesion mechanism in Saccharomyces cerevisiae.

    Science.gov (United States)

    Goossens, Katty; Willaert, Ronnie

    2010-11-01

    Cell-cell adhesion occurs in a broad spectrum of biological processes, of which yeast flocculation is an area of interest for evolutionary scientists to brewers and winemakers. The flocculation mechanism is based on a lectin-carbohydrate interaction but is not yet fully understood, although the first model dates back to the 1950s. This review will update the current understanding of the complex mechanism behind yeast flocculation. Moreover, modern technologies to measure the forces involved in single carbohydrate-lectin interactions, are discussed. The Flo1 protein has been extensively described as the protein responsible for strong flocculation. Recently, more research has been directed to the detailed analysis of this flocculin. Due to the advances in the field of bioinformatics, more information about Flo1p could be obtained via structurally or functionally related proteins. Here, we review the current knowledge of the Flo1 protein, with a strong emphasis towards its structure.

  9. 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.

  10. 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.

  11. WHEY PROTEIN-BASED WATER RESISTANT AND ENVIRONMENTALLY SAFE ADHESIVES FOR PLYWOOD

    Directory of Open Access Journals (Sweden)

    Zongyan Zhao

    2011-06-01

    Full Text Available Whey protein is a renewable and environmentally safe biomaterial, a by-product of cheese production. It can be utilized for non-food applications for value-added products. The substances glyoxal (GO, glutaraldehyde (GA, polymeric methylene biphenyl diisocyanate (p-MDI, urea-formaldehyde (UF resin, and phenol-formaldehyde oligomer (PFO that contain reactive groups were applied together with whey protein as modifier in order to increase crosslinking density and molecular weight for improving the bond strength and water resistance of whey protein. A water-resistant and environmentally safe whey protein-based wood adhesive for plywood was developed by evaluating the effects of these modifiers on the bond strength, bond durability, and free formaldehyde emission of the resulting plywood panels. Results of FTIR and SEM analyses and bond evaluation indicated that GO, GA, and p-MDI were not suitable to modify whey proteins due to their high reactivity with whey proteins, causing phase separation. UF resin was not a good modifier for whey proteins because of either its poor water-resistance or higher emission of hazardous formaldehyde. Whey protein adhesives modified with PFO had a dry shear bond strength of 1.98 MPa and a 28h-boiling-dry-boiling wet shear strength of 1.73 MPa, which were both much higher than the required values for structural use according to standard JIS K6806-2003, while its formaldehyde emission was 0.067mg/L, much lower than the required value for green plywood according to standard JIS A5908.

  12. 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.

  13. Direct fabrication of nanoscale bio-adhesive patterns by electron beam surface modification of plasma polymerized poly ethylene oxide-like coatings.

    Science.gov (United States)

    Brétagnol, Frédéric; Sirghi, Lucel; Mornet, Stéphane; Sasaki, Takao; Gilliland, Douglas; Colpo, Pascal; Rossi, Francois

    2008-03-26

    In this study we present a method to produce nanostructured surfaces containing bio-adhesive features inside a non bio-adhesive matrix. The strategy is based on the combination of low pressure plasma polymerization and electron beam lithography processes and allows the fabrication of the structured materials in just two steps without using any solvents. In a first step, a thin protein-and-cell-repelling coating (∼10 nm) is obtained by plasma polymerization of Di-glyme. Then, in a second step, the bio-adhesive properties of the layer are tuned by monitoring the concentration of ether bonds of the film by irradiating it locally by different irradiation doses with an electron beam. Time-of-flight secondary ion mass spectroscopy and atomic force microscopy analysis have been used to characterize the produced surfaces. Experiments with a model protein (bovine serum albumin) on the patterned surfaces show preferential adhesion to the irradiated regions, indicating the potential of this simple technique for the development of highly compacted sensitive bio-sensing devices.

  14. Protein Modifications after Foxtail Millet Extrusion: Solubility and Molecular Weight

    Directory of Open Access Journals (Sweden)

    Xuewei Zhao

    2015-03-01

    Full Text Available With the aim of illustrating the effects of extrusion cooking on the solubility of proteins in foxtail millet and their molecular basis, foxtail millet was extruded at five barrel temperature profiles and feed moisture contents. The proteins of raw and extrudate samples were extracted with six solutions sequentially. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE of total protein and Starch Granule-Associate Protein (SGAP was performed. Extrusion caused a significant decrease in globulin, setarin and glutelin fractions with a corresponding increase in SDS- and SDS+2-ME-soluble and residual fractions. Increasing extrusion temperature or moisture content all led to SDS-soluble fraction decrease, while SDS+2-ME-soluble fraction increase. SDS-PAGE demonstrated that disulfide bond cross-linking occurred among glutelin and with setarin subunits. Extrusion had a less pronounced impact on the 60 kDa SGAP than the other middle-high molecular weight subunits. It is the protein-protein interaction shift from electrostatic force to hydrophobic and/or hydrogen forces and covalent disulfide cross-links that contributed to the decreased solubility of protein in foxtail millet extrudates.

  15. 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 (require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured 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.

  16. TiO2 nanotubes functionalized with regions of bone morphogenetic protein-2 increases osteoblast adhesion.

    Science.gov (United States)

    Balasundaram, Ganesan; Yao, Chang; Webster, Thomas J

    2008-02-01

    Titanium (Ti) and its alloys are widely used in orthopedic and dental applications. However, the native TiO2 layer is not bioactive enough to form a direct bond with bone, which sometimes translates into a lack of osseointegration into juxtaposed bone that might lead to long term implant failure. In this study, the 20 amino acid peptide sequence (the so-called "knuckle epitope") of bone morphogenetic protein-2 (BMP-2) was immobilized onto Ti nanotubes created by electrochemical anodization. Further, human osteoblast (bone-forming cell) responses to such anodic Ti oxides functionalized with the BMP-2 knuckle epitope was examined in vitro. Materials were characterized by scanning electron and atomic force microscopy. Results of this in vitro study continued to provide evidence of increased osteoblast adhesion on Ti anodized to possess nanotubes compared to unanodized Ti. However, for the first time, results also showed that the immobilization of the BMP-2 knuckle epitope onto Ti anodized to possess nanotubes increased osteoblast adhesion compared to non-functionalized anodized Ti, anodized Ti functionalized with amine (NH2) groups, and unanodized Ti after 4 h. Results also showed increased osteoblast adhesion on amine terminated anodized Ti compared to respective non-functionalized anodized Ti and unanodized Ti. In summary, results of this in vitro study provided evidence that Ti anodized to possess nanotubes and then further functionalized with the BMP-2 knuckle epitope should be further studied for improved orthopedic applications.

  17. Surface modification using interfacial assembly of the Streptomyces chaplin proteins

    NARCIS (Netherlands)

    Ekkers, David Matthias; Claessen, Dennis; Galli, Federica; Stamhuis, Eize

    The chaplin proteins are instrumental in the formation of reproductive aerial structures by the filamentous bacterium Streptomyces coelicolor. They lower the water surface tension thereby enabling aerial growth. In addition, chaplins provide surface hydrophobicity to the aerial hyphae by assembling

  18. Modification of tooth development by heat shock protein 60

    OpenAIRE

    Papp Tamás; Polyák Angéla; Papp Krisztina; Mészár Zoltán (1977-) (állatorvos); Zákány Róza (1963-) (anatómus-, kötőszövetbiológus); Mészár-Katona Éva (1986-) (Ph.D hallgató); Terdik Tünde (1969-) (analitikus); Chang, Hwa Ham; Felszeghy Szabolcs Béla (1972-) (fogorvos, anatómus, kötőszövetbiológus)

    2016-01-01

    Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). H...

  19. Fluorescent biphenyl derivatives of phenylalanine suitable for protein modification.

    Science.gov (United States)

    Chen, Shengxi; Fahmi, Nour Eddine; Bhattacharya, Chandrabali; Wang, Lin; Jin, Yuguang; Benkovic, Stephen J; Hecht, Sidney M

    2013-11-26

    In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from Escherichia coli (ecDHFR) with minimal disruption of protein structure or function, even when the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR and afforded modified proteins that consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational

  20. 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

    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...

  1. Adhesion of Candida albicans to various dental implant surfaces and the influence of salivary pellicle proteins.

    Science.gov (United States)

    Bürgers, Ralf; Hahnel, Sebastian; Reichert, Torsten E; Rosentritt, Martin; Behr, Michael; Gerlach, Till; Handel, Gerhard; Gosau, Martin

    2010-06-01

    Dental implants may be considered a potential reservoir for (re)infection with oral Candida albicans. Our aim was to evaluate initial fungal adhesion to three differentially textured titanium and one zirconia implant surface, and to correlate these findings to differences in specific surface characteristics (surface roughness (R(a)) and surface free energy (SFE)). Additionally, we investigated the influence of salivary protein films and two pellicle proteins (mucin and albumin). Implant surfaces were characterized by perthometer (R(a)) and goniometer (SFE) measurements. Implant specimens were rinsed with human whole saliva, mucin, albumin, or phosphate buffered saline and incubated in C. albicans suspension for 2.5h. Adherent fungi were quantified by means of a bioluminometric assay. The lowest amount of fungal cells was found on sand-blasted titanium, whereas zirconia implants did not show any reduced potential to adhere C. albicans. The influence of the implant SFE on fungal biofilm formation appears to be more important than the influence of R(a). The protein mucin enhanced C. albicans accumulation. In contrast, albumin is unlikely to be involved in the adhesion process of C. albicans. Copyright 2010. Published by Elsevier Ltd.

  2. A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito.

    Science.gov (United States)

    Pradel, Gabriele; Hayton, Karen; Aravind, L; Iyer, Lakshminarayan M; Abrahamsen, Mitchell S; Bonawitz, Annemarie; Mejia, Cesar; Templeton, Thomas J

    2004-06-07

    The recent sequencing of several apicomplexan genomes has provided the opportunity to characterize novel antigens essential for the parasite life cycle that might lead to the development of new diagnostic and therapeutic markers. Here we have screened the Plasmodium falciparum genome sequence for genes encoding extracellular multidomain putative adhesive proteins. Three of these identified genes, named PfCCp1, PfCCp2, and PfCCp3, have multiple adhesive modules including a common Limulus coagulation factor C domain also found in two additional Plasmodium genes. Orthologues were identified in the Cryptosporidium parvum genome sequence, indicating an evolutionary conserved function. Transcript and protein expression analysis shows sexual stage-specific expression of PfCCp1, PfCCp2, and PfCCp3, and cellular localization studies revealed plasma membrane-associated expression in mature gametocytes. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged microgametes and macrogametes. PfCCp expression markedly decreased after formation of zygotes. To begin to address PfCCp function, the PfCCp2 and PfCCp3 gene loci were disrupted by homologous recombination, resulting in parasites capable of forming oocyst sporozoites but blocked in the salivary gland transition. Our results describe members of a conserved apicomplexan protein family expressed in sexual stage Plasmodium parasites that may represent candidates for subunits of a transmission-blocking vaccine.

  3. Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain*

    Science.gov (United States)

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A.; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-01-01

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. PMID:24904058

  4. Focal Adhesion Kinase Regulates Expression of Thioredoxin-interacting Protein (TXNIP) in Cancer Cells

    OpenAIRE

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter ac...

  5. Molekulare Charakterisierung und Identifizierung eines Aktivierungsmechanismus von Adhesion-G-Protein-gekoppelten Rezeptoren

    OpenAIRE

    Schön, Julia

    2015-01-01

    Die Familie der Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR) stellt die zweitgrößte Gruppe der GPCR dar. Ein strukturelles Charakteristikum der aGPCR ist der modular aufgebaute N-Terminus, welcher eine GPCR-proteolytic site (GPS) mit einem konservierten Spaltungsmotiv enthält. Trotz der hohen medizinischen Relevanz dieser Rezeptorgruppe sind für die meisten der 33 humanen Vertreter der aGPCR bis heute weder Funktion noch endogener Agonist bekannt. Um sie jedoch zukünftig als potentielle ...

  6. 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

    Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human...... 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...

  7. 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 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...

  8. Protein micro patterned lattices to probe a fundamental lengthscale involved in cell adhesion

    CERN Document Server

    Guillou, Herve; Chaussy, Jacques; Block, Marc R

    2009-01-01

    Cell adhesion, a fundamental process of cell biology is involved in the embryo development and in numerous pathologies especially those related to cancers. We constrained cells to adhere on extracellular matrix proteins patterned in a micro lattices. The actin cytoskeleton is particularly sensitive to this constraint and reproducibly self organizes in simple geometrical patterns. Such highly organized cells are functional and proliferate. We performed statistical analysis of spread cells morphologies and discuss the existence of a fundamental lengthscale associated with active processes required for spreading.

  9. 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

  10. 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.

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

    Science.gov (United States)

    Olspert, Allan; Hosmillo, Myra; Chaudhry, Yasmin; Peil, Lauri; Truve, Erkki; Goodfellow, Ian

    2016-01-01

    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.

  12. Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth*♦

    Science.gov (United States)

    Cheng, Zhaokang; Zhu, Qiang; Dee, Rachel; Opheim, Zachary; Mack, Christopher P.; Cyr, Douglas M.; Taylor, Joan M.

    2017-01-01

    Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo. Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux. PMID:27994061

  13. Protein-protein-interaction network organization of the hypusine modification system

    National Research Council Canada - National Science Library

    Sievert, Henning; Venz, Simone; Platas-Barradas, Oscar; Dhople, Vishnu M; Schaletzky, Martin; Nagel, Claus-Henning; Braig, Melanie; Preukschas, Michael; Pällmann, Nora; Bokemeyer, Carsten; Brümmendorf, Tim H; Pörtner, Ralf; Walther, Reinhard; Duncan, Kent E; Hauber, Joachim; Balabanov, Stefan

    2012-01-01

    Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases...

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

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    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...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in 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...

  15. Multienzyme Modification of Hemp Protein for Functional Peptides Synthesis

    Directory of Open Access Journals (Sweden)

    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.

  16. Site-selective protein-modification chemistry for basic biology and drug development

    Science.gov (United States)

    Krall, Nikolaus; da Cruz, Filipa P.; Boutureira, Omar; Bernardes, Gonçalo J. L.

    2016-02-01

    Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

  17. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    Science.gov (United States)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Werneburg

    2015-05-01

    Full Text Available 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.

  19. Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

    Science.gov (United States)

    Mayer, Magnus C; Schauenburg, Linda; Thompson-Steckel, Greta; Dunsing, Valentin; Kaden, Daniela; Voigt, Philipp; Schaefer, Michael; Chiantia, Salvatore; Kennedy, Timothy E; Multhaup, Gerhard

    2016-04-01

    The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

  20. Photoinduced protein modifications by methylene blue and naproxen.

    Science.gov (United States)

    Bracchitta, Giuseppina; Catalfo, Alfio; De Guidi, Guido

    2012-12-01

    HPLC and emission spectroscopy were used to investigate UVA photosensitization of methylene blue (MB) or naproxen (NAP) towards bovine serum albumin (BSA). In addition, time resolved singlet oxygen measurements were carried out. The most stable drug : protein adducts stoichiometry of MB-BSA (1 : 1) and NAP-BSA (9 : 1) were verified by means of binding constant determination. UVA photosensitization of MB or NAP on BSA was studied by monitoring tryptophan (Trp) residue integrity. The sensitized photodegradation of the BSA resulted in different degrees of Trp damage. Thus, protein damage was determined by quantitative measurements of the different Trp (photo)-products. Indeed, many of these Trp derivatives are diagnostic for the photosensitization mechanism and some of them, for the first time in this work, were obtained by UVA photosensitization in proteins. The analysis of quantum yields of the photoproduct distribution allowed to weigh up the type I/II contribution to the UVA photosensitization mechanism. As expected, additional experiments in deuterated solvent resulted in an increase of the photodegradation quantum yields for those species where a singlet oxygen mechanism was involved. The UVA mediated generation of these Trp derivatives is consistent with the occurrence of singlet oxygen formation (almost dominant in MB), and photoionization (significant in NAP) within the protein matrix. Additional experiments at lower NAP concentration, as well as with human serum albumin (which differs for Trp content and, partially, localization), support further the molecular mechanism of photosensitization proposed. The results obtained in the case of this more complex system are in agreement with the free Trp model, even if, in almost all cases, the Trp photoproduct formation quantum yields are lower, due to the higher number of sensitization targets in the proteins.

  1. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    Science.gov (United States)

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  2. Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

    DEFF Research Database (Denmark)

    Roepstorff, P; Larsen, Martin Røssel

    2001-01-01

    dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational...... modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated....

  3. 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.

  4. 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.

  5. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    Science.gov (United States)

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells.

  6. Sensitivity of protein adsorption to architectural variations in a protein-resistant polymer brush containing engineered nanoscale adhesive sites.

    Science.gov (United States)

    Gon, Saugata; Santore, Maria M

    2011-12-20

    Patchy polymer brushes contain nanoscale (5-15 nm) adhesive elements, such as polymer coils or nanoparticles, embedded at their base at random positions on the surface. The competition between the brush's steric (protein resistant) repulsions and the attractions from the discrete adhesive elements provides a precise means to control bioadhesion. This differs from the classical approach, where functionality is placed on the brush's periphery. The current study demonstrates the impact of poly(etheylene glycol) (PEG) brush architecture and ionic strength on fibrinogen adsorption on brushes containing embedded poly-l-lysine (PLL, 20K MW) coils or "patches". The consistent appearance of a fibrinogen adsorption threshold, a minimum loading of patches on the surface, below which protein adsorption does not occur, suggests multivalent protein capture: Adsorbing proteins simultaneously engage several patches. The surface composition (patch loading) at the threshold is extremely sensitive to the brush height and ionic strength, varying up to a factor of 5 in the surface loading of the PLL patches (~50% of the range of possible surfaces). Variations in ionic strength have a similar effect, with the smallest thresholds seen for the largest Debye lengths. While trends with brush height were the clearest and most dominant, consideration of the PEG loading within the brush or its persistence length did not reveal a critical brush parameter for the onset of adsorption. The lack of straightforward correlation on brush physics was likely a result of multivalent binding, (producing an additional dependence on patch loading), and might be resolved for univalent adsorption onto more strongly binding patches. While studies with similar brushes placed uniformly on a surface revealed that the PEG loading within the brush is the best indicator of protein resistance, the current results suggest that brush height is more important for patchy brushes. Likely the interactions producing brush

  7. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    DEFF Research Database (Denmark)

    Refsgaard, Hanne; Tsai, Lin; Stadtman, Earl

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(lll)/O-2] depends on the degree of unsaturation of the fatty acid. The fatty acid......-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate fatty acids were oxidized in the presence...... in the formation of protein carbonyls, These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues...

  8. Regulator of G protein signaling 20 enhances cancer cell aggregation, migration, invasion and adhesion.

    Science.gov (United States)

    Yang, Lei; Lee, Maggie M K; Leung, Manton M H; Wong, Yung H

    2016-11-01

    Several RGS (regulator of G protein signaling) proteins are known to be upregulated in a variety of tumors but their roles in modulating tumorigenesis remain undefined. Since the expression of RGS20 is elevated in metastatic melanoma and breast tumors, we examined the effects of RGS20 overexpression and knockdown on the cell mobility and adhesive properties of different human cancer cell lines, including cervical cancer HeLa, breast adenocarcinoma MDA-MB-231, and non-small cell lung carcinoma H1299 and A549 cells. Expression of RGS20 enhanced cell aggregation, migration, invasion and adhesion as determined by hanging drop aggregation, wound healing, transwell chamber migration and invasion assays. Conversely, shRNA-mediated knockdown of endogenous RGS20 impaired these responses. In addition, RGS20 elevated the expression of vimentin (a mesenchymal cell marker) but down-regulated the expression of E-cadherin, two indicators commonly associated with metastasis. These results suggest that the expression of RGS20 may promote metastasis of tumor cells.

  9. 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.

  10. The DNA damage response pathways: at the crossroad of protein modifications

    Institute of Scientific and Technical Information of China (English)

    Michael SY Huen; Junjie Chen

    2008-01-01

    Post-translational modifications play a crucial role in coordinating cellular response to DNA damage. Recent evidence suggests an interplay between multiple protein modifications, including phosphorylation, ubiquitylation, acetylation and sumoylation, that combine to propagate the DNA damage signal to elicit cell cycle arrest, DNA repair, apoptosis and senescence. Utility of specific post-translational modifiers allows temporal and spatial control over protein relo-calization and interactions, and may represent a means for trans-regulatory activation of protein activities. The abil-ity to recognize these specific modifiers also underscores the capacity for signal amplification, a crucial step for the maintenance of genomic stability and tumor prevention. Here we have summarized recent findings that highlight the complexity of post-translational modifications in coordinating the DNA damage response, with emphasis on the DNA damage signaling cascade.

  11. Surface modification of graphene nanopores for protein translocation

    Science.gov (United States)

    Shan, Y. P.; Tiwari, P. B.; Krishnakumar, P.; Vlassiouk, I.; Li, W.Z.; Wang, X.W.; Darici, Y.; Lindsay, S.M.; Wang, H. D.; Smirnov, S.; He, J.

    2014-01-01

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5-20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pore occur during the process. PMID:24231385

  12. 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.

  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

    2016-06-07

    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. Adhesive modification of indium-tin-oxide surface for template attachment for deposition of highly ordered nanostructure arrays

    Energy Technology Data Exchange (ETDEWEB)

    Gu, W. [Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123 (China); Liao, L.S., E-mail: lsliao@suda.edu.cn [Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123 (China); Cai, S.D.; Zhou, D.Y.; Jin, Z.M.; Shi, X.B.; Lei, Y.L. [Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, Jiangsu 215123 (China)

    2012-08-01

    Polyvinyl alcohol (PVA), a very cheap polymer with one hydroxyl group in each repeating unit, was spun coated on the surface of an indium-tin-oxide (ITO) substrate to improve the adhesion between the substrate and a nanoporous anodic aluminum oxide (AAO) template layer for a template-directed fabrication of nanostructures. Compared with dihydroxy-terminated polystyrene (PS-dOH) and a silane coupling agent (KH550), PVA was a superior binder because of its abundant hydroxyl groups for adhesion enhancement and its low cost for applications. As an example, a highly ordered CdSe nanorod array free standing on the ITO substrate was electrochemically deposited by using an ultrathin AAO layer as the template on the PVA modified surface. It was demonstrated that the PVA modified ITO can be reliably used for the template-directed fabrication of nanostructures.

  15. 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.

  16. Surface modification of carbon fibers by a polyether sulfone emulsion sizing for increased interfacial adhesion with polyether sulfone

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Haojie [National Engineering Laboratory for Carbon Fiber Technology, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Shouchun, E-mail: zschun@sxicc.ac.cn [National Engineering Laboratory for Carbon Fiber Technology, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001 (China); Lu, Chunxiang [National Engineering Laboratory for Carbon Fiber Technology, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001 (China)

    2014-10-30

    Highlights: • A polyether sulfone emulsion (PES) sizing was prepared for the first time. • The sizing enhanced the surface activity and wettability of carbon fibers. • Compared to the original sizing, the PES emulsion sizing resulted in an 18.4% increase in the interlaminar shear strength of carbon fiber/PES composites. • Important influences of emulsifier on the fiber surface and composite interface were demonstrated. • The reinforcing mechanisms are the improved fiber surface wettability and interfacial compatibility in composites. - Abstract: Interests on carbon fiber-reinforced thermoplastic composites are growing rapidly, but the challenges with poor interfacial adhesion have slowed their adoption. In this work, a polyether sulfone (PES) emulsion sizing was prepared successfully for increased interfacial adhesion of carbon fiber/PES composites. To obtain a high-quality PES emulsion sizing, the key factor, emulsifier concentration, was studied by dynamic light scattering technique. The results demonstrated that the suitable weight ratio of PES to emulsifier was 8:3, and the resulting PES emulsion sizing had an average particle diameter of 117 nm and Zeta potential of −52.6 mV. After sizing, the surface oxygen-containing functional groups, free energy and wettability of carbon fibers increased significantly, which were advantageous to promote molecular-level contact between carbon fiber and PES. Finally, short beam shear tests were performed to evaluate the interfacial adhesion of carbon fiber/PES composites. The results indicated that PES emulsion sizing played a critical role for the enhanced interfacial adhesion in carbon fiber/PES composites, and a 26% increase of interlaminar shear strength was achieved, because of the improved fiber surface wettability and interfacial compatibility between carbon fiber and PES.

  17. A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1.

    Science.gov (United States)

    Choi, Sunga; Park, Myoung Soo; Lee, Yu Ran; Lee, Young Chul; Kim, Tae Woo; Do, Seon-Gil; Kim, Dong Seon; Jeon, Byeong Hwa

    2013-02-01

    Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-α)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 µg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-α-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-α-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-α-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis.

  18. Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

    Directory of Open Access Journals (Sweden)

    Thomas Wurster

    Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  19. Modification of fluorous substrates with oligo(ethylene glycol) via "click" chemistry for long-term resistance of cell adhesion.

    Science.gov (United States)

    Contreras-Caceres, Rafael; Santos, Catherine M; Li, Siheng; Kumar, Amit; Zhu, Zhiling; Kolar, Satya S; Casado-Rodriguez, Miguel A; Huang, Yongkai; McDermott, Alison; Lopez-Romero, Juan Manuel; Cai, Chengzhi

    2015-11-15

    In this work perfluorinated substrates fabricated from SiO2 glass slides are modified with oligo(ethylene glycol) (OEG) units for long-term resistance of cell adhesion purposes, based on fluorous interactions and click chemistry. Specifically, fluorous substrates, prepared by treatment of glass slides with 1H, 1H, 2H, 2H-perfluorodecyltrimethoxysilane (FAS17), were coated with ethynyl-OEG-C8F17, followed by covalent attachment of an azido-OEG via copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click" reaction. We demonstrate that the resultant surface avoid fibrinogen adsorption and resisted cell adhesion for over 14days. X-ray photoemission spectroscopy (XPS) analysis and contact angle goniometry measurements confirm the presence of the OEG molecules on the fluorous substrates. Bright field optical images show total absence of 3T3 fibroblast cells on the OEG modified fluorinated substrate for 1 and 5days, and a remarkably decrease of cell adhesion at 14days.

  20. Effects of oxidative modification on gel properties of isolated porcine myofibrillar protein by peroxyl radicals.

    Science.gov (United States)

    Zhou, Feibai; Zhao, Mouming; Zhao, Haifeng; Sun, Weizheng; Cui, Chun

    2014-04-01

    AAPH-derived (2,2'-azobis (2-amidinopropane) dihydrochloride) peroxyl radicals were selected as representative free radicals of lipid peroxidation to investigate the effects of oxidative modifications on isolated porcine myofibrillar protein structures as well as their rheological and gelling properties. Incubation of myofibrillar protein with increasing concentrations of AAPH resulted in a gradual increase (p3 mM) concentrations of AAPH induced aggregation of myosin and denaturation of myosin, troponin and tropomyosin, respectively. These structural changes resulted in changes on gelation of myofibrillar protein. Low level protein oxidation (AAPH≤0.5 mM) had no remarkable effect (p>0.05) on the viscoelastic pattern of myofibrillar protein gelation. Moderate oxidative modification (AAPH~1mM) enhanced the water-holding capacity (WHC) and texture properties of gels, while further oxidation (AAPH>3mM) significantly reduced the gel quality.

  1. 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.

  2. Modification of tooth development by heat shock protein 60

    Institute of Scientific and Technical Information of China (English)

    Tamas Papp; Angela Polyak; Krisztina Papp; Zoltan Meszar; Roza Zakany; Eva Meszar-Katona; Palne Terdik Tu nde; Chang Hwa Ham; Szabolcs Felszeghy

    2016-01-01

    Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2- deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

  3. Modification of tooth development by heat shock protein 60.

    Science.gov (United States)

    Papp, Tamas; Polyak, Angela; Papp, Krisztina; Meszar, Zoltan; Zakany, Roza; Meszar-Katona, Eva; Tünde, Palne Terdik; Ham, Chang Hwa; Felszeghy, Szabolcs

    2016-03-30

    Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

  4. Platelet adhesion and protein adsorption on silicone rubber surface by ozone-induced grafted polymerization with carboxybetaine monomer.

    Science.gov (United States)

    Zhou, Jun; Yuan, Jiang; Zang, Xiaopeng; Shen, Jian; Lin, Sicong

    2005-03-10

    Platelet adhesion and protein adsorption on the silicone rubber film grafted with N,N'-dimethyl-N-methacryloyloxyethyl-N-(2-carboxyethyl) ammonium (DMMCA) was studied. The grafting was carried out by means of ozone-induced method and was confirmed by ATR-FTIR and XPS investigations. The grafted films possessed relatively hydrophilic surface revealed by contact angle measurement. The blood compatibility of the grafted film was evaluated in vitro by platelet adhesion in platelet-rich plasma (PRP) and protein absorption in bovine fibrinogen (BFG) using silicone film as the reference. No substantial platelet adhesion was observed for the grafted films incubated in PRP for 60 and 180 min. The protein absorption was also significantly reduced after incubated in bovine fibrinogen for 60 min. Both the results indicated that the blood compatibility of silicone rubber was greatly improved by ozone-induced grafting of carboxybetaine zwitterionic polymer onto its surface.

  5. Differential protein levels and post-translational modifications in spinal cord injury of the rat.

    Science.gov (United States)

    Afjehi-Sadat, Leila; Brejnikow, Mika; Kang, Sung Ung; Vishwanath, Vinay; Walder, Nadja; Herkner, Kurt; Redl, Heinz; Lubec, Gert

    2010-03-05

    Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

  6. 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.

  7. Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment

    Science.gov (United States)

    Byron, Adam; Humphries, Jonathan D; Craig, Sue E; Knight, David; Humphries, Martin J

    2012-01-01

    Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events. PMID:22623428

  8. 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

    fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and alpha-actinin at every stage that we can detect by IRM or by double staining to reveal...... 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......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...

  9. Identification of novel splice variants of Adhesion G protein-coupled receptors.

    Science.gov (United States)

    Bjarnadóttir, Thóra K; Geirardsdóttir, Kristín; Ingemansson, Malena; Mirza, Majd A I; Fredriksson, Robert; Schiöth, Helgi B

    2007-01-31

    Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

  10. 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

    2011-01-01

    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 electroncapture 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. PMID:21982782

  11. 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

  12. 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.

  13. Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Diptiman Chanda

    Full Text Available Anterior Gradient Protein (AGR-2 is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

  14. Functional roles of mannose-binding protein in the adhesion, cytotoxicity and phagocytosis of Acanthamoeba castellanii.

    Science.gov (United States)

    Kim, Jong-Hyun; Matin, Abdul; Shin, Ho-Joon; Park, Hyun; Yoo, Kyung-Tae; Yuan, Xi-Zhe; Kim, Kwang Sik; Jung, Suk-Yul

    2012-10-01

    Acanthamoeba castellanii is a single-celled protozoan that is widely distributed in the environment and is a well-known of causing human keratitis, a vision-threatening infection. In this study, an ethyl methane sulfonate (EMS) and a selection of saccharide were applied to A. castellanii by chemical mutagenesis. To understand the functional roles of a mannose-binding protein (MBP). A. castellanii were treated with methyl-alpha-D-mannopyranoside abbreviated Man, with and without the EMS pre-treatment, and their adhesion and cytotoxicity were analyzed, using a human brain microvascular endothelial cell (HBMEC) as the target cell. Both EMS and Man mutants exhibited significantly decreased levels of MBP expression and cytotoxicity to HBMEC, but showed similar levels of binding to HBMEC, as compared with the wild type. Of interest was that the exogenous mannose inhibited amoebae (i.e., Man mutant) binding to the HBMEC by <20%. Only the mutant Man exhibited a significant decrease in bacterial uptake, as compared to the wild type, 0.020 vs 0.032 (p<0.05) and proteolytic activity. The results showed that MBP should be clearly provided as the pathogenic target candidate, to further target-based therapy, but EMS mutation should not be associated with initial adhesion and phagocytosis of A. castellanii.

  15. Direct covalent coupling of proteins to nanostructured plasma polymers: a route to tunable cell adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Melnichuk, Iurii, E-mail: iurii.melnichuk@gmail.com [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); Choukourov, Andrei, E-mail: choukourov@kmf.troja.mff.cuni.cz [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); Bilek, Marcela, E-mail: m.bilek@physics.usyd.edu.au [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); School of Physics, University of Sydney, NSW 2006 (Australia); Weiss, Anthony, E-mail: tony.weiss@sydney.edu.au [School of Molecular Bioscience, University of Sydney, NSW 2006 (Australia); Vandrovcová, Marta, E-mail: Marta.Vandrovcova@fgu.cas.cz [Institute of Physiology of Czech Academy of Science, Prague 14220 (Czech Republic); Bačáková, Lucie, E-mail: Lucie.Bacakova@fgu.cas.cz [Institute of Physiology of Czech Academy of Science, Prague 14220 (Czech Republic); Hanuš, Jan, E-mail: jan.hanus@gmail.com [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); Kousal, Jaroslav, E-mail: jarda@kmf.troja.mff.cuni.cz [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); Shelemin, Artem, E-mail: artem.shelemin@gmail.com [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); Solař, Pavel, E-mail: pawell.solar@seznam.cz [Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague 18000 (Czech Republic); and others

    2015-10-01

    Highlights: • Flat and nanostructured interfaces were overcoated by hydrocarbon plasma polymer. • Linker-free covalent attachment of proteins to resultant surfaces was validated. • Ultra-thin hydrocarbon overcoat (<2 nm) secured prolonged effective binding. • Pre-adsorbed tropoelastin promoted proliferation of osteoblast-like MG-63 cells. • Nanostructured films were multi-affine and impeded cell adhesion. - Abstract: Flat and nanostructured thin films were fabricated by deposition of ultra-thin (<2 nm) layer of hydrocarbon plasma polymer over polished silicon and over a pattern of 8 nm-thick poly(ethylene) islands on silicon. Linker-free radical-based covalent binding of bovine serum albumin and tropoelastin was confirmed for both types of films. The binding capability of albumin was found to be stable over many days of ambient air storage time. Tropoelastin-mediated flat plasma polymers favored adhesion and proliferation of osteoblast-like MG-63 cells. Nanostructured plasma polymers were multi-affine and their hierarchical surface represented an additional barrier for cell attachment.

  16. 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.

  17. 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

    2013-10-22

    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.

  18. 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

    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...

  19. The adhesion of mussel foot protein-3 to TiO2 surfaces: the effect of pH

    Science.gov (United States)

    Yu, Jing; Wei, Wei; Menyo, Matthew S.; Masic, Admir; Waite, J. Herbert; Israelachvili, Jacob N.

    2013-01-01

    The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3, 4-dihydroxyphenylalanine (Dopa). As a side-chain, Dopa is capable of strong bidentate interactions with a variety of surfaces, including many minerals and metal oxides. Titanium is among the most widely used medical implant material and quickly forms a TiO2 passivation layer under physiological conditions. Understanding the binding mechanism of Dopa to TiO2 surfaces is therefore of considerable theoretical and practical interest. Using a surface forces apparatus, we explored the force-distance profiles and adhesion energies of mussel foot protein 3 (mfp-3) to TiO2 surfaces at three different pHs (pH3, 5.5 and 7.5). At pH3, mfp-3 showed the strongest adhesion force on TiO2, with an adhesion energy of ~ −7.0 mJ/m2. Increasing the pH gives rise to two opposing effects: (1) increased oxidation of Dopa, thus decreasing availability for the Dopa-mediated adhesion, and (2) increased bidentate Dopa-Ti coordination, leading to the further stabilization of the Dopa group and thus an increasing of adhesion force. Both effects were reflected in the resonance-enhanced Raman spectra obtained at the three deposition pHs. The two competing effects give rise to a higher adhesion force of mfp-3 on TiO2 surface at pH 7.5 than at pH 5.5. Our results suggest that Dopa-containing proteins and synthetic polymers have great potential as coating materials for medical implant materials, particularly if redox activity can be controlled. PMID:23452271

  20. Surface modification of polyethylene by radiation-induced grafting for adhesive bonding. III. Oxidative degradation and stabilization of grafted layer

    Energy Technology Data Exchange (ETDEWEB)

    Yamakawa, S.; Yamamoto, F.

    1978-09-01

    Vapor-phase mutual grafting of methyl acrylate (MA) onto polyethylene (PE) and subsequent saponification treatment produce a surface graft having a high adhesive bondability, which results from the presence of a hydrolized homopolymer layer (consisting of only monomer componenet) on an inner graft copolymer layer consisting of both PE and monomer components. The oxidative deterioration and the stabilization of the grated surface layer have been investigated to clarify the long-term stability of the adhesive bondability. The bondability rapidly disappears with accelerated weatherly followed by acetone extraction treatment, whereas it is kept unchanged during thermal-oxidative aging at 100/sup 0/C. Microscopic and attenuated total resonance (ATR) infrared spectroscopic observations of the degreaded surfaces show that the bondability loss is due to degradiative removal of the surface homopolymer layer. The addition of combinations of conventional antioxidants and ultraviolet absorbers stabilizes the grafted surface layer against thermal-oxidative and photo-oxidative degradation and thus extends the bondability rentention time. The stabilization is more effective in the grafts of carbon black-containing PE, where carbon black is present in the inner-graft copolymer layer.

  1. Surface modification with poly(sulfobetaine methacrylate-co-acrylic acid) to reduce fibrinogen adsorption, platelet adhesion, and plasma coagulation.

    Science.gov (United States)

    Kuo, Wei-Hsuan; Wang, Meng-Jiy; Chien, Hsiu-Wen; Wei, Ta-Chin; Lee, Chiapyng; Tsai, Wei-Bor

    2011-12-12

    Zwitterionic sulfobetaine methacrylate (SBMA) polymers were known to possess excellent antifouling properties due to high hydration capacity and neutral charge surface. In this study, copolymers of SBMA and acrylic acid (AA) with a variety of compositions were synthesized and were immobilized onto polymeric substrates with layer-by-layer polyelectrolyte films via electrostatic interaction. The amounts of platelet adhesion and fibrinogen adsorption were determined to evaluate hemocompatibility of poly(SBMA-co-AA)-modified substrates. Among various deposition conditions by modulating SBMA ratio in the copolymers and pH of the deposition solution, poly(SBMA(56)-co-AA(44)) deposited at pH 3.0 possessed the best hemocompatibility. This work demonstrated that poly(SBMA-co-AA) copolymers adsorbed on polyelectrolyte-base films via electrostatic interaction improve hemocompatibility effectively and are applicable for various substrates including TCPS, PU, and PDMS. Furthermore, poly(SBMA-co-AA)-coated substrate possesses great durability under rigorous conditions. The preliminary hemocompatibility tests regarding platelet adhesion, fibrinogen adsorption, and plasma coagulation suggest the potential of this technique for the application to blood-contacting biomedical devices.

  2. The role of the thiol group in protein modification with methylglyoxal

    Directory of Open Access Journals (Sweden)

    JELENA M. AĆIMOVIĆ

    2009-08-01

    Full Text Available Methylglyoxal is a highly reactive α-oxoaldehyde with elevated production in hyperglycemia. It reacts with nucleophilic Lys and Arg side-chains and N-terminal amino groups causing protein modification. In the present study, the importance of the reaction of the Cys thiol group with methylglyoxal in protein modification, the competitiveness of this reaction with those of amino and guanidine groups, the time course of these reactions and their role and contribution to protein cross-linking were investigated. Human and bovine serum albumins were used as model systems. It was found that despite the very low levels of thiol groups on the surface of the examined protein molecules (approx. 80 times lower than those of amino and guanidino groups, a very high percentage of it reacts (25–85 %. The amount of reacted thiol groups and the rate of the reaction, the time for the reaction to reach equilibrium, the formation of a stable product and the contribution of thiol groups to protein cross-linking depend on the methylglyoxal concentration. The product formed in the reaction of thiol and an insufficient quantity of methylglyoxal (compared to the concentrations of the groups accessible for modification participates to a significant extent (4 % to protein cross-linking. Metformin applied in equimolar concentration with methylglyoxal prevents its reaction with amino and guanidino groups but, however, not with thiol groups.

  3. Proteomic studies on protein modification by cyclopentenone prostaglandins: expanding our view on electrophile actions.

    Science.gov (United States)

    Garzón, Beatriz; Oeste, Clara L; Díez-Dacal, Beatriz; Pérez-Sala, Dolores

    2011-10-19

    Cyclopentenone prostaglandins (cyPG) are lipid mediators that participate in the mechanisms regulating inflammation and tumorigenesis. cyPG are electrophilic compounds that act mainly through the covalent modification of cellular proteins. The stability of many cyPG-protein adducts makes them suitable for proteomic analysis. Indeed, methodological advances in recent years have allowed identifying many cyPG targets, including components of pro-inflammatory transcription factors, cytoskeletal proteins, signaling kinases and proteins involved in redox control. Insight into the diversity of cyPG targets is providing a better understanding of their mechanism of action, uncovering novel links between resolution of inflammation, proliferation and redox regulation. Moreover, identification of the target residues has unveiled the selectivity of protein modification by these electrophiles, providing valuable information for potential pharmacological applications. Among the challenges ahead, the detection of proteins modified by endogenous cyPG and the quantitative aspects of the modification require further efforts. Importantly, only a few years after the appearance of the first proteomic studies, research on cyPG targets is yielding new paradigms for redox and electrophilic signaling.

  4. 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.

  5. Adsorption and adhesion of blood proteins and fibroblasts on multi-wall carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    LI Dedun; YUAN Li; YANG Ying; DENG XiangYun; Lü XiaoYing; HUANG Yan; CAO Zheng; LIU Hao; SUN XueLiang

    2009-01-01

    This article concerns the investigation of blood protein adsorption on carbon paper and multi-wall carbon nanotubes (MWCNTs). Mouse fibroblast cell adhesion and growth on MWCNTs was also studied. The results showed that fibrinogen adsorption on carbon paper was much lower than that on MWCNTs, which means that platelets readily aggregate on the surface of MWCNTs. Mouse fibroblast cells im-planted on MWCNTs tended to grow more prolifically than those implanted on carbon paper. The cell concentration observed on MWCNTs increased from 1.2×105/mL for a single day culture to 2×105/mL for a 7-day culture. No toxicity reaction was observed during the culturing period. These results indi-cated that MWCNTs possessed excellent tissue compatibility.

  6. Adsorption and adhesion of blood proteins and fibroblasts on multi-wall carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    This article concerns the investigation of blood protein adsorption on carbon paper and multi-wall carbon nanotubes (MWCNTs). Mouse fibroblast cell adhesion and growth on MWCNTs was also studied. The results showed that fibrinogen adsorption on carbon paper was much lower than that on MWCNTs, which means that platelets readily aggregate on the surface of MWCNTs. Mouse fibroblast cells implanted on MWCNTs tended to grow more prolifically than those implanted on carbon paper. The cell concentration observed on MWCNTs increased from 1.2×105/mL for a single day culture to 2×105/mL for a 7-day culture. No toxicity reaction was observed during the culturing period. These results indicated that MWCNTs possessed excellent tissue compatibility.

  7. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Xuchen Xu; Ming Li; Qian Liu; Zhaojun Jia; Yuying Shi; Yan Cheng; Yufeng Zheng; L.Q. Ruan

    2014-01-01

    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.

  9. A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

    Science.gov (United States)

    Mayer, T; Meyer, M; Janning, A; Schiedel, A C; Barnekow, A

    1999-03-25

    The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

  10. 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.

  11. 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.

  12. 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...

  13. Redox Proteomics: A Key Tool for New Insights into Protein Modification with Relevance to Disease

    Science.gov (United States)

    Perluigi, Marzia

    2017-01-01

    Abstract Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the “triangle of death” composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277–279. PMID:27835924

  14. Surface modification of cotton fabrics by gas plasmas for color strength and adhesion by inkjet ink printing

    Energy Technology Data Exchange (ETDEWEB)

    Pransilp, Porntapin, E-mail: lookpad_hae@hotmail.com [Program of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University (Thailand); Pruettiphap, Meshaya, E-mail: pruettiphap_m@hotmail.com [Program of Petrochemistry, Faculty of science, Chulalongkorn University (Thailand); Bhanthumnavin, Worawan, E-mail: worawan.b@chula.ac.th [Department of Chemistry, Faculty of Science, Chulalongkorn University (Thailand); Paosawatyanyong, Boonchoat, E-mail: paosawat@sc.chula.ac.th [Department of Physics, Faculty of Science, Chulalongkorn University (Thailand); Kiatkamjornwong, Suda, E-mail: ksuda@chula.ac.th [Program of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University (Thailand); Department of Imaging and Printing Technology, Faculty of Science, Chulalongkorn University (Thailand); Academy of Science, The Royal Society of Thailand, Sueapa, Dusit, Bangkok 10300 (Thailand)

    2016-02-28

    Graphical abstract: - Highlights: • Both O{sub 2} and N{sub 2} plasma increased cotton surface wettability and higher K/S. • SF6 plasma gave hydrophobicity on cotton surface and increased contact angle to 138°. • Plasma treatment on cotton fabric produced surface roughness. • XPS confirmed the generation of new functional groups on cotton fabric. • Wettability and surface roughness controlled K/S and good ink adhesion. - Abstract: Surface properties of cotton fabric were modified by three types of gas plasma pretreatment, namely, oxygen (O{sub 2}), nitrogen (N{sub 2}) and sulfur hexafluoride (SF{sub 6}), to improve ink absorption of water-based pigmented inkjet inks and color reproduction of the treated surfaces. Effects of gas plasma exposure parameters of power, exposure time and gas pressure on surface physical and chemical properties of the treated fabrics were investigated. XPS (X-ray photoelectron spectroscopy) was used to identify changes in functional groups on the fabric surface while AFM (atomic force microscopy) and SEM (scanning electron microscopy) were used to reveal surface topography of the fabric. Color spectroscopic technique was used to investigate changes in color strength caused by different absorptions of the printed fabrics. The O{sub 2} plasma treatments produced new functional groups, −O−C−O/C=O and O−C=O while N{sub 2} plasma treatments produced additionally new functional groups, C−N and O=C−NH, onto the fabric surface which increased hydrophilic properties and surface energy of the fabric. For cotton fabric treated with SF{sub 6} plasma, the fluorine functionalization was additionally found on the surface. Color strength values (K/S) increased when compared with those of the untreated fabrics. SF{sub 6} plasma-treated fabrics were hydrophobic and caused less ink absorption. Fabric surface roughness caused by plasma etching increased fabric surface areas, captured more ink, and enhanced a larger ink color gamut and

  15. 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.

  16. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    Science.gov (United States)

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. © 2011 IEEE

  17. Adhesion to porcelain and metal.

    Science.gov (United States)

    Bertolotti, Raymond L

    2007-04-01

    Some compelling clinical benefits of porcelain and metal adhesion are presented. Current concepts for metal adhesion are reviewed, including modifications of metal surface and resin chemistry. Porcelain adhesion is reviewed, including little-known methods that use silane but no hydrofluoric acid etching. Clinical protocols for use of metal and porcelain adhesives are presented.

  18. Surface modification of cotton fabrics by gas plasmas for color strength and adhesion by inkjet ink printing

    Science.gov (United States)

    Pransilp, Porntapin; Pruettiphap, Meshaya; Bhanthumnavin, Worawan; Paosawatyanyong, Boonchoat; Kiatkamjornwong, Suda

    2016-02-01

    Surface properties of cotton fabric were modified by three types of gas plasma pretreatment, namely, oxygen (O2), nitrogen (N2) and sulfur hexafluoride (SF6), to improve ink absorption of water-based pigmented inkjet inks and color reproduction of the treated surfaces. Effects of gas plasma exposure parameters of power, exposure time and gas pressure on surface physical and chemical properties of the treated fabrics were investigated. XPS (X-ray photoelectron spectroscopy) was used to identify changes in functional groups on the fabric surface while AFM (atomic force microscopy) and SEM (scanning electron microscopy) were used to reveal surface topography of the fabric. Color spectroscopic technique was used to investigate changes in color strength caused by different absorptions of the printed fabrics. The O2 plasma treatments produced new functional groups, sbnd Osbnd Csbnd O/Cdbnd O and Osbnd Cdbnd O while N2 plasma treatments produced additionally new functional groups, Csbnd N and Odbnd Csbnd NH, onto the fabric surface which increased hydrophilic properties and surface energy of the fabric. For cotton fabric treated with SF6 plasma, the fluorine functionalization was additionally found on the surface. Color strength values (K/S) increased when compared with those of the untreated fabrics. SF6 plasma-treated fabrics were hydrophobic and caused less ink absorption. Fabric surface roughness caused by plasma etching increased fabric surface areas, captured more ink, and enhanced a larger ink color gamut and ink adhesion. Cotton fabrics exhibited higher ink adhesion and wider color gamut after the O2 plasma treatment comparing with those after N2 plasma treatment.

  19. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    Science.gov (United States)

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  20. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5.

    Science.gov (United States)

    Tan, Minjia; Peng, Chao; Anderson, Kristin A; Chhoy, Peter; Xie, Zhongyu; Dai, Lunzhi; Park, Jeongsoon; Chen, Yue; Huang, He; Zhang, Yi; Ro, Jennifer; Wagner, Gregory R; Green, Michelle F; Madsen, Andreas S; Schmiesing, Jessica; Peterson, Brett S; Xu, Guofeng; Ilkayeva, Olga R; Muehlbauer, Michael J; Braulke, Thomas; Mühlhausen, Chris; Backos, Donald S; Olsen, Christian A; McGuire, Peter J; Pletcher, Scott D; Lombard, David B; Hirschey, Matthew D; Zhao, Yingming

    2014-04-01

    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. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

  1. KSHV latent protein LANA2 inhibits sumo2 modification of p53

    Science.gov (United States)

    Laura, Marcos-Villar; de la Cruz-Herrera, Carlos F; Ferreirós, Alba; Baz-Martínez, Maite; Lang, Valerie; Vidal, Anxo; Muñoz-Fontela, Cesar; Rodríguez, Manuel S; Collado, Manuel; Rivas, Carmen

    2015-01-01

    Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation. PMID:25607652

  2. Posttranslational Protein Modification in the Salivary Glands of Sjögren's Syndrome Patients.

    Science.gov (United States)

    Herrera-Esparza, Rafael; Rodríguez-Rodríguez, Mayra; Pérez-Pérez, María Elena; Badillo-Soto, Martha Adriana; Torres-Del-Muro, Felipe; Bollain-Y-Goytia, Juan José; Pacheco-Tovar, Deyanira; Avalos-Díaz, Esperanza

    2013-01-01

    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.

  3. Applications of post-translational modifications of FoxO family proteins in biological functions

    Institute of Scientific and Technical Information of China (English)

    Ying Zhao; Yachen Wang; Wei-Guo Zhu

    2011-01-01

    The functions of the FoxO family proteins, in particular their transcriptional activities, are modulated by post-translational modifications (PTMs), including phosphorylation, acetylation, ubiquitination, methylation and glycosylation. These PTMs occur in response to different cellular stresses, which in turn regulate the subcellular localization of FoxO family proteins, as well as their half-life, DNA binding, transcriptional activity and ability to interact with other cellular proteins. In this review, we summarize the role of PTMs of FoxO family proteins in linking their biological and functional relevance with various diseases.%The functions of the FoxO family proteins,in particular their transcriptional activities,are modulated by post-translational modifications (PTMs),including phosphorylation,acetylation,ubiquitination,methylation and glycosylation.These PTMs occur in response to different cellular stresses,which in turn regulate the subceilular localization of FoxO family proteins,as well as their half-life,DNA binding,transcriptional activity and ability to interact with other cellular proteins.In this review,we summarize the role of PTMs of FoxO family proteins in linking their biological and functional relevance with various diseases.

  4. 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.

  5. Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

    Science.gov (United States)

    Hummerich, René; Costina, Victor; Findeisen, Peter; Schloss, Patrick

    2015-07-15

    Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bβ, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

  6. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

    Science.gov (United States)

    Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

  7. 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 ...

  8. 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.

  9. The role of protein modifications in senescence of freeze-dried Acetobacter senegalensis during storage

    Science.gov (United States)

    2014-01-01

    Background Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (−21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis. Results Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at −21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred. Conclusions High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced

  10. 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.; Mei, van der 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

  11. 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

  12. Staphylococcus aureus-fibronectin interactions with and without fibronectin-binding proteins and their role in adhesion and desorption

    NARCIS (Netherlands)

    Xu, Chun; Boks, Niels P; de Vries, Jacob; Kaper, Harm; Norde, Willem; Busscher, Hendrik; van der Mei, Henderina

    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 wi

  13. 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 wi

  14. Site-Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome.

    Science.gov (United States)

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

    2016-11-21

    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 Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications.

  15. 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.

  16. 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.

  17. Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses.

    Science.gov (United States)

    Gevaert, Kris; Impens, Francis; Van Damme, Petra; Ghesquière, Bart; Hanoulle, Xavier; Vandekerckhove, Joël

    2007-12-01

    Numerous gel-free proteomics techniques have been reported over the past few years, introducing a move from proteins to peptides as bits of information in qualitative and quantitative proteome studies. Many shotgun proteomics techniques randomly sample thousands of peptides in a qualitative and quantitative manner but overlook the vast majority of protein modifications that are often crucial for proper protein structure and function. Peptide-based proteomic approaches have thus been developed to profile a diverse set of modifications including, but not at all limited, to phosphorylation, glycosylation and ubiquitination. Typical here is that each modification needs a specific, tailor-made analytical procedure. In this minireview, we discuss how one technique - diagonal reverse-phase chromatography - is applied to study two different types of protein modification: protein processing and protein N-glycosylation. Additionally, we discuss an activity-based proteome study in which purine-binding proteins were profiled by diagonal chromatography.

  18. 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.

  19. Enhanced Interfacial Adhesion in HDPE/HA Composites by Surface Modification of HA Particles via in situ Polymerization and Copolymerization

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Hydroxyapatite ( HA )-reinforced high density polyethylene (HDPE) was developed as a bone replacement material. In order to enhance the interfacial bondiag between HA and polyethylene and improve the mechanical properties of HDPE/ HA composites, the surface of the micron-sized HA particles was modified by in situ polymerization of butyl acrylate ( BA ) and in situ copolynerization of vinyl triethoxyl silane (VTES) and BA ,then the modified HA particles were compounded with HDPE. The effects of the surface modification of HA on morphology and mechanical properties of HDPE/ HA composites were investigated. The experimental results show that the presence of HA particles does not inhibit the polymerization of BA . The poly( butyl acrylate) ( PBA ) segments on the HA surface enhance the compatibility between HA and HDPE, improve the dispersion of HA particles in HDPE matrix, and enhance the interfacial ndhesion between HA and matrix. Surface modifications, especially by in situ copolymerization of VTES and BA , significantly increase notch impact strengths and marginal stiffness and tensile strengths of HDPE/ HA composites. And it is found that there is a critical thickness of PBA coating on HA particles for optimum mechanical properties of HDPE / HA composites.

  20. 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.

  1. 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.

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

    Science.gov (United States)

    Margreitter, Christian; Petrov, Drazen; Zagrovic, Bojan

    2013-01-01

    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. PMID:23703210

  3. Hydrophobic recovery of UV/ozone treated poly(dimethylsiloxane): adhesion studies by contact mechanics and mechanism of surface modification

    Science.gov (United States)

    Oláh, Attila; Hillborg, Henrik; Vancso, G. Julius

    2005-01-01

    Silicone elastomers (Sylgard 184 and 170), based on poly(dimethylsiloxane) (PDMS), were surface treated by a combined exposure to UV and ozone. The effects of the treatments were analyzed as a function of time elapsed after stopping the treatments using different standard surface characterization techniques, such as water contact angle measurements, XPS and atomic force microscopy (AFM). However, the primary focus of this study was to apply the Johnson-Kendall-Roberts (JKR) contact mechanics approach to investigate PDMS samples prior to and following UV/ozone surface treatment. A gradual formation of a hydrophilic, silica-like surface layer with increasing modulus was observed with increasing UV/ozone exposure. A subsequent hydrophobic recovery after UV/ozone exposure was observed, as indicated by increasing contact angles. This supports the hypothesis that the hydrophobic recovery is mainly caused by the gradual coverage of a permanent silica-like structure with free siloxanes and/or reorientation of polar groups. PDMS containing a homogenously dispersed filler (Sylgard 184), exhibited a decreasing surface roughness (by AFM) when the oxidized surface region "collapsed" into a smooth SiO x layer (final surface roughness Sylgard 170), exhibited an increasing surface roughness with treatment dose, which was attributed to the "collapse" of the oxidized surface region thus exposing the contours of the underlying filler aggregates (final surface roughness ˜140 nm). A dedicated device was designed and built to study the contact mechanics behavior of PDMS prior to, and following surface treatment. The value of the combined elastic modulus obtained for PDMS lens and semi-infinite flat surface system showed an increase in full agreement with the formation of a silica-like layer exhibiting a high elastic modulus (compared with untreated PDMS). The work of adhesion observed in JKR experiments exhibited an increasing trend as a function of treatment done in agreement with

  4. Control of high affinity interactions in the talin C terminus: how talin domains coordinate protein dynamics in cell adhesions.

    OpenAIRE

    2009-01-01

    In cell-extracellular matrix junctions (focal adhesions), the cytoskeletal protein talin is central to the connection of integrins to the actin cytoskeleton. Talin is thought to mediate this connection via its two integrin, (at least) three actin, and several vinculin binding sites. The binding sites are cryptic in the head-to-rod autoinhibited cytoplasmic form of the protein and require (stepwise) conformational activation. This activation process, however, remains poorly understood, and the...

  5. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    Science.gov (United States)

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

  6. Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.

    Directory of Open Access Journals (Sweden)

    Ran Zichel

    Full Text Available Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Emulsifying and Foaming Properties of Soy Protein Isolates with Covalent Modification by (--Epigallocatechin-3-Gallate

    Directory of Open Access Journals (Sweden)

    M. Zheng

    2014-02-01

    Full Text Available Soy Protein Isolates (SPI with covalent modification by (--Epigallocatechin-3-Gallate (EGCG were prepared under the alkaline condition. The effects of covalent modification on the emulsifying and foaming properties of SPI were evaluated. The Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE profiles of the modified SPI revealed that EGCG treatment caused cross-linking of subunits. Emulsifying activity of modified SPI significantly increased when compared with that of control (p<0.05 at the concentration of 35 mg/mL. Modification of SPI by EGCG caused a decrease in the foam volume initially. In the range of 15 to 35 mg/mL, the foaming activities of modified SPI were found to be less than those of the control (p<0.05. The foaming stabilities of modified SPI were significantly higher when compared to those of control (p<0.05. The results obtained in this study indicated the modification by EGCG resulted in the enhancement of emulsifying activity and foaming stability of SPI. This modification may serve as a promising approach for improving functional properties of SPI.

  9. Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Wirth, Christophe; Rospert, Sabine; Hu, Zehan; Dengjel, Jörn; Tzivelekidis, Tina; Andersen, Gregers Rom; Hunte, Carola; Schlosser, Andreas; Aktories, Klaus

    2017-08-11

    Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A), delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the alpha amino group of mono-L-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe, but not in various other eukaryotic organisms tested despite strict conservation of the Glu-45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification, which appears to influence protein translation. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  10. Design Methods of Cell Adhesion Proteins Based on ELISA Usable in-vitro in Gene Therapy

    Directory of Open Access Journals (Sweden)

    E Hosseini

    2016-07-01

    Full Text Available Background & aim: One of the strategies to improve the therapeutic gene is targeting gene therapy. A method which can be considered, is adding code sequences peptide or protein with high tendency to target cells and secreting the therapeutic gene encodes a protein. However, evaluating the effectiveness of such changes in the targeted cell binding protein gene product with the usual therapeutic methods produced in prokaryotic system is directly impossible. The purpose of this study was to evaluate the design methods of cell adhesion proteins based on ELISA usable in-vitro in gene therapy. Methods: In order to target the therapeutic gene Mda-7 by using genetic engineering, peptide coding sequence RGD4C with the tendency to cancerous cell surface integrin were inserted shortly after the artificial signal peptide sequence and the N-terminal coding region of the protein. Then, the modified and unmodified cDNA eukaryotic expression vector pCDNA3.1 were matched. Vectors were transfected in HEK-293 cell line. Then Mda-7 secreted expression levels were measured in cell culture by ELISA. After adjusting the protein concentration of Mda-7 and RGD.Mda-7, in cells transfected media, they were used as a source of protein. Reduce the concentration of these genes was assessed two hours after exposure to the integrin cell lines with HepG2, M21 and lacking integrin Saos-2  were also determined by ELISA. The present study was conducted three times independently.  Data were analyzed using t-test. Results: Statistical analysis of the results suggested that the gene product of the gene product RGD.Mda-7 and Mda-7 to connect to HepG2 cells and M21 were more likely to have integrin. While binding to the cell lines of Saos-2, no significant difference were observed. Conclusions: It seems the present ELISA based method was a suitable strategy for cell attachment assay in gene therapy research.  

  11. Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion.

    Science.gov (United States)

    Bulanova, Daria R; Akimov, Yevhen A; Rokka, Anne; Laajala, Teemu D; Aittokallio, Tero; Kouvonen, Petri; Pellinen, Teijo; Kuznetsov, Sergey G

    2016-10-07

    G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

  12. 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.

  13. Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification.

    Science.gov (United States)

    Li, Shi-Yan; Du, Min; Dolence, E Kurt; Fang, Cindy X; Mayer, Gabriele E; Ceylan-Isik, Asli F; LaCour, Karissa H; Yang, Xiaoping; Wilbert, Christopher J; Sreejayan, Nair; Ren, Jun

    2005-04-01

    Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24-26-month) mice using a MyoCam system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, +/- dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was approximately 2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4-7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.

  14. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    Science.gov (United States)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  15. Towards an understanding of regulating Cajal body activity by protein modification.

    Science.gov (United States)

    Hebert, Michael D; Poole, Aaron R

    2016-10-07

    The biogenesis of small nuclear ribonucleoproteins (snRNPs), small Cajal body-specific RNPs (scaRNPs), small nucleolar RNPs (snoRNPs) and the telomerase RNP involves Cajal bodies (CBs). Although many components enriched in the CB contain post-translational modifications (PTMs), little is known about how these modifications impact individual protein function within the CB and, in concert with other modified factors, collectively regulate CB activity. Since all components of the CB also reside in other cellular locations, it is also important that we understand how PTMs affect the subcellular localization of CB components. In this review, we explore the current knowledge of PTMs on the activity of proteins known to enrich in CBs in an effort to highlight current progress as well as illuminate paths for future investigation.

  16. Toward an understanding of regulating Cajal body activity by protein modification.

    Science.gov (United States)

    Hebert, Michael D; Poole, Aaron R

    2016-10-07

    The biogenesis of small nuclear ribonucleoproteins (snRNPs), small Cajal body-specific RNPs (scaRNPs), small nucleolar RNPs (snoRNPs) and the telomerase RNP involves Cajal bodies (CBs). Although many components enriched in the CB contain post-translational modifications (PTMs), little is known about how these modifications impact individual protein function within the CB and, in concert with other modified factors, collectively regulate CB activity. Since all components of the CB also reside in other cellular locations, it is also important that we understand how PTMs affect the subcellular localization of CB components. In this review, we explore the current knowledge of PTMs on the activity of proteins known to enrich in CBs in an effort to highlight current progress as well as illuminate paths for future investigation.

  17. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    Science.gov (United States)

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  18. Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior

    Directory of Open Access Journals (Sweden)

    M Bongio

    2011-12-01

    Full Text Available The ultimate goal of this work was to develop a biocompatible and biomimetic in situ crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells and calcium phosphate (CaP nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue. Rat bone marrow osteoblast-like cells (OBLCs were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol fumarate (OPF, RGD-modified OPF, OPF enriched with CaP nanoparticles and RGD-modified OPF enriched with CaP nanoparticles and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles significantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better biological response than that of the individual components. Specifically, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems.

  19. Molecular Modification of a HSV-1 Protein and Its Associated Gene Transcriptional Regulation

    Institute of Scientific and Technical Information of China (English)

    Yan-chun CHE; Li JIANG; Qi-han LI

    2008-01-01

    The molecular modifications of Herpes Simplex Virus Type Ⅰ (HSV-1) proteins represented by acetylation and phosphorylation are essential to its biological functions.The cellular chromatin-remodeling/assembly is involved in HSV-1 associated gene transcriptional regulation in human cells harboring HSV-1 lytic or latent infections.Further investigation on these biological events would provide a better understanding of the mechanisms of HSV- 1 viral gene transcriptional regulation.

  20. Dissection of the DNA mimicry of the bacteriophage T7 Ocr protein using chemical modification

    OpenAIRE

    Stephanou, Augoustinos S.; Roberts, Gareth A; Cooper, Laurie P.; Clarke, David J.; Thomson, Andrew R; MacKay, C. Logan; Nutley, Margaret; Cooper, Alan; Dryden, David T.F

    2009-01-01

    The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modi...

  1. Covalent modifications of ribosomal proteins in growing and aggregation-competent dictyostelium discoideum: phosphorylation and methylation.

    Science.gov (United States)

    Ramagopal, S

    1991-04-01

    Phosphorylated and methylated ribosomal proteins were identified in vegetatively growing amoebae and in the starvation-induced, aggregation-competent cells of Dictyostelium discoideum. Of the 15 developmentally regulated cell-specific ribosomal proteins reported earlier, protein A and the acidic proteins A1, A2, and A3 were identified as phosphoproteins, and S5, S6, S10, and D were identified as methylated proteins. Three other ribosomal proteins were phosphorylated and 19 others methylated. S19, L13, A1, A2, and A3 were the predominant phosphoproteins in growing amoebae, whereas S20 and A were the predominant ones in the aggregation-competent cells. Among the methylated proteins, eight (S6, S10, S13, S30, D, L1, L2, and L31) were modified only during growth phase, six (S5, S7, S8, S24, S31, and L36) were altered only during aggregation-competent phase, and nine (S9, S27, S28, S29, S34, L7, L35, L41, and L42) were modified under both phases. Five proteins (S6, S24, L7, L41, and L42) were heavily methylated and of these, the large subunit proteins were present in both growing amoebae and aggregation-competent cells. These findings demonstrate that covalent modification of specific ribosomal proteins is regulated during cell differentiation in D. discoideum.

  2. 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.

  3. Protein adsorption mechanisms determine the efficiency of thermally controlled cell adhesion on poly(N-isopropyl acrylamide) brushes.

    Science.gov (United States)

    Choi, Sangwook; Choi, Byung-Chan; Xue, Changying; Leckband, Deborah

    2013-01-14

    This study investigated the impact of the protein adsorption mechanism(s) on the efficiency of thermally controlled cell adhesion and release from poly(N-isopropyl acrylamide) brushes. Large format polymer gradients were used to screen for grafting densities and substrate chemistries that alter both cell adhesion at 37 °C and rapid cell release at 25 °C. In particular, the grafting conditions investigated allowed protein adsorption to the underlying substrate, penetration of the brush only, or adsorption to the outer edge of the film. At an average molecular weight of 30 kDa (degree of polymerization N ∼ 270), the results show that robust protein adsorption to polymer brushes impairs rapid cell release below the lower critical solution temperature. Conversely, grafting conditions that permit protein penetration of the brush but block strong adsorption to the underlying substrate support cell adhesion above the transition temperature and ensure efficient cell recovery at lower temperature. These findings demonstrate the impact of protein adsorption mechanisms, surface chemistry, and polymer properties on thermally controlled cell capture and release.

  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. 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

    antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination...

  6. Influence of surface modification and static pressure on microdialysis protein extraction efficiency.

    Science.gov (United States)

    Chu, Jiangtao; Undin, Torgny; Lind, Sara Bergström; Hjort, Klas; Dahlin, Andreas P

    2015-10-01

    There is growing interest in using microdialysis (MD) for monitoring larger and more complex molecules such as neuropeptides and proteins. This promotes the use of MD membranes with molecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of the membrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure. In the meantime, despite the large pore size, studies have shown that membrane biofouling still lead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextran and Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency (EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests, using as MD sample a protein standard with similar concentrations as in human cerebral spinal fluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysate fractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for total protein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. The FR results suggested that the surface modified catheters were less sensitive to the pressure and provide higher precision, and provided a FR closer to 100%. The surface modification did not show a significant effect on the protein EE. The average total protein EE of surface modified catheters was slightly higher than that of the native ones. The MS EE data of individual proteins showed a clear trend of complex response in EE with pressure.

  7. Modifications in nanoparticle-protein interactions by varying the protein conformation

    Science.gov (United States)

    Kumar, Sugam; Yadav, I.; Aswal, V. K.; Kohlbrecher, J.

    2017-05-01

    Small-angle neutron scattering has been used to study the interaction of silica nanoparticle with Bovine Serum Albumin (BSA) protein without and with a protein denaturing agent urea. The measurements have been carried out at pH 7 where both the components (nanoparticle and protein) are similarly charged. We show that the interactions in nanoparticle-protein system can be modified by changing the conformation of protein through the presence of urea. In the absence of urea, the strong electrostatic repulsion between the nanoparticle and protein prevents protein adsorption on nanoparticle surface. This non-adsorption, in turn gives rise to depletion attraction between nanoparticles. However, with addition of urea the depletion attraction is completely suppressed. Urea driven denaturation of protein is utilized to expose the positively charged patched of the BSA molecules which eventually leads to adsorption of BSA on nanoparticles eliminating the depletion interaction.

  8. 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......, 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......, these data suggest that 1) IL-2R ligation induces homotypic adhesion through a wortmannin/LY294002-sensitive, rapamycin-resistant pathway, 2) tyrosine kinases play a critical role in cytokine-induced adhesion, and 3) adhesion, but not mitogenesis, correlates with enhanced tyrosine phosphorylation...

  9. Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

    Science.gov (United States)

    Papoian, Garegin

    2012-02-01

    Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.

  10. Comparison of two different plasma surface-modification techniques for the covalent immobilization of protein monolayers.

    Science.gov (United States)

    Cifuentes, Anna; Borrós, Salvador

    2013-06-04

    The immobilization of biologically active species is crucial for the fabrication of smart bioactive surfaces. For this purpose, plasma polymerization is frequently used to modify the surface nature without affecting the bulk properties of the material. Thus, it is possible to create materials with surface functional groups that can promote the anchoring of all kinds of biomolecules. Different methodologies in protein immobilization have been developed in recent years, although some drawbacks are still not solved, such as the difficulties that some procedures involve and/or the denaturalization of the protein due to the immobilization process. In this work, two different strategies to covalently attach bovine serum albumin (BSA) protein are developed. Both techniques are compared in order to understand how the nature of the surface modification affects the conformation of the protein upon immobilization.

  11. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion.

    Science.gov (United States)

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei

    2013-07-19

    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

  12. Cross talk between tetanus neurotoxin-insensitive vesicle-associated membrane protein-mediated transport and L1-mediated adhesion.

    Science.gov (United States)

    Alberts, Philipp; Rudge, Rachel; Hinners, Ina; Muzerelle, Aude; Martinez-Arca, Sonia; Irinopoulou, Theano; Marthiens, Veronique; Tooze, Sharon; Rathjen, Fritz; Gaspar, Patricia; Galli, Thierry

    2003-10-01

    The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.

  13. 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...... to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity....

  14. 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.

  15. C1q/TNF-related protein-9 inhibits cytokine-induced vascular inflammation and leukocyte adhesiveness via AMP-activated protein kinase activation in endothelial cells.

    Science.gov (United States)

    Jung, Chang Hee; Lee, Min Jung; Kang, Yu Mi; Lee, Yoo La; Seol, So Mi; Yoon, Hae Kyeong; Kang, Sang-Wook; Lee, Woo Je; Park, Joong-Yeol

    2016-01-05

    Although recent studies have reported cardioprotective effects of C1q/TNF-related protein 9 (CTRP9), the closet adiponectin paralog, its role on cytokine-induced endothelial inflammation is unknown. We investigated whether CTRP9 prevented inflammatory cytokine-induced nuclear factor-kappa B (NF-κB) activation and inhibited the expression of adhesion molecules and a chemokine in the vascular endothelial cell. We used human aortic endothelial cells (HAECs) to examine the effects of CTRP9 on NF-κB activation and the expression of NF-κB-mediated genes, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1). Tumor necrosis factor alpha (TNFα) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNFα-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNFα-induced activation of NF-κB, as well as the expression of ICAM-1, VCAM-1, and MCP-1. In addition, treatment with CTRP9 significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the downstream target of AMPK. The inhibitory effect of CTRP9 on the expression of ICAM-1, VCAM-1, and MCP-1 and monocyte adhesion to HAECs was abolished after transfection with an AMPKα1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation.

  16. Modification of cysteine residues by cyclopentenone prostaglandins: interplay with redox regulation of protein function.

    Science.gov (United States)

    Oeste, Clara L; Pérez-Sala, Dolores

    2014-01-01

    Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Adhesive properties of Clostridium perfringens to extracellular matrix proteins collagens and fibronectin.

    Science.gov (United States)

    Hitsumoto, Yasuo; Morita, Naomi; Yamazoe, Ryosuke; Tagomori, Mika; Yamasaki, Tsutomu; Katayama, Seiichi

    2014-02-01

    The adhesive properties of Clostridium perfringens to collagens, gelatin, fibronectin (Fn), Fn-prebound collagens, and Fn-prebound gelatin were investigated. C. perfringens could bind to Fn-prebound collagen type II, type III, and gelatin, but not to gelatin or collagens except for collagen type I directly. Recombinant Fn-binding proteins of C. perfringens, rFbpA and rFbpB, were used to examine Fn-mediated bacterial adherence to collagen type I. In the presence of rFbps, C. perfringens adherence to Fn-prebound collagen type I was inhibited in a dose-dependent manner. Fn was not released from the coated collagen type I by the presence of rFbps, and rFbps did not bind to collagen type I. Thus, the inhibition of C. perfringens binding to Fn-prebound collagen type I by rFbps could not be explained by the removal of Fn from collagen or by the competitive binding of rFbps to collagen. Instead, both rFbps were found to bind to C. perfringens. These results suggest the possibility that rFbps may bind to the putative Fn receptor expressed on C. perfringens and competitively inhibit Fn binding to C. perfringens.

  20. Modification of an apparatus for tumor-suppressor protein crystal growth in the International Space Station

    Science.gov (United States)

    de Morais Mendonca Teles, Antonio

    Some human diseases as tumors are being studied continuously for the development of vaccines against them. And a way of doing that is by means of proteins research. There are some kinds of proteins, like the p53 and p73 proteins, which are tumor suppressors. There are other diseases such as A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases which are protein-related. The determination of how proteins geometrically order themselves, during its biological functions is very necessary to understand how a protein's structure affects its function, to design vaccines that intercede in tumor-protein activities and in other proteins related to those other diseases. The protein crystal growth in microgravity environment produces purer crystallization than on the ground, and it is a powerful tool to produce better vaccines. Several data have already been acquired using ground-based research and in spaceflight experiments aboard the Spacelab and Space Shuttle missions, and in the MIR and in the International Space Station (ISS). Here in this paper, I propose to be performed in the ISS Biological Research Facility (which is being developed), multiple crystal growth of proteins related to cancer (as tumors suppressors and oncoproteins), A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases, for the future obtaining of possible vaccines against them. I also propose a simple and practical equipment, a modification of the crystallization plates (which use a vapor diffusion technique) inside each cylinder of the Protein Crystallization Apparatus in Microgravity (PCAM), with multiple chambers with different sizes. Instead of using some chambers with the same size it is better to use several chambers with different sizes. Why is that? The answer is: the energy associated with the surface tension of the liquid in the chamber is directly related to the circle area of it. So, to minimize the total energy of the surface tension of a proteins liquid -making it more stable

  1. Preparation and properties of plywood adhesives with soy protein isolate modified by SDS%胶合板用SDS改性大豆分离蛋白胶粘剂的制备及性能

    Institute of Scientific and Technical Information of China (English)

    李娜; 谢建军; 曾念; 丁出; 冉德龙

    2012-01-01

    The effects of mass concentration, the reaction temperature and the reaction time of soy protein isolate(SPI) and sodium dodecyl sulfate(SDS) on the adhesive strength of the modified SPI adhesives has been studied through orthogonal experiments. And the adhesive mechanism of the modified SPI adhesives was discussed. The results show that the optimum formula conditions are as follows: mass concentration of SPI was 14%, that of SDS 1%, reaction temperature 35 ℃ and the reaction time 3 hours. Under the optimum conditions, the dry adhesive strength of the modified SPI adhesives was 1.82 MPa, the wet adhesive strength was 0.82 MPa, the viscosity was 5.8 Pa's, the solid content was 12.96%. After SDS was added into SPI, the composites of SDS-SPI were formed, the internal hydrophobic groups among the SPI molecule structure were turned out and the water resistance of the modified SPI adhesives was enhanced with an increase of the modification time. When the concentration of SDS was over a defined value, the disulfide bond was broken and the modification effect of SDS went bad.%采用正交试验方法研究了大豆分离蛋白(SPI)及十二烷基磺酸钠(SDS)质量浓度、反应温度、反应时间对胶粘剂粘接强度的影响;并对改性产物的机理进行了探讨.结果表明:SDS改性大豆分离蛋白胶粘剂最佳工艺条件为:14%SPI、1%SDS、反应温度35℃、反应时间3h,并测得该胶的干态粘接强度为1.82 MPa,湿态粘接强度为0.82 MPa,粘度为5.8 Pa·s,固含量为12.96%.大豆分离蛋白分子经SDS改性后形成了SDS-SPI 复合物,使包围在内部的疏水性基团转而向外,改性时间增加,其耐水性增加;SDS超过某浓度后,二硫键断裂增加,改性效果变差.

  2. Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

    Science.gov (United States)

    Stephanou, Augoustinos S.; Roberts, Gareth A.; Cooper, Laurie P.; Clarke, David J.; Thomson, Andrew R.; MacKay, C. Logan; Nutley, Margaret; Cooper, Alan; Dryden, David T.F.

    2009-01-01

    The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼ 50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼ 86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼ 800-fold. PMID:19523474

  3. 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

  4. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    Directory of Open Access Journals (Sweden)

    Odgerel Oidovsambuu

    Full Text Available VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs. Although Vsig1(-/Y ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  5. Adhesion protein VSIG1 is required for the proper differentiation of glandular gastric epithelia.

    Science.gov (United States)

    Oidovsambuu, Odgerel; Nyamsuren, Gunsmaa; Liu, Shuai; Göring, Wolfgang; Engel, Wolfgang; Adham, Ibrahim M

    2011-01-01

    VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3'untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5' flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1(-/Y) ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1(-/Y) cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

  6. 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.

  7. Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance.

    Science.gov (United States)

    Luo, Quanzhou; Wypych, Jette; Jiang, Xinzhao Grace; Zhang, Xin; Luo, Shun; Jerums, Matthew; Lewis, Jeffrey; Iii, Ronald Keener; Huang, Gang; Apostol, Izydor

    2012-02-15

    In this report, we examine the performance of a mass spectrometry (MS)-based method for quantification of protein posttranslational modifications (PTMs) using stable isotope labeled internal standards. Uniform labeling of proteins and highly similar behavior of the labeled vs nonlabeled analyte pairs during chromatographic separation and electrospray ionization (ESI) provide the means to directly quantify a wide range of PTMs. In the companion report (Jiang et al., Anal. Biochem., 421 (2012) 506-516.), we provided principles and example applications of the method. Here we show satisfactory accuracy and precision for quantifying protein modifications by using the SILIS method when the analyses were performed on different types of mass spectrometers, such as ion-trap, time-of-flight (TOF), and quadrupole instruments. Additionally, the stable isotope labeled internal standard (SILIS) method demonstrated an extended linear range of quantification expressed in accurate quantification up to at least a 4 log concentration range on three different types of mass spectrometers. We also demonstrate that lengthy chromatographic separation is no longer required to obtain quality results, offering an opportunity to significantly shorten the method run time. The results indicate the potential of this methodology for rapid and large-scale assessment of multiple quality attributes of a therapeutic protein in a single analysis.

  8. Profiling of Protein N-Termini and Their Modifications in Complex Samples.

    Science.gov (United States)

    Demir, Fatih; Niedermaier, Stefan; Kizhakkedathu, Jayachandran N; Huesgen, Pitter F

    2017-01-01

    Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.

  9. Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils.

    Science.gov (United States)

    Wilkie-Grantham, Rachel P; Magon, Nicholas J; Harwood, D Tim; Kettle, Anthony J; Vissers, Margreet C; Winterbourn, Christine C; Hampton, Mark B

    2015-04-10

    Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.

  10. Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications.

    Science.gov (United States)

    Steffensen, Charlotte L; Andersen, Mogens L; Degn, Peter E; Nielsen, Jacob H

    2008-12-24

    Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.

  11. 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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Bade, Veronika N; Nickels, Jochen; Keusekotten, Kirstin; Praefcke, Gerrit J K

    2012-01-01

    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 reducing agent

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  15. Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

    DEFF Research Database (Denmark)

    Sasaki, T; Brakebusch, C; Engel, J

    1998-01-01

    in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

  16. The effect of temperature on adhesion forces between surfaces and model foods containing whey protein and sugar

    OpenAIRE

    Goode, K. R.; Bowen, James; Akhtar, N.; Robbins, P. T.; Fryer, P. J.

    2013-01-01

    The formation of fouling deposit from foods and food components is a severe problem in food processing and leads to frequent cleaning. The design of surfaces that resist fouling may decrease the need for cleaning and thus increase efficiency. Atomic force microscopy has been used to measure adhesion forces between stainless steel (SS) and fluoro-coated glass (FCG) microparticles and the model food deposits (i) whey protein (WPC), (ii) sweetened condensed milk, and (iii) caramel. Measurements ...

  17. Chemical surface modification of parylene C for enhanced protein immobilization and cell proliferation.

    Science.gov (United States)

    Zhang, Changhong; Thompson, Mark E; Markland, Frank S; Swenson, Steve

    2011-10-01

    To introduce the adhesion site of proteins and/or cells on parylene C (PC)-coated medical devices that can be used as implantable biosensors or drug delivery capsules, the PC surfaces were initially modified by the Friedel-Crafts acylation reaction to generate active chlorines. These chlorines were then employed to initiate the atom transfer radical polymerization of tert-butyl acrylate (TBA) and form a polymer brush layer of polyTBA on PC; the acrylate groups in the polymer brushes were hydrolyzed to carboxylic acid groups and further activated into succinimidyl ester groups via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide coupling reaction. The PC surface grafted with polymer brushes and activated by succinimide showed efficient attachment of proteins, including gelatin, contortrostatin (CN) and bovine serum albumin (BSA), all at high density on the PC surface. The CN density on the surface was evaluated for both monolayer and polymer brush-based coatings. Based on fluorescence measurements, the polymer brush gives a 60-fold higher surface protein density than the monolayer-based system. Gelatin was used as a model protein and covalently coated onto the modified PC surface for cell culture study. Substrates with gelatin coating showed a significantly higher cell attachment and proliferation in 7 days cultures as compared to the uncoated substrates. In addition, a conventional photolithography technique was coupled with the surface chemistry to successfully pattern the BSA labeled with fluorescein isothiocyanate on the modified PC surfaces.

  18. Effects of ovarian cancer G protein coupled receptor 1 on the proliferation, migration, and adhesion of human ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    REN Juan; ZHANG Long

    2011-01-01

    Background OGR1 was found as a G-protein coupled receptor (GPCR) and proton sensor. Our previous studies have found that OGR1 has inhibitory effect on the metastasis of prostate cancer. In order to investigate the roles of OGR1 gene in the biological activities of ovarian cancer, we studied the OGR1 effects on ovarian cancer cells, HEY cells.Methods OGR1 gene was transfected into HEY cell, in which endogenous expression is low. OGR1-overxepressed cells and vector-transfected cells were compared in different assays. Western blotting was employed to confirm the high expression level of OGR1. Cell proliferation was determined by MTT assay and cell doubling time assay. Cell migration assay (transwell assay) and cell adhesion assay were performed to determine the migration and adhesion potential of cells. Student's t test was employed for statistical analysis.Results Proliferation of OGR1-overexpressed cells was significantly reduced (P <0.01); cell migration was significantly inhibited in the OGR1-transfected cells (P <0.01); cell adhesion to extracellular matrix including fibronectin, vitronectin,collagen Ⅰ/Ⅳ was significantly increased (P <0.01).Conclusions OGR1 expression in human ovarian cancer cells significantly inhibited the cell proliferation and migration,but significantly enhanced cell adhesion to the extracellular matrix. It indicated that OGR1 may be a tumor suppressor gene for ovarian cancer.

  19. 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.

  20. 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

    Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration...

  1. Recombinant S-layer proteins of Lactobacillus brevis mediating antibody adhesion to calf intestine alleviated neonatal diarrhea syndrome.

    Science.gov (United States)

    Khang, Yong-Ho; Park, Hee-Young; Jeong, Yoo-Seok; Kim, Jung-Ae; Kim, Young-Hwan

    2009-05-01

    A chimeric gene encoding enhanced green fluorescent protein (EGFP) and a S-layer protein from Lactobacillus brevis KCTC3102, and/or two copies of the Fc-binding Z-domain, a synthetic analog of the B-domain of protein A, was constructed and expressed in Escherichia coli BL21(DE3). The S-layer fusion proteins produced in a 500-l fermentor were likely to be stable in the range of pH 5 to 8 and 0 degree to 40 degrees . Their adhesive property enabled an easy and rapid immobilization of enzymes or antibodies on solid materials such as plastics, glass, sol-gel films, and intestinal epithelial cells. Owing to their affinity towards intestinal cells and immunoglobulin G, the Slayer fusion proteins enabled the adhesion of antibodies to human epithelial cells. In addition, feeding a mixture of the S-layer fusion proteins and antibodies against neonatal calf diarrhea (coronavirus, rotavirus, Escherichia coli, and Salmonella typhimurium) to Hanwoo calves resulted in 100% prevention of neonatal calf diarrhea syndrome (p<0.01),whereas feeding antibodies only resulted in 56% prevention.

  2. Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.

    Science.gov (United States)

    Mölleken, Katja; Schmidt, Eleni; Hegemann, Johannes H

    2010-11-01

    Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. Adhesion of the infectious elementary body to the eukaryotic host cell is a pivotal step in chlamydial pathogenesis. Here we describe the characterization of members of the polymorphic membrane protein family (Pmp), the largest protein family (with up to 21 members) unique to Chlamydiaceae. We show that yeast cells displaying Pmp6, Pmp20 or Pmp21 on their surfaces, or beads coated with the recombinant proteins, adhere to human epithelial cells. A hallmark of the Pmp protein family is the presence of multiple repeats of the tetrapeptide motifs FxxN and GGA(I, L, V) and deletion analysis shows that at least two copies of these motifs are needed for adhesion. Importantly, pre-treatment of human cells with recombinant Pmp6, Pmp20 or Pmp21 protein reduces infectivity upon subsequent challenge with Chlamydia pneumoniae and correlates with diminished attachment of Chlamydiae to target cells. Antibodies specific for Pmp21 can neutralize infection in vitro. Finally, a combination of two different Pmp proteins in infection blockage experiments shows additive effects, possibly suggesting similar functions. Our findings imply that Pmp6, Pmp20 and Pmp21 act as adhesins, are vital during infection and thus represent promising vaccine candidates.

  3. Systems analysis of protein modification and cellular responses induced by electrophile stress.

    Science.gov (United States)

    Jacobs, Aaron T; Marnett, Lawrence J

    2010-05-18

    Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile-DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood. In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived alpha,beta-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation. Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a

  4. Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

    Energy Technology Data Exchange (ETDEWEB)

    Scalise, F.W.

    1988-01-01

    Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca{sup 2+} concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca{sup 2+}-mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca{sup 2+}-dependent phosphorylation of the {alpha}subunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca{sup 2+} are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that ({sup 3}H) spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development.

  5. 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.

  6. Characterization of in vivo phosphorylation modification of differentially accumulated proteins in cotton fiber-initiation process.

    Science.gov (United States)

    Liu, Wenying; Zhang, Bing; He, Wenying; Wang, Zi; Li, Guanqiao; Liu, Jinyuan

    2016-08-01

    Initiation of cotton fiber from ovule epidermal cells determines the ultimate number of fibers per cotton ovule, making it one of the restriction factors of cotton fiber yield. Previous comparative proteomics studies have collectively revealed 162 important differentially accumulated proteins (DAPs) in cotton fiber-initiation process, however, whether and how post-translational modifications, especially phosphorylation modification, regulate the expression and function of the DAPs are still unclear. Here we reported the successful identification of 17 phosphopeptides from 16 phosphoproteins out of the 162 DAPs using the integrated bioinformatics analyses of peptide mass fingerprinting data and targeted MS/MS identification method. In-depth analyses indicated that 15 of the 17 phosphorylation sites were novel phosphorylation sites first identified in plants, whereas 6 of the 16 phosphoproteins were found to be the phosphorylated isoforms of 6 proteins. The phosphorylation-regulated dynamic protein network derived from this study not only expanded our understanding of the cotton fiber-initiation process, but also provided a valuable resource for future functional studies of the phosphoproteins. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Double sequential modifications of composite cryogel beds for enhanced ion-exchange capacity of protein.

    Science.gov (United States)

    Wang, Chuan; Sun, Yan

    2013-09-13

    Composite cryogel monoliths based on poly(2-hydroxyethylmethacrylate) (pHEMA) were fabricated by incorporating polymeric resin particles. The monoliths were sequentially modified by polyethylenimine (PEI) and diethylaminoethyl (DEAE). The novel composite material had rough pore walls and extended anion-exchange tentacles, which provided more binding sites for protein molecules. The dynamic adsorption capacity of bovine serum albumin (BSA) on the novel cryogel bed reached 11.2mg/mL bed volume at a flow velocity of 8cm/min, which was about 1.5-4.6 times higher than the cryogel beds obtained by single modifications. The capacity value was also much higher than the BSA capacities of cryogel beds reported in literature (1-6mg/mL). The capacity decreased only slightly with increasing flow rate from 0.6 to 12cm/min. The height equivalent to a theoretical plate of the composite beds was in the range 2-2.5mm, changed indistinctively in a flow rate range 0.6-18cm/min. Hence, the work has proved that the double-modification strategy was promising for enhancing protein adsorption capacity of cryogel monolith for high-speed protein chromatography.

  8. 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.

  9. Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

    Science.gov (United States)

    Demberg, Lilian M; Winkler, Jana; Wilde, Caroline; Simon, Kay-Uwe; Schön, Julia; Rothemund, Sven; Schöneberg, Torsten; Prömel, Simone; Liebscher, Ines

    2017-03-17

    Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel-derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel-derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Cell Adhesion Induced Using Surface Modification with Cell-Penetrating Peptide-Conjugated Poly(ethylene glycol)-Lipid: A New Cell Glue for 3D Cell-Based Structures.

    Science.gov (United States)

    Teramura, Yuji; Asif, Sana; Ekdahl, Kristina N; Gustafson, Elisabet; Nilsson, Bo

    2017-01-11

    We synthesized a novel material, cell-penetrating peptide-conjugated poly(ethylene glycol)-lipid (CPP-PEG-lipid), that can induce the adhesion of floating cells. Firm cell adhesion with spreading could be induced by cell surface modification with the CPP-PEG-lipids. Cell adhesion was induced by CPPs but not by any other cationic short peptides we tested. Here, we demonstrated adherence using the floating cell line CCRF-CEM as well as primary human T cells, B cells, erythrocytes, and hepatocytes. As compared to cells grown in suspension, adherent cells were more rapidly induced to attach to substrates with the cell-surface modification. The critical factor for attachment was localization of CPPs at the cell membrane by PEG-lipids with PEG > 20 kDa. These cationic CPPs on PEG chains were able to interact with substrate surfaces such as polystyrene (PS) surfaces, glass surfaces, and PS microfibers that are negatively charged, inducing firm cell adhesion and cell spreading. Also, as opposed to normal cationic peptides that interact strongly with cell membranes, CPPs were less interactive with the cell surfaces because of their cell-penetrating property, making them more available for adhering cells to the substrate surface. No effects on cell viability or cell proliferation were observed after the induction of cell adhesion. With this technique, cells could be easily immobilized onto PS microfibers, an important step in fabricating 3D cell-based structures. Cells immobilized onto 3D PS microfibers were alive, and human hepatocytes showed normal production of urea and albumin on the microfibers. This method is novel in inducing firm cell adhesion via a one-step treatment.

  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

    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. 环境友好型胶合板用胶黏剂及其改性技术的研究进展%Overview on Adhesives and Relevant Modification for Environmentally-Friendly Plywood

    Institute of Scientific and Technical Information of China (English)

    方露; 常亮; 郭文静; 王正

    2012-01-01

    The authors reviewed recent progress of environmentally-friendly plywood and focused on the modification of current adhesives used in plywood. There were three important methods used to reduce the emissions of formaldehyde, including modification of traditional formaldehyde-based adhesives, application of biomass-based adhesives and thermo-plastic adhesives as well as pretreatment of veneer. Based on the analysis on these processing methods, the development trend and research directions of the environmentally-friendly plywood were also discussed.%总结国内外环境友好型胶合板用胶改性技术的研究成果,重点分析传统“三醛胶”的改性处理、无甲醛生物质胶黏剂和化学胶黏剂的应用以及单板预处理等可以降低胶合板中甲醛释放量的技术措施,讨论上述处理技术在应用中存在的问题及未来研究方向.

  13. 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.

  14. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    Energy Technology Data Exchange (ETDEWEB)

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  15. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

    Science.gov (United States)

    Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

    2012-04-10

    Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

  16. Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

    Directory of Open Access Journals (Sweden)

    Hyun-Il Shin

    2013-10-01

    Full Text Available Turnip yellow mosaic virus (TYMV is a positive strand RNAvirus. We have modified TYMV coat protein (CP by inserting ac-Myc epitope peptide at the N- or C-terminus of the CP, andhave examined its effect on assembly. We introduced therecombinant CP constructs into Nicotiana benthamiana leavesby agroinfiltration. Examination of the leaf extracts by agarosegel electrophoresis and Western blot analysis showed that theCP modified at the N-terminus produced a band co-migratingwith wild-type virions. With C-terminal modification, however,the detected bands moved faster than the wild-type virions. Tofurther examine the effect, TYMV constructs producing themodified CPs were prepared. With N-terminal modification,viral RNAs were protected from RNase A. In contrast, the viralRNAs were not protected with C-terminal modification.Overall, the results suggest that virion assembly and RNApackaging occur properly when the N-terminus of CP ismodified, but not when the C-terminus is modified. [BMBReports 2013; 46(10: 495-500

  17. Mass Spectrometry Analysis of Lysine Posttranslational Modifications of Tau Protein from Alzheimer's Disease Brain.

    Science.gov (United States)

    Thomas, Stefani N; Yang, Austin J

    2017-01-01

    Recent advances in mass spectrometry (MS)-based proteomics have greatly facilitated the robust identification and quantification of posttranslational modifications (PTMs), including those that are present at substoichiometric site occupancies. The abnormal posttranslational modification and accumulation of the microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD), and it is thought that the primary mode of regulation of tau occurs through PTMs. Several studies have been published regarding tau phosphorylation; however, other tau PTMs such as ubiquitylation, acetylation, methylation, oxidation, sumoylation, nitration, and glycosylation have not been analyzed as extensively. The comprehensive detection and delineation of these PTMs is critical for drug target discovery and validation. Lysine-directed PTMs including ubiquitylation, acetylation, and methylation play key regulatory roles with respect to the rates of tau turnover and aggregation. MS-based analytical approaches have been used to gain insight into the tau lysine-directed PTM signature that is most closely associated with neurofibrillary lesion formation. This chapter provides details pertaining to the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based analysis of the lysine-directed posttranslational modification of tau.

  18. Plasma treated polyethylene grafted with adhesive molecules for enhanced adhesion and growth of fibroblasts.

    Science.gov (United States)

    Rimpelová, Silvie; Kasálková, Nikola Slepičková; Slepička, Petr; Lemerová, Helena; Švorčík, Václav; Ruml, Tomáš

    2013-04-01

    The cell-material interface plays a crucial role in the interaction of cells with synthetic materials for biomedical use. The application of plasma for tailoring polymer surfaces is of abiding interest and holds a great promise in biomedicine. In this paper, we describe polyethylene (PE) surface tuning by Ar plasma irradiating and subsequent grafting of the chemically active PE surface with adhesive proteins or motives to support cell attachment. These simple modifications resulted in changed polymer surface hydrophilicity, roughness and morphology, which we thoroughly characterized. The effect of our modifications on adhesion and growth was tested in vitro using mouse embryonic fibroblasts (NIH 3T3 cell line). We demonstrate that the plasma treatment of PE had a positive effect on the adhesion, spreading, homogeneity of distribution and moderately on proliferation activity of NIH 3T3 cells. This effect was even more pronounced on PE coated with biomolecules.

  19. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    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.

  20. Activated PTHLH Coupling Feedback Phosphoinositide to G-Protein Receptor Signal-Induced Cell Adhesion Network in Human Hepatocellular Carcinoma by Systems-Theoretic Analysis

    Directory of Open Access Journals (Sweden)

    Lin Wang

    2012-01-01

    Full Text Available Studies were done on analysis of biological processes in the same high expression (fold change ≥2 activated PTHLH feedback-mediated cell adhesion gene ontology (GO network of human hepatocellular carcinoma (HCC compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection. Activated PTHLH feedback-mediated cell adhesion network consisted of anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport in HCC. We proposed activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Our hypothesis was verified by the different activated PTHLH feedback-mediated cell adhesion GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network included BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, and VCAN in HCC from GEO dataset using gene regulatory network inference method and our programming.

  1. Underwater adhesion: The barnacle way

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Anil, A.C.

    of substrates in aqueous environment. The adhesive properties of the barnacle adhesive proteins have been utilized for various dental and medical purposes. These polyphenolic proteins are currently in demand as they are non-toxic biomaterial, highly effective...

  2. 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.

  3. 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.

  4. Advances in identification and validation of protein targets of natural products without chemical modification.

    Science.gov (United States)

    Chang, J; Kim, Y; Kwon, H J

    2016-05-04

    Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

  5. REDUCING NONSELECTIVE PROTEIN ADSORPTION AND CELL ADHESION ON POLYACRYLONITRILE FILMS BY COPOLYMERIZATION OF ACRYLONITRILE WITH α-ALLYL GLUCOSIDE

    Institute of Scientific and Technical Information of China (English)

    Rui-qiang Kou; Chao Qu; Zhi-kang Xu; You-yi Xu; Ke Yao

    2003-01-01

    In this work, the surface properties of novel sugar-containing polymers, α-allyl glucoside (AG)/acrylonitrile (AN)copolymers, were studied by contact angle, protein adsorption and cell adhesion measurements. It was found that the contact angle of the copolymer films decreased from 68° to 30° with the increase of AG content in the copolymer. The adsorption amount of bovine serum albumin (BSA) and the adhesive macrophage onto the film surface also decreased significantly with increasing α-allyl glucoside content from 0 to 42 wt% in the copolymer. These preliminary results reveal that both the hydrophilicity and the biocompatibility of polyacrylonitrile-based membranes could be improved by copolymerizing acrylonitrile with vinyl carbohydrates.

  6. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have...... demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown...... of specific focal adhesion proteins previously identified as substrates for this enzyme. We show that stimulation also leads to an increase in calpain activity in these cells. These data support the pivotal role for m-calpain in the control of muscle precursor cell differentiation and thus strengthen the idea...

  7. Study on the Soy Protein-Based Wood Adhesive Modified by Hydroxymethyl Phenol

    Directory of Open Access Journals (Sweden)

    Hong Lei

    2016-07-01

    Full Text Available To explain the reason why using phenol-formaldehyde (PF resin improves the water resistance of soy-based adhesive, the performance of soy-based adhesive cross-linked with hydroxymethyl phenol (HPF and the reaction between HPF and a common dipeptide N-(2-l-alanyl-l-glutamine (AG being used as a model compound were studied in this paper. The DSC and DMA results indicated the reaction between HPF and soy-based adhesive. The soy-based adhesive cross-linked with HPF cured at a lower temperature than the adhesive without HPF. The former showed better mechanical performance and heat resistance than the latter. The ESI-MS, FT-IR and 13C-NMR results proved the reaction between HPF and AG. Because of the existence of branched ether groups in the 13C-NMR results of HPF/AG, the reaction between HPF and AG might mainly happened between hydroxymethyl groups and amino groups under a basic condition.

  8. Mussel-inspired modification of dextran for protein-resistant coatings of titanium oxide.

    Science.gov (United States)

    Park, Jae Yoon; Kim, Jee Seon; Nam, Yoon Sung

    2013-09-12

    Surface modification of inorganic materials to prevent non-specific protein adsorption is critically important for developing a biocompatible materials' platform for medical implantation, diagnostics, and therapeutics. Here we report mussel-inspired chemical modification of dextran for anti-fouling coatings of metal oxide. Catechols are conjugated to dextran via a carbamate ester linkage, producing catechol-grafted dextran with a grafting density of 7.3 mol.%. Titanium dioxide (TiO₂) is coated with the catechol-grafted dextran, and the anti-fouling effect of dextran coatings is examined by using the adsorption of human serum albumin. The mussel-inspired dextran coatings show excellent resistance to non-specific protein adsorption: the adsorption equilibrium constant (K) is 0.69 Lg(-1) for dextran-coated TiO₂ while that for pristine TiO₂ surface is 3.53 Lg(-1). This study suggests that catechol-grafted dextran is a promising material for effective anti-fouling coatings of implantable inorganic materials. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Characterization of a novel posttranslational modification in polypyrimidine tract-binding proteins by SUMO1.

    Science.gov (United States)

    Han, Wei; Wang, Lin; Yin, Bin; Peng, Xiaozhong

    2014-04-01

    Polypyrimidine tract-binding protein 1 (PTBP1) and its brainspecific homologue, PTBP2, are associated with pre-mRNAs and influence pre-mRNA processing, as well as mRNA metabolism and transport. They play important roles in neural differentiation and glioma development. In our study, we detected the expression of the two proteins in glioma cells and predicted that they may be sumoylated using SUMOplot analyses. We confirmed that PTBP1 and PTBP2 can be modified by SUMO1 with co-immunoprecipitation experiments using 293ET cells transiently co-expressing SUMO1 and either PTBP1 or PTBP2. We also found that SUMO1 modification of PTBP2 was enhanced by Ubc9 (E2). The mutation of the sumoylation site (Lys137) of PTBP2 markedly inhibited its modification by SUMO1. Interestingly, in T98G glioma cells, the level of sumoylated PTBP2 was reduced compared to that of normal brain cells. Overall, this study shows that PTBP2 is posttranslationally modified by SUMO1.

  11. Surface modification on silicon with chitosan and biological research

    Energy Technology Data Exchange (ETDEWEB)

    Lue Xiaoying; Cui Wei; Huang Yan; Zhao Yi [State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096 (China); Wang Zhigong, E-mail: luxy@seu.edu.c [Institute of RF- and OE-ICs, Southeast University, Nanjing, 210096 (China)

    2009-08-15

    The aim of the present study was to investigate the effect of chitosan modification of silicon (Si) on protein adsorption, cell adhesion and cell proliferation. Chitosan was first immobilized on the Si surface through a (3-aminopropyl)triethoxysilane (APTES) bridge. The surface was then characterized by contact angle measurement, atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS) and energy dispersive x-ray spectroscopy (EDX). The amount of protein adsorbed on the native Si and chitosan-modified Si surface was evaluated by a modified Coomassie brilliant blue (CBB) protein assay. The adhesion and proliferation behavior of L-929 and pc12 cells were then assessed by microscopy and methylthiazoltetrazolium (MTT) tests. The results showed that the chitosan modification could resist protein adsorption and inhibit the adhesion and proliferation of two kinds of cells on Si.

  12. 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

    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...... 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...... eDNA and proteins are the most important adhesins for initiating S. aureus biofilms. S. epidermidis was strongly affected by all enzyme treatments, which in addition to impairing adhesion to glass, also prevented the formation of aggregates and streamers observed abundantly in control samples. One...

  13. Adhesions of extracellular surface-layer associated proteins in Lactobacillus M5-L and Q8-L.

    Science.gov (United States)

    Zhang, Yingchun; Xiang, Xinling; Lu, Qianhui; Zhang, Lanwei; Ma, Fang; Wang, Linlin

    2016-02-01

    Surface-layer associated proteins (SLAP) that envelop Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L cell surfaces are involved in the adherence of these strain to the human intestinal cell line HT-29. To further elucidate some of the properties of these proteins, we assessed the yields and expressions of SLAP under different incubation conditions. An efficient and selective extraction of SLAP was obtained when cells of Lactobacillus were treated with 5 M LiCl at 37°C in aerobic conditions. The SLAP of Lactobacillus M5-L and Q8-L in cell extracts were visualized by SDS-PAGE and identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin-labeled HT-29 cells as adhesion proteins. Atomic force microscopy contact imaging revealed that Lactobacillus strains M5-L and Q8-L normally display a smooth, homogeneous surface, whereas the surfaces of M5-L and Q8-L treated with 5 M LiCl were rough and more heterogeneous. Analysis of adhesion forces revealed that the initial adhesion forces of 1.41 and 1.28 nN obtained for normal Lactobacillus M5-L and Q8-L strains, respectively, decreased to 0.70 and 0.48 nN, respectively, following 5 M LiCl treatment. Finally, the dominant 45-kDa protein bands of Lactobacillus Q8-L and Lactobacillus M5-L were identified as elongation factor Tu and surface antigen, respectively, by liquid chromatography-tandem mass spectrometry.

  14. The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

    Science.gov (United States)

    Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

    2017-01-01

    Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut. PMID:28281568

  15. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  16. The influence of cross-linking on protein-protein interactions in a marine adhesive: the case of two byssus plaque proteins from the blue mussel.

    Science.gov (United States)

    Fant, Camilla; Elwing, Hans; Höök, Fredrik

    2002-01-01

    The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was further investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO(4)), Cu(2+), or mushroom tyrosinase. With this coupling strategy it is shown that a requirement for attraction between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp-1 as well as for the subsequent binding of Mefp-2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.

  17. 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 PTM1 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. PMID:25389185

  18. 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.

  19. Development and validation of a method for profiling post-translational modification activities using protein microarrays.

    Directory of Open Access Journals (Sweden)

    Sonia V Del Rincón

    Full Text Available BACKGROUND: Post-translational modifications (PTMs impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens. METHODOLOGY/PRINCIPAL FINDINGS: In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8, ATP regenerating system, and other required components (depending on the assay that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay. CONCLUSIONS/SIGNIFICANCE: This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.

  20. 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

  1. Phosphoproteome Reveals an Atlas of Protein Signaling Networks During Osteoblast Adhesion

    NARCIS (Netherlands)

    Milani, Renato; Ferreira, Carmen V.; Granjeiro, Jose M.; Paredes-Gamero, Edgar J.; Silva, Rodrigo A.; Justo, Giselle Z.; Nader, Helena B.; Galembeck, Eduardo; Peppelenbosch, Maikel P.; Aoyama, Hiroshi; Zambuzzi, Willian F.

    2010-01-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important r

  2. Phosphoproteome Reveals an Atlas of Protein Signaling Networks During Osteoblast Adhesion

    NARCIS (Netherlands)

    Milani, Renato; Ferreira, Carmen V.; Granjeiro, Jose M.; Paredes-Gamero, Edgar J.; Silva, Rodrigo A.; Justo, Giselle Z.; Nader, Helena B.; Galembeck, Eduardo; Peppelenbosch, Maikel P.; Aoyama, Hiroshi; Zambuzzi, Willian F.

    2010-01-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important

  3. Differential Impact of Plasma Proteins on the Adhesion Efficiency of Vascular-Targeted Carriers (VTCs) in Blood of Common Laboratory Animals.

    Science.gov (United States)

    Namdee, Katawut; Sobczynski, Daniel J; Onyskiw, Peter J; Eniola-Adefeso, Omolola

    2015-12-16

    Vascular-targeted carrier (VTC) interaction with human plasma is known to reduce targeted adhesion efficiency in vitro. However, the role of plasma proteins on the adhesion efficiency of VTCs in laboratory animals remains unknown. Here, in vitro blood flow assays are used to explore the effects of plasma from mouse, rabbit, and porcine on VTC adhesion. Porcine blood exhibited a strong negative plasma effect on VTC adhesion while no significant plasma effect was found with rabbit and mouse blood. A brush density poly(ethylene glycol) (PEG) on VTCs was effective at improving adhesion of microsized, but not nanosized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as better models in preclinical research for predicting the in vivo functionality of VTCs for use in humans. These considerations hold great importance for the design of various pharmaceutical products and development of reliable drug delivery systems.

  4. Surface Modification of Poly(dimethylsiloxane) Using Ionic Complementary Peptides to Minimize Nonspecific Protein Adsorption.

    Science.gov (United States)

    Yu, Xiaoling; Xiao, Junzhu; Dang, Fuquan

    2015-06-02

    Poly(dimethylsiloxane) (PDMS) has become a widely used material for microfluidic and biological applications. However, PDMS has unacceptably high levels of nonspecific protein adsorption, which significantly lowers the performance of PDMS-based microfluidic chips. Most existing methods to reduce protein fouling of PDMS are to make the surface more hydrophilic by surface oxidization, polymer grafting, and physisorbed coatings. These methods suffer from the relatively short-term stability, the multistep complex treatment procedure, or the insufficient adsorption reduction. Herein, we developed a novel and facile modification method based on self-assembled peptides with well-tailored amino acid composition and sequence, which can also interact strongly with the PDMS surface in the same way as proteins, for suppressing the nonspecific protein fouling and improving the biocompatibility of PDMS-based microfluidic chips. We first demonstrated that an ionic complementary peptide, EAR16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2], can readily self-assemble into an amphipathic film predominantly composed of tightly packed β-sheets on the native hydrophobic and plasma-oxidized hydrophilic PDMS surfaces upon low concentrations of carbohydrates. The self-assembled EAR16-II amphipathic film exposed its hydrophobic side to the solution and thus rendered the PDMS surface hydrophobic with water contact angles (WCAs) of around 110.0°. However, the self-assembled EAR16-II amphipathic film exhibited excellent protein-repelling and blood compatibility properties comparable to or better than those obtained with previously reported methods. A schematic model has been proposed to explain the interactions of EAR16-II with the PDMS surface and the antifouling capability of EAR16-II coatings at a molecular level. The current work will pave the way to the development of novel coating materials to address the nonspecific protein adsorption on PDMS, thereby broadening the

  5. Modifications and oxidation of lipids and proteins in human serum detected by thermochemiluminescence.

    Science.gov (United States)

    Shnizer, Sergei; Kagan, Tamara; Lanir, Amos; Maor, Irit; Reznick, Abraham Z

    2003-01-01

    Detection of electronically excited species (EES) in body fluids may constitute an important diagnostic tool in various pathologies. Examples of such products are triplet excited carbonyls (TEC), which can be a source for photon emission in the 400-550 nm range. The aim of the present study was to determine the actual contribution of lipid and protein components (protein carbonyls) to photon emission generated by thermochemiluminescence (TCL) during the heating of biological fluids. In this study, a new TCL Photometer device, designed by Lumitest Ltd, Israel, was used. Samples were heated to a constant temperature of 80 +/- 0.5 degrees C for 280 s and photon emission was measured at several time points. In order to compare the results of TCL measurements to conventional methods of detecting lipid and protein oxidation, each examined sample was also heated in a waterbath at 80 degrees C for 10-280 s. Lipid and protein oxidation were subsequently measured using conventional methods. The TCL of four polyunsaturated fatty acids (PUFA) with three to six double bonds was measured. The elevation of the PUFA TCL amplitude correlated with the increase in the number of double bonds of PUFA. A correlation between the increase in TCL intensity and protein carbonyl generation in bovine serum albumin (BSA) was also observed. In the venous blood serum, our study showed that an increase of TCL intensity during heating reflected the cleavage of TEC of lipid origin. Our study suggests that biological molecules such as proteins, lipids and other molecules, which may become unstable during heating, are capable of generating EES. We demonstrated that a TCL curve can be used as a kinetic model for measuring oxidative processes, which reflects modifications of different molecules involved in the oxidative stress phenomena.

  6. 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

    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.

  7. 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

  8. β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

    Science.gov (United States)

    Maiorova, Mariia A; Odintsova, Nelly A

    2015-08-01

    In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion.

  9. The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein.

    Science.gov (United States)

    Burzio, L A; Saéz, C; Pardo, J; Waite, J H; Burzio, L O

    2000-06-15

    The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure.

  10. Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

    Science.gov (United States)

    Alcaide, María; Papaioannou, Stavros; Taylor, Andrew; Fekete, Ladislav; Gurevich, Leonid; Zachar, Vladimir; Pennisi, Cristian Pablo

    2016-05-01

    Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.

  11. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

    Science.gov (United States)

    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  12. 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

  13. Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeongkwon; Zhang, Rui; Strittmatter, Eric F.; Smith, Richard D.; Zand, Robert

    2008-07-11

    Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the posttranslational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial.

  14. Quantification of protein posttranslational modifications using stable isotope and mass spectrometry I: principles and applications.

    Science.gov (United States)

    Jiang, Xinzhao Grace; Apostol, Izydor; Luo, Quanzhou; Lewis, Jeffrey; Keener, Ronald; Luo, Shun; Jerums, Matthew; Zhang, Xin; Wypych, Jette; Huang, Gang

    2012-02-15

    With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where ¹⁵N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of ¹⁵N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm.

  15. Modification of foaming properties of soy protein isolate by high ultrasound intensity: Particle size effect.

    Science.gov (United States)

    Morales, Rocío; Martínez, Karina D; Pizones Ruiz-Henestrosa, Víctor M; Pilosof, Ana M R

    2015-09-01

    The effect of high intensity ultrasound (HIUS) may produce structural modifications on proteins through a friendly environmental process. Thus, it can be possible to obtain aggregates with a determined particle size, and altering a defined functional property at the same time. The objective of this work was to explore the impact of HIUS on the functionality of a denatured soy protein isolate (SPI) on foaming and interfacial properties. SPI solutions at pH 6.9 were treated with HIUS for 20 min, in an ultrasonic processor at room temperature, at 75, 80 and 85°C. The operating conditions were: 20 kHz, 4.27 ± 0.71 W and 20% of amplitude. It was determined the size of the protein particles, before and after the HIUS treatment, by dynamic light scattering. It was also analyzed the interfacial behavior of the different systems as well as their foaming properties, by applying the whipping method. The HIUS treatment and HIUS with temperature improved the foaming capacity by alteration of particle size whereas stability was not modified significantly. The temperature of HIUS treatment (80 and 85°C) showed a synergistic effect on foaming capacity. It was found that the reduction of particle size was related to the increase of foaming capacity of SPI. On the other hand, the invariable elasticity of the interfacial films could explain the stability of foams over time.

  16. Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis.

    Science.gov (United States)

    Qin, C; Baba, O; Butler, W T

    2004-06-04

    The extracellular matrix (ECM) of bone and dentin contains several non-collagenous proteins. One category of non-collagenous protein is termed the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, that includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix