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Sample records for profiling protein thiol

  1. Protein Thiols as an Indication of Oxidative Stress

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    Yousef Rezaei Chianeh

    2014-06-01

    Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

  2. Multiplexed Thiol Reactivity Profiling for Target Discovery of Electrophilic Natural Products.

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    Tian, Caiping; Sun, Rui; Liu, Keke; Fu, Ling; Liu, Xiaoyu; Zhou, Wanqi; Yang, Yong; Yang, Jing

    2017-11-16

    Electrophilic groups, such as Michael acceptors, expoxides, are common motifs in natural products (NPs). Electrophilic NPs can act through covalent modification of cysteinyl thiols on functional proteins, and exhibit potent cytotoxicity and anti-inflammatory/cancer activities. Here we describe a new chemoproteomic strategy, termed multiplexed thiol reactivity profiling (MTRP), and its use in target discovery of electrophilic NPs. We demonstrate the utility of MTRP by identifying cellular targets of gambogic acid, an electrophilic NP that is currently under evaluation in clinical trials as anticancer agent. Moreover, MTRP enables simultaneous comparison of seven structurally diversified α,β-unsaturated γ-lactones, which provides insights into the relative proteomic reactivity and target preference of diverse structural scaffolds coupled to a common electrophilic motif and reveals various potential druggable targets with liganded cysteines. We anticipate that this new method for thiol reactivity profiling in a multiplexed manner will find broad application in redox biology and drug discovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Fast and Selective Modification of Thiol Proteins/Peptides by N-(Phenylseleno)phthalimide

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    Wang, Zhengfang; Zhang, Yun; Zhang, Hao; Harrington, Peter B.; Chen, Hao

    2012-03-01

    We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1-2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiol peptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri- n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.

  4. Orented immobilization of farnesylated proteins by the thiol-ene reaction

    NARCIS (Netherlands)

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

    2010-01-01

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

  5. Proteomic detection of oxidized and reduced thiol proteins in cultured cells.

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    Cuddihy, Sarah L; Baty, James W; Brown, Kristin K; Winterbourn, Christine C; Hampton, Mark B

    2009-01-01

    The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

  6. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

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    Aslı Neslihan Avan

    2016-08-01

    Full Text Available Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET-based total antioxidant capacity (TAC assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC, and ferric reducing antioxidant power (FRAP, were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol and (phenol + protein mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II compounds were added to stabilize the thiol components in the form of Hg(II-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols mixtures.

  7. Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

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    Terrill, Jessica R; Radley-Crabb, Hannah G; Iwasaki, Tomohito; Lemckert, Frances A; Arthur, Peter G; Grounds, Miranda D

    2013-09-01

    The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice. © 2013 The Authors Journal compilation © 2013 FEBS.

  8. Differential Labeling of Free and Disulfide-Bound Thiol Functions in Proteins

    NARCIS (Netherlands)

    Seiwert, B.; Hayen, H.; Karst, U.

    2008-01-01

    A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid

  9. Peptidoglycan recognition proteins kill bacteria by inducing oxidative, thiol, and metal stress.

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    Des Raj Kashyap

    2014-07-01

    Full Text Available Mammalian Peptidoglycan Recognition Proteins (PGRPs are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS, a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.

  10. Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Østergaard, Henrik; Nørgaard, Per

    2007-01-01

    Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify...... the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient...... sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high...

  11. Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels

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    M. Ryan Smith

    2016-08-01

    Full Text Available Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP, decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231 breast adenocarcinoma cells up to 6 days after an initial 24 h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10 µM of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC protein levels, although other protein levels were

  12. Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

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    Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

    2015-01-01

    Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

  13. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    International Nuclear Information System (INIS)

    Ou Wu; Silver, Jonathan

    2006-01-01

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

  14. The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

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    Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

    2018-04-01

    Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

  15. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

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    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

    2015-08-25

    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

  16. Modification of nanoelectrode ensembles by thiols and disulfides to prevent non specific adsorption of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Silvestrini, M. [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy); Schiavuta, P.; Scopece, P. [Associazione CIVEN, via delle Industrie 5, 30175 Marghera - Venice (Italy); Pecchielan, G.; Moretto, L.M. [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy); Ugo, P., E-mail: ugo@unive.it [Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Santa Marta 2137, 30123 Venice (Italy)

    2011-09-01

    Highlights: > Complex nanostructures are built on the gold surface of ensembles of nanoelectrodes. > Gold surface of nanoelectrodes was functionalized with SAM of organic sulphurs. > The polycarbonate surrounding nanoelectrodes was functionalized with proteins. > SAMs protect the nanoelectrodes from undesired proteins adsorption. - Abstract: The possibility to functionalize selectively with thiols or disulfides the surface of the gold nanoelectrodes of polycarbonate templated nanoelectrode ensembles (NEEs) is studied. It is shown that the Au nanoelectrodes can be coated by a self assembled monolayer (SAM) of thioctic acid (TA) or 2-mercaptoethanesulfonic (MES) acid. The study of the electrochemical behavior of SAM-modified NEEs by cyclic voltammetry (CV) at different solution pH, using ferrocenecarboxylate as an anionic redox probe (FcCOO{sup -}) and (ferrocenylmethyl)trimethylammonium (FA{sup +}) as a cationic redox probe, demonstrate that the SAM-modified nanoelectrodes are permselective, in that only cationic or neutral probes can access the SAM-coated nanoelectrode surface. CV, AFM and FTIR-ATR data indicate that proteins such as casein or bovine serum albumin, which are polyanionic at pH 7, adsorb on the surface of NEEs untreated with thiols, tending to block the electron transfer of the ferrocenyl redox probes. On the contrary, the pre-treatment of the NEE with an anionic SAM protects the nanoelectrodes from protein fouling, allowing the detection of well shaped voltammetric patterns for the redox probe. Experimental results indicate that, in the case of MES treated NEEs, the protein is bound only onto the polycarbonate surface which surrounds the nanoelectrodes, while the tips of the gold nanoelectrodes remain protein free.

  17. Modification of nanoelectrode ensembles by thiols and disulfides to prevent non specific adsorption of proteins

    International Nuclear Information System (INIS)

    Silvestrini, M.; Schiavuta, P.; Scopece, P.; Pecchielan, G.; Moretto, L.M.; Ugo, P.

    2011-01-01

    Highlights: → Complex nanostructures are built on the gold surface of ensembles of nanoelectrodes. → Gold surface of nanoelectrodes was functionalized with SAM of organic sulphurs. → The polycarbonate surrounding nanoelectrodes was functionalized with proteins. → SAMs protect the nanoelectrodes from undesired proteins adsorption. - Abstract: The possibility to functionalize selectively with thiols or disulfides the surface of the gold nanoelectrodes of polycarbonate templated nanoelectrode ensembles (NEEs) is studied. It is shown that the Au nanoelectrodes can be coated by a self assembled monolayer (SAM) of thioctic acid (TA) or 2-mercaptoethanesulfonic (MES) acid. The study of the electrochemical behavior of SAM-modified NEEs by cyclic voltammetry (CV) at different solution pH, using ferrocenecarboxylate as an anionic redox probe (FcCOO - ) and (ferrocenylmethyl)trimethylammonium (FA + ) as a cationic redox probe, demonstrate that the SAM-modified nanoelectrodes are permselective, in that only cationic or neutral probes can access the SAM-coated nanoelectrode surface. CV, AFM and FTIR-ATR data indicate that proteins such as casein or bovine serum albumin, which are polyanionic at pH 7, adsorb on the surface of NEEs untreated with thiols, tending to block the electron transfer of the ferrocenyl redox probes. On the contrary, the pre-treatment of the NEE with an anionic SAM protects the nanoelectrodes from protein fouling, allowing the detection of well shaped voltammetric patterns for the redox probe. Experimental results indicate that, in the case of MES treated NEEs, the protein is bound only onto the polycarbonate surface which surrounds the nanoelectrodes, while the tips of the gold nanoelectrodes remain protein free.

  18. Relationship of non-protein thiol pools and accumulated Cd or Hg in the marine macrophyte Posidonia oceanica (L.) Delile

    International Nuclear Information System (INIS)

    Maserti, B.E.; Ferrillo, V.; Avdis, O.; Nesti, U.; Di Garbo, A.; Catsiki, A.; Maestrini, P.L.

    2005-01-01

    The accumulation of cadmium or mercury and the effect of these elements on the levels of non-protein thiols in the blades of the marine macrophyte Posidonia oceanica were investigated. A significant accumulation of cadmium or mercury, dependent on metal concentration supplied, was observed in metal-treated blades. In the blades treated either with cadmium or mercury, a significant increase in the levels of non-protein thiols (other than glutathione) and a marked depletion of the reduced glutathione content as a function of the metal, exposure time and metal concentration supplied were found. This investigation provides first experimental report on the relationship between non-protein thiol pools and accumulated cadmium or mercury in P. oceanica

  19. Quantification of protein-derived thiols during atmosphere-controlled brewing in laboratory scale

    DEFF Research Database (Denmark)

    Murmann, Anne Nordmark; Andersen, Preben; Mauch, Alexander

    2016-01-01

    . Fermentation caused an increase in free thiols, and the balance between free and total thiols was shifted toward a higher degree of free thiols. This was explained by either a reducing effect of fermentation or secretion of thiol-containing compounds from yeast. The efficiency of sulfite to reduce reversibly...... was more pronounced at longer incubation times. However, the reduction of the pool of oxidized thiols by sulfite was inefficient for sulfite concentrations typically found in beer, and the reaction was found to be relatively slow compared with reduction by tris(carboxyethyl)phosphine....

  20. Modulating the physicochemical and structural properties of gold-functionalized protein nanotubes through thiol surface modification.

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    Carreño-Fuentes, Liliana; Plascencia-Villa, Germán; Palomares, Laura A; Moya, Sergio E; Ramírez, Octavio T

    2014-12-16

    Biomolecules are advantageous scaffolds for the synthesis and ordering of metallic nanoparticles. Rotavirus VP6 nanotubes possess intrinsic affinity to metal ions, a property that has been exploited to synthesize gold nanoparticles over them. The resulting nanobiomaterials have unique properties useful for novel applications. However, the formed nanobiomaterials lack of colloidal stability and flocculate, limiting their functionality. Here we demonstrate that it is possible to synthesize thiol-protected gold nanoparticles over VP6 nanotubes, which resulted in soluble nanobiomaterials. With this strategy, it was possible to modulate the size, colloidal stability, and surface plasmon resonance of the synthesized nanoparticles by controlling the content of the thiolated ligands. Two types of water-soluble ligands were tested, a small linear ligand, sodium 3-mercapto-1-propanesulfonate (MPS), and a bulky ligand, 5-mercaptopentyl β-D-glucopyranoside (GlcC5SH). The synthesized nanobiomaterials had a higher stability in suspension, as determined by Z-potential measurements. To the extent of our knowledge, this is the first time that a rational strategy is developed to modulate the particular properties of metal nanoparticles in situ synthesized over a protein bioscaffold through thiol coating, achieving a high spatial and structural organization of nanoparticles in a single integrative hybrid structure.

  1. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Kwapiszewski, Radoslaw; Jensen, Thomas Glasdam

    2012-01-01

    ” and “ene” monomers present in the microfluidic chip bulk material provides a simple and efficient way of tuning the chip’s surface chemistry. Here, thiol-ene chips displaying an excess of functional thiol groups at their surfaces are functionalized with biotin and streptavidin in a controlled fashion using...

  2. High-energy intermediates in protein unfolding characterized by thiol labeling under nativelike conditions.

    Science.gov (United States)

    Malhotra, Pooja; Udgaonkar, Jayant B

    2014-06-10

    A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.

  3. Thiol oxidation of hemolymph proteins in oysters Crassostrea brasiliana as markers of oxidative damage induced by urban sewage exposure.

    Science.gov (United States)

    Trevisan, Rafael; Flores-Nunes, Fabrício; Dolores, Euler S; Mattos, Jacó J; Piazza, Clei E; Sasaki, Sílvio T; Taniguchi, Satie; Montone, Rosalinda C; Bícego, Márcia C; Dos Reis, Isis M M; Zacchi, Flávia L; Othero, Bárbara N M; Bastolla, Camila L V; Mello, Danielle F; Fraga, Ana Paula M; Wendt, Nestor; Toledo-Silva, Guilherme; Razzera, Guilherme; Dafre, Alcir L; de Melo, Cláudio M R; Bianchini, Adalto; Marques, Maria R F; Bainy, Afonso C D

    2017-07-01

    Urban sewage is a concerning issue worldwide, threatening both wildlife and human health. The present study investigated protein oxidation in mangrove oysters (Crassostrea brasiliana) exposed to seawater from Balneário Camboriú, an important tourist destination in Brazil that is affected by urban sewage. Oysters were exposed for 24 h to seawater collected close to the Camboriú River (CAM1) or 1 km away (CAM2). Seawater from an aquaculture laboratory was used as a reference. Local sewage input was marked by higher levels of coliforms, nitrogen, and phosphorus in seawater, as well as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), linear alkylbenzenes (LABs), and fecal steroid in sediments at CAM1. Exposure of oysters to CAM1 caused marked bioaccumulation of LABs and decreased PAH and PCB concentrations after exposure to both CAM1 and CAM2. Protein thiol oxidation in gills, digestive gland, and hemolymph was evaluated. Lower levels of reduced protein thiols were detected in hemolymph from CAM1, and actin, segon, and dominin were identified as targets of protein thiol oxidation. Dominin susceptibility to oxidation was confirmed in vitro by exposure to peroxides and hypochlorous acid, and 2 cysteine residues were identified as potential sites of oxidation. Overall, these data indicate that urban sewage contamination in local waters has a toxic potential and that protein thiol oxidation in hemolymph could be a useful biomarker of oxidative stress in bivalves exposed to contaminants. Environ Toxicol Chem 2017;36:1833-1845. © 2016 SETAC. © 2016 SETAC.

  4. N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis.

    Science.gov (United States)

    Terrill, Jessica R; Radley-Crabb, Hannah G; Grounds, Miranda D; Arthur, Peter G

    2012-05-01

    Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Exploring the Lean Phenotype of Glutathione-Depleted Mice: Thiol, Amino Acid and Fatty Acid Profiles.

    Directory of Open Access Journals (Sweden)

    Amany K Elshorbagy

    Full Text Available Although reduced glutathione (rGSH is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys is positively associated with stearoyl-coenzyme A desaturase (SCD activity and adiposity in humans and animal models.To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice.Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis.Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001, and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH and rGSH (by ~70%, and liver tGSH (by 82%. Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all, but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all.Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.

  6. In vitro glutathione peroxidase mimicry of ebselen is linked to its oxidation of critical thiols on key cerebral suphydryl proteins - A novel component of its GPx-mimic antioxidant mechanism emerging from its thiol-modulated toxicology and pharmacology.

    Science.gov (United States)

    Kade, I J; Balogun, B D; Rocha, J B T

    2013-10-25

    The antioxidant mechanism of ebselen in rats brain is largely linked with its glutathione peroxidase (GPx) rather than its peroxiredoxin mimicry ability. However, the precise molecular dynamics between the GPx-mimicry of ebselen and thiol utilization is yet to be fully clarified and thus still open. Herein, we investigated the influence of dithiothreitol (DTT) on the antioxidant action of ebselen against oxidant-induced cerebral lipid peroxidation and deoxyribose degradation. Furthermore, the critical inhibitory concentrations of ebselen on the activities of sulphydryl enzymes such as cerebral sodium pump, δ-aminolevulinic acid dehydratase (δ-ALAD) and lactate dehydrogenase (LDH) were also investigated. We observe that ebselen (at ≥42 μM) markedly inhibited lipid peroxidation in the presence and absence of DTT, whereas it inhibited deoxyribose degradation only in the presence of DTT. Furthermore, under in vitro conditions, ebselen inhibited the thiol containing enzymes; cerebral sodium pump (at ≥40 μM), δ-ALAD (≥10 μM) and LDH (≥1 μM) which were either prevented or reversed by DTT. However, the inhibition of the activities of these sulphydryl proteins in diabetic animals was prevented by ebselen. Summarily, it is apparent that the effective in vitro inhibitory doses of ebselen on the activity of the sulphydryl proteins are far less than its antioxidant doses. In addition, the presence of DTT is evidently a critical requirement for ebselen to effect its antioxidant action against deoxyribose degeradation and not lipid peroxidation. Consequently, we conclude that ebselen possibly utilizes available thiols on sulphydryl proteins to effect its GPx mimicry antioxidant action against lipid peroxidation in rat brain homogenate. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

    Science.gov (United States)

    Zanotto-Filho, Alfeu; Masamsetti, V Pragathi; Loranc, Eva; Tonapi, Sonal S; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C F; Chen, Yidong; Bishop, Alexander J R

    2016-12-01

    Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR. ©2016 American Association for Cancer Research.

  8. Alkylating agent induced NRF2 blocks endoplasmic reticulum stress-mediated apoptosis via control of glutathione pools and protein thiol homeostasis

    Science.gov (United States)

    Zanotto-Filho, Alfeu; Masamsetti, V. Pragathi; Loranc, Eva; Tonapi, Sonal S.; Gorthi, Aparna; Bernard, Xavier; Gonçalves, Rosângela Mayer; Moreira, José C. F.; Chen, Yidong; Bishop, Alexander J. R.

    2016-01-01

    Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival; an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine (BSO) caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine (NAC) or glutathione-ethyl-ester (GSH-E), decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 targets overexpression and poor survival. In KEAP1 mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. PMID:27638861

  9. Global Structural Flexibility of Metalloproteins Regulates Reactivity of Transition Metal Ion in the Protein Core: An Experimental Study Using Thiol-subtilisin as a Model Protein.

    Science.gov (United States)

    Matsuo, Takashi; Kono, Takamasa; Shobu, Isamu; Ishida, Masaya; Gonda, Katsuya; Hirota, Shun

    2018-02-21

    The functions of metal-containing proteins (metalloproteins) are determined by the reactivities of transition metal ions at their active sites. Because protein macromolecular structures have several molecular degrees of freedom, global structural flexibility may also regulate the properties of metalloproteins. However, the influence of this factor has not been fully delineated in mechanistic studies of metalloproteins. Accordingly, we have investigated the relationship between global protein flexibility and the characteristics of a transition metal ion in the protein core using thiol-subtilisin (tSTL) with a Cys-coordinated Cu 2+ ion as a model system. Although tSTL has two Ca 2+ -binding sites, the Ca 2+ -binding status hardly affects its secondary structure. Nevertheless, guanidinium-induced denaturation and amide H/D exchange indicated the increase in the structural flexibility of tSTL by the removal of bound Ca 2+ ions. Electron paramagnetic resonance and absorption spectral changes have revealed that the protein flexibility determines the characteristics of a Cu 2+ ion in tSTL. Therefore, global protein flexibility should be recognized as an important factor that regulates the properties of metalloproteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Effect of the cooking method (grilling, roasting, frying and sous-vide) on the oxidation of thiols, tryptophan, alkaline amino acids and protein cross-linking in jerky chicken.

    Science.gov (United States)

    Silva, Fábio A P; Ferreira, Valquíria C S; Madruga, Marta S; Estévez, Mario

    2016-08-01

    Broiler breast ( pectoralis major ) meat was submitted to salting with NaCl + NaNO 3 followed by a drying process to produce jerky-type chicken. The final product (raw broiler charqui) was desalted and then cooked using grilled, roasted, fried and sous-vide techniques. Sous-vide cooked samples showed lowest results of moisture loss compared to roasted and fried ones. Fatty acid profile suffered minor changes after cooking of broiler charqui. Regarding to protein oxidation, tryptophan fluorescence, protein carbonylation and disulphide bonds formation of chicken charqui were affected by cooking temperature while free thiol groups, Schiff base formation and hardness were mostly impacted by the length of cooking. Instrumental color of broiler charqui was affected by the type of cooking, being closely related with Maillard products formation. In conclusion, sous-vide technique seems to be the most advantageous cooking method to obtain high-quality ready-to-eat chicken charqui.

  11. Selection of protease for increased solubilization of protein-derived thiols during mashing with limited release of free amino acids in beer

    DEFF Research Database (Denmark)

    Murmann, Anne Nordmark; Lunde, Christina; Lund, Marianne Nissen

    2016-01-01

    Extraction of protein-derived thiols by protease treatment during mashing for improvement of flavor stability in beer has previously been shown to cause concomitant increase in free amino acid concentrations and thereby increased levels of unwanted Maillard reaction products during aging. The pre......Extraction of protein-derived thiols by protease treatment during mashing for improvement of flavor stability in beer has previously been shown to cause concomitant increase in free amino acid concentrations and thereby increased levels of unwanted Maillard reaction products during aging...... of a protease with a higher temperature optimum dosed at only 3 mg of enzyme/kg of malt, it is possible to increase thiol concentrations in wort by 30% and with only a maximum 10% increase in amino acid concentration compared with a control. Pilot brewing showed that beer brewed with addition of protease...... stability during storage could not be evaluated. Overall, similar brewing and sensory characteristics were obtained compared with a control beer brewed without addition of protease. Foam stability was decreased by protease treatment, and formation of haze was reduced by protease treatment....

  12. PROFIL PROTEIN SUSU DAN PRODUK OLAHANNYA

    Directory of Open Access Journals (Sweden)

    R. Susanti

    2017-03-01

    Full Text Available Penelitian ini bertujuan untuk menganalisis kadar protein dan profil protein pada beberapa susu (susu kedelai, susu kambing dan olahannya (yogurt, tofu. Kadar protein diukur dengan metode Lowry, sedangkan profil protein dianalisis menggunakan SDS PAGE. Data yang diperoleh dianalisis secara deskriptif. Kadar protein tertinggi pada sampel yang dianalisis terdapat pada produk yogurt A (579,5 mg/ml, disusul susu kedelai (289,99 mg/ml dan susu kambing (133,1 mg/ml. Analisis profil protein terlihat pita protein dengan mobilitas terendah sampai tertinggi terletak pada berat molekul 14-150 KDa. Pita protein khas yang hanya dimiliki susu kambing adalah pita 150kDa. Sementara pita protein khas yang hanya dimiliki susu kedelai adalah pita 44 kDa dan 55kDa. Pita protein yang khas hanya dimiliki yogurt A (dengan bakteri Lactobacillus bulgaricus dan Streptococcus thermophillus adalah pita 65Da. Semua jenis susu dan olahannya memiliki pita 70kDa, kecuali susu kedelai. Profil protein susu kedelai dan tofu menunjukkan profil protein yang sangat berbeda, namun keduanya memiliki pita 18kDa.This study aimed to observe protein level and profiles on some milks (soy milk, goat's milk and dairy (yogurt, tofu product. Protein content was observed by Lowry method, whereas the protein profiles were analyzed by polyacrylamide gel electrophoresis. Data were analyzed descriptively. The highest protein content of the observed sample was in yogurt A products (579,5 mg/ml, followed by soy milk (289,99 mg/ml and goat's milk (133,1 mg/ml. Analysis of protein profiles showed protein bands with lowest to highest mobility lies in the molecular weight of 14-150 KDa. Typical protein band of goat's milk was a 150kDa band. While the typical protein bands of soy milk were 44 kDa and 55kDa band. The typical protein band of yogurt A (with Lactobacillus bulgaricus and Streptococcus thermophillus bacterium was 65Da. All types of milks and dairy had 70kDa band, except for soy milk. Protein

  13. Quantification of thiols and disulfides

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Thorpe, Colin

    2014-01-01

    lengths to regulate thiol-disulfide bond homeostasis, typically with several, apparently redundant, systems working in parallel. Dissecting the extent of oxidation and reduction of disulfides is an ongoing challenge due, in part, to the facility of thiol/disulfide exchange reactions.......Disulfide bond formation is a key posttranslational modification, with implications for structure, function and stability of numerous proteins. While disulfide bond formation is a necessary and essential process for many proteins, it is deleterious and disruptive for others. Cells go to great...

  14. Hypochlorite-induced oxidation of thiols

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan; Hawkins, C L

    2000-01-01

    -molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion......-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative...... to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented...

  15. Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

    Science.gov (United States)

    Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

    2013-01-01

    Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

  16. Quantifying the global cellular thiol-disulfide status

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Roth, Doris; Winther, Jakob R

    2009-01-01

    It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data...... cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active...

  17. Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

    Directory of Open Access Journals (Sweden)

    Kuljit Singh

    2017-08-01

    Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

  18. Total Thiols: Biomedical Importance And Their Alteration In Various Disorders

    Directory of Open Access Journals (Sweden)

    Mungli Prakash

    2009-09-01

    Full Text Available Thiols are the organic compounds that contain a sulphydryl group. Among all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and they play a significant role in defense against reactive oxygen species. Total thiols composed of both intracellular and extracellular thiols either in the free form as oxidized or reduced glutathione, or thiols bound to proteins. Among the thiols that are bound to proteins, albumin makes the major portion of the protein bound thiols, which binds to sufhydryl group at its cysteine-34 portion. Apart from their role in defense against free radicals, thiols share significant role in detoxification, signal transduction, apoptosis and various other functions at molecular level. The thiol status in the body can be assessed easily by determining the serum levels of thiols. Decreased levels of thiols has been noted in various medical disorders including chronic renal failure and other disorders related to kidney, cardiovascular disorders, stroke and other neurological disorders, diabetes mellitus, alcoholic cirrhosis and various other disorders. Therapy using thiols has been under investigation for certain disorders.

  19. Protein profiling of cerebrospinal fluid

    DEFF Research Database (Denmark)

    Simonsen, Anja H

    2012-01-01

    The cerebrospinal fluid (CSF) perfuses the brain and spinal cord. CSF contains proteins and peptides important for brain physiology and potentially also relevant for brain pathology. Hence, CSF is the perfect source to search for new biomarkers to improve diagnosis of neurological diseases as well...

  20. Chemistry and stability of thiol based polyethylene glycol surface coatings on colloidal gold and their relationship to protein adsorption and clearance in vivo

    Science.gov (United States)

    Carpinone, Paul

    Nanomaterials have presented a wide range of novel biomedical applications, with particular emphasis placed on advances in imaging and treatment delivery. Of the many particulate nanomaterials researched for biomedical applications, gold is one of the most widely used. Colloidal gold has been of great interest due to its chemical inertness and its ability to perform multiple functions, such as drug delivery, localized heating of tissues (hyperthermia), and imaging (as a contrast agent). It is also readily functionalized through the use of thiols, which spontaneously form sulfur to gold bonds with the surface. Polyethylene glycol (PEG) is the most widely used coating material for these particles as it provides both steric stability to the suspension and protein resistance. These properties extend the circulation time of the particles in blood, and consequently the efficacy of the treatment. Despite widespread use of PEG coated gold particles, the coating chemistry and stability of these particles are largely unknown. The goal of this work was to identify the mechanisms leading to degradation and stability of thiol based polyethylene glycol coatings on gold particles and to relate this behavior to protein adsorption and clearance in vivo. The results indicate that the protective PEG coating is susceptible to sources of oxidation (including dissolved oxygen) and competing adsorbates, among other factors. The quality of commercially available thiolated PEG reagents was also found to play a key role in the quality and protein resistance of the final PEG coating. Analysis of the stability of these coatings indicated that they rapidly degrade under physiological conditions, leading to the onset of protein adsorption when exposed to plasma or blood. Paralleling the protein adsorption behavior and onset of coating degradation observed in vitro, blood clearance of parenterally administered PEG coated particles in mice began after approximately 2h of circulation time. Taken

  1. The first decade of MALDI protein profiling

    DEFF Research Database (Denmark)

    Albrethsen, Jakob

    2011-01-01

    MALDI protein profiling has identified several important challenges in omics-based biomarker research. First, research into the analytical performance of a novel omics-platform of potential diagnostic impact must be carried out in a critical manner and according to common guidelines. Evaluation s...

  2. The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control

    International Nuclear Information System (INIS)

    Myers, Judith M.; Antholine, William E.; Myers, Charles R.

    2011-01-01

    Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.

  3. Protein profiles of hatchery egg shell membrane.

    Science.gov (United States)

    Rath, N C; Liyanage, R; Makkar, S K; Lay, J O

    2016-01-01

    Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM). Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins. Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic. The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

  4. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    Science.gov (United States)

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  5. Thiol biochemistry of prokaryotes

    Science.gov (United States)

    Fahey, Robert C.

    1986-01-01

    The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject.

  6. Pharmacology profiling of chemicals and proteins

    DEFF Research Database (Denmark)

    Kringelum, Jens Vindahl

    between pharmaceuticals and proteins in vivo potential leads to unwanted adverse effects, toxicity and reduced half-life, but can also lead to novel therapeutic effects of already approved pharmaceuticals. Hence identification of in vivo targets is of importance in discovery, development and repurposing....... This limitation complicates adverse effect assessment in the early drug-development phase, thus contributing to drugattrition. Prediction models offer the possibility to close these gaps and provide more complete pharmacology profiles, however improvements in performances are required for these tools to serve...... to its nonself origin, which potentially alters the pharmacology profile of the substance. The neutralization of biopharmaceuticals by antidrug antibodies (ADAs) is an important element in the immune response cascade, however studies of ADA binding site on biopharmaceuticals, referred to as B...

  7. Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

    NARCIS (Netherlands)

    Kouwen, Thijs R. H. M.; Dubois, Jean-Yves F.; Freudl, Roland; Quax, Wim J.; van Dijl, Jan Maarten

    2008-01-01

    Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of

  8. Oxidative protein folding: from thiol-disulfide exchange reactions to the redox poise of the endoplasmic reticulum.

    Science.gov (United States)

    Hudson, Devin A; Gannon, Shawn A; Thorpe, Colin

    2015-03-01

    This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI(red):PDI(ox). The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC-MS-MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Analysis of protein profiles using fuzzy clustering methods

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya

    The tissue protein profiles of healthy volunteers and volunteers with cervical cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence  technique  (HPLC-LIF)  developed  in  our  lab.      We analyzed      the protein profile data using different...

  10. Changes in the protein profile of Habanero pepper ( Capsicum ...

    African Journals Online (AJOL)

    Protein profile was studied during the development of Capsicum chinense somatic embryos. The total protein content and profile of polypeptides (by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of somatic embryos at different developmental stages (globular, heart-shaped, torpedo and cotyledonary stages) ...

  11. A neutral polyacrylate copolymer coating for surface modification of thiol-ene microchannels for improved performance of protein separation by microchip electrophoresis

    DEFF Research Database (Denmark)

    Mesbah, Kiarach; Mai, T.D.; Jensen, Thomas Glasdam

    2016-01-01

    We have investigated the behavior of thiol-ene substrates that is a class of promising materials for lab-on-a-chip electrophoresis applications. Two polymeric materials were prepared by copolymerization of N, N-dimethylacrylamide (DMA), (3-(methacryloyl-oxy)propyl)trimethoxysilane (PMA) and 3-tri...

  12. Impaired Thiol-Disulfide Balance in Acute Brucellosis.

    Science.gov (United States)

    Kolgelier, Servet; Ergin, Merve; Demir, Lutfi Saltuk; Inkaya, Ahmet Cagkan; Aktug Demir, Nazlim; Alisik, Murat; Erel, Ozcan

    2017-05-24

    The objective of this study was to examine a novel profile: thiol-disulfide homeostasis in acute brucellosis. The study included 90 patients with acute brucellosis, and 27 healthy controls. Thiol-disulfide profile tests were analyzed by a recently developed method, and ceruloplasmin levels were determined. Native thiol levels were 256.72 ± 48.20 μmol/L in the acute brucellosis group and 461.13 ± 45.37 μmol/L in the healthy group, and total thiol levels were 298.58 ± 51.78 μmol/L in the acute brucellosis group and 504.83 ± 51.05 μmol/L in the healthy group (p brucellosis than in the healthy controls (p brucellosis. The strong associations between thiol-disulfide parameters and a positive acute-phase reactant reflected the disruption of the balance between the antioxidant and oxidant systems. Since thiol groups act as anti-inflammatory mediators, the alteration in the thiol-disulfide homeostasis may be involved in brucellosis.

  13. Novel thiols of prokaryotes.

    Science.gov (United States)

    Fahey, R C

    2001-01-01

    Glutathione metabolism is associated with oxygenic cyanobacteria and the oxygen-utilizing purple bacteria, but is absent in many other prokaryotes. This review focuses on novel thiols found in those bacteria lacking glutathione. Included are glutathione amide and its perthiol, produced by phototrophic purple sulfur bacteria and apparently involved in their sulfide metabolism. Among archaebacteria, coenzyme M (2-mercaptoethanesulfonic acid) and coenzyme B (7-mercaptoheptanoylthreonine phosphate) play central roles in the anaerobic production of CH4 and associated energy conversion by methanogens, whereas the major thiol in the aerobic phototrophic halobacteria is gamma-glutamylcysteine. The highly aerobic actinomycetes produce mycothiol, a conjugate of N-acetylcysteine with a pseudodisaccharide of glucosamine and myo-inositol, AcCys-GlcNalpha(1 --> 1)Ins, which appears to play an antioxidant role similar to glutathione. Ergothioneine, also produced by actinomycetes, remains a mystery despite many years of study. Available data on the biosynthesis and metabolism of these and other novel thiols is summarized and key areas for additional study are identified.

  14. Protein Profiling of Preeclampsia Placental Tissues

    OpenAIRE

    Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y.; He, Jin

    2014-01-01

    Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expres...

  15. Protein profiling of preeclampsia placental tissues.

    Science.gov (United States)

    Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y; He, Jin; Ye, Fei

    2014-01-01

    Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

  16. Protein profiling of preeclampsia placental tissues.

    Directory of Open Access Journals (Sweden)

    Chang Shu

    Full Text Available Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A and adverse outcomes (Flt-1 in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

  17. Protein Profiling of Preeclampsia Placental Tissues

    Science.gov (United States)

    Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y.

    2014-01-01

    Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia. PMID:25392996

  18. Differences of protein profile before and after orthodontic treatment

    Science.gov (United States)

    Nasri, Farah Amirah Mohd; Wahab, Rohaya Megat Abdul; Karsani, Saiful Anuar; Ariffin, Shahrul Hisham Zainal

    2016-11-01

    Mechanical forces in orthodontic treatment used to treat malocclusion can cause inflamed gingival tissue and the process of tooth movement may resorb dental root. Root resorption is an iatrogenic effect of orthodontic treatment but it can be monitored using protein biomarker. This study aims to investigate the differences of protein profile before and after orthodontic treatment using different staining methods. Human gingival crevicular fluid and saliva were collected from orthodontic patients before and after treatment. Protein profile were observed using SDS-PAGE. Our study shows down regulation of proteins after 3 months of treatment. Hence, there are potential values from this study to aid in investigation for specific biomarkers for root resorption.

  19. Investigating homology between proteins using energetic profiles.

    Science.gov (United States)

    Wrabl, James O; Hilser, Vincent J

    2010-03-26

    Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may

  20. Investigating homology between proteins using energetic profiles.

    Directory of Open Access Journals (Sweden)

    James O Wrabl

    2010-03-01

    Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

  1. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms

    DEFF Research Database (Denmark)

    Maeda, K.; Finnie, Christine; Svensson, Birte

    2004-01-01

    search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase...

  2. Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non ...

    African Journals Online (AJOL)

    Effects of Pineal Proteins on Biochemical, Enzyme Profile and Non-Specific Immune Response of Indian Goats under Thermal Stress. ... Total precipitated pineal proteins successfully and significantly relieved the animals from adverse effects of heat stress and metyrapone treatment. There is evidence that most of the ...

  3. Proteomic characterization of the human centrosome by protein correlation profiling

    DEFF Research Database (Denmark)

    Andersen, Jens S; Wilkinson, Christopher J; Mayor, Thibault

    2003-01-01

    chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human...... centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel...... components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity....

  4. Antioxidant activities of the synthesized thiol-contained peptides derived from computer-aided pepsin hydrolysis of yam tuber storage protein, dioscorin.

    Science.gov (United States)

    Han, Chuan-Hsiao; Liu, Ju-Chi; Fang, Sheng-Uei; Hou, Wen-Chi

    2013-06-01

    Our previous report showed that yam dioscorin and its peptic hydrolysates exhibit radical scavenging activities; however, the functions of these peptic hydrolases are still under investigation. In this study, the thiol-containing peptides derived from computer-aided simulation of pepsin hydrolysis of dioscorin, namely, KTCGNGME (diotide1), PPCSE (diotide2), CDDRVIRTPLT (diotide3), KTCGY (diotide4), and PPCTE (diotide5) were synthesized to compare their antioxidant activities with GSH and/or carnosine by examining hydroxyl radical scavenging activity by electron spin resonance spectrometry, anti-low-density lipoprotein peroxidation, anti-AAPH-induced hemolysis, and oxygen radical absorbance capacity activity. We found that while all the synthesized diotides showed antioxidant activity, diotide4 exhibited the highest levels. Moreover, all diotides (100 μM) showed protective effects against methylglyoxal-induced human umbilical vein endothelial cell death. These results suggest that thiol-containing diotides derived from dioscorin hydrolysis exhibit antioxidant activities and reveal the benefits of yam tuber as an antioxidant-rich food. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

    Directory of Open Access Journals (Sweden)

    Cuncong Zhong

    2016-07-01

    Full Text Available Analyses of metagenome data (MG and metatranscriptome data (MT are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE genes. Finally, HMM

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

    Science.gov (United States)

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

    2014-01-01

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

  7. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    Directory of Open Access Journals (Sweden)

    Rom William N

    2005-08-01

    Full Text Available Abstract Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD. Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

  8. Protein profiles of serum, brain regions and hypophyses of pubertal ...

    African Journals Online (AJOL)

    The effects of dietary fumonisin B1 (FB1 ), a toxin produced mainly by Fusarium verticillioides and F. proliferatum that grow on maize worldwide, on protein profiles of serum, brain regions and hypophyses were studied in 24 male Large White weanling pigs randomly divided into four groups (n = 6). In a completely ...

  9. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Czech Academy of Sciences Publication Activity Database

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

  10. Thiol/disulphide homeostasis in celiac disease

    Science.gov (United States)

    Kaplan, Mustafa; Ates, Ihsan; Yuksel, Mahmut; Ozderin Ozin, Yasemin; Alisik, Murat; Erel, Ozcan; Kayacetin, Ertugrul

    2017-01-01

    AIM To determine dynamic thiol/disulphide homeostasis in celiac disease and to examine the associate with celiac autoantibodies and gluten-free diet. METHODS Seventy three patients with celiac disease and 73 healthy volunteers were enrolled in the study. In both groups, thiol/disulphide homeostasis was examined with a new colorimetric method recently developed by Erel and Neselioglu. RESULTS In patients with celiac disease, native thiol (P = 0.027) and total thiol (P = 0.031) levels were lower, while disulphide (P < 0.001) level, disulphide/native thiol (P < 0.001) and disulphide/total thiol (P < 0.001) ratios were higher compared to the control group. In patients who do not comply with a gluten-free diet, disulphide/native thiol ratio was found higher compared to the patients who comply with the diet (P < 0.001). In patients with any autoantibody-positive, disulphide/native thiol ratio was observed higher compared to the patients with autoantibody-negative (P < 0.05). It is found that there is a negative correlation between celiac autoantibodies, and native thiol, total thiol levels and native thiol/total thiol ratio, while a positive correlation is observed between disulphide, disulphide/native thiol and disulphide/total thiol levels. CONCLUSION This study is first in the literature which found that the patients with celiac disease the dynamic thiol/disulphide balance shifts through disulphide form compared to the control group. PMID:28533921

  11. O-GlcNAc profiling: from proteins to proteomes

    Science.gov (United States)

    2014-01-01

    O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

  12. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    International Nuclear Information System (INIS)

    Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

    2005-01-01

    Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

  13. Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

    Science.gov (United States)

    Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon

    2016-01-01

    Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

    DEFF Research Database (Denmark)

    Callesen, Anne K; Madsen, Jonna S; Vach, Werner

    2009-01-01

    Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and ...

  15. Thiol/disulfide redox states in signaling and sensing

    Science.gov (United States)

    Go, Young-Mi; Jones, Dean P.

    2015-01-01

    Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady-states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling, and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities. PMID:23356510

  16. Automatic selection of reference taxa for protein-protein interaction prediction with phylogenetic profiling

    DEFF Research Database (Denmark)

    Simonsen, Martin; Maetschke, S.R.; Ragan, M.A.

    2012-01-01

    Motivation: Phylogenetic profiling methods can achieve good accuracy in predicting protein–protein interactions, especially in prokaryotes. Recent studies have shown that the choice of reference taxa (RT) is critical for accurate prediction, but with more than 2500 fully sequenced taxa publicly......: We present three novel methods for automating the selection of RT, using machine learning based on known protein–protein interaction networks. One of these methods in particular, Tree-Based Search, yields greatly improved prediction accuracies. We further show that different methods for constituting...... phylogenetic profiles often require very different RT sets to support high prediction accuracy....

  17. Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

    Science.gov (United States)

    Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

    2012-01-01

    Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

  18. Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.

    Science.gov (United States)

    Manickam, Nagaraj; Sun, Xiuhua; Hakala, Kevin W; Weintraub, Susan T; Essex, David W

    2008-07-01

    Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

  19. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

    Science.gov (United States)

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

    1999-01-29

    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  20. Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

    Directory of Open Access Journals (Sweden)

    Rundle William T

    2008-06-01

    Full Text Available Abstract Background Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase, but at body temperature (37°C, a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei. Results Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein

  1. Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts

    DEFF Research Database (Denmark)

    Bennett, K L; Kussmann, M; Björk, P

    2000-01-01

    The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vit...

  2. Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

    Science.gov (United States)

    Wible, Ryan S; Sutter, Thomas R

    2017-03-20

    The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

  3. Bioorthogonal chemistry: applications in activity-based protein profiling.

    Science.gov (United States)

    Willems, Lianne I; van der Linden, Wouter A; Li, Nan; Li, Kah-Yee; Liu, Nora; Hoogendoorn, Sascha; van der Marel, Gijs A; Florea, Bogdan I; Overkleeft, Herman S

    2011-09-20

    of chemical biology research include contributions from many areas of the multifaceted discipline of chemistry, and particularly from organic chemistry. Researchers apply knowledge inherent to organic chemistry, such as reactivity and selectivity, to the manipulation of specific biomolecules in biological samples (cell extracts, living cells, and sometimes even animal models) to gain insight into the biological phenomena in which these molecules participate. In this Account, we highlight some of the recent developments in chemical biology research driven by organic chemistry, with a focus on bioorthogonal chemistry in relation to activity-based protein profiling. The rigorous demands of bioorthogonality have not yet been realized in a truly bioorthogonal reagent pair, but remarkable progress has afforded a range of tangible contributions to chemical biology research. Activity-based protein profiling, which aims to obtain information on the workings of a protein (or protein family) within the larger context of the full biological system, has in particular benefited from these advances. Both activity-based protein profiling and bioorthogonal chemistry have been around for approximately 15 years, and about 8 years ago the two fields very profitably intersected. We expect that each discipline, both separately and in concert, will continue to make important contributions to chemical biology research. © 2011 American Chemical Society

  4. Protein profile of mouse ovarian follicles grown in vitro.

    Science.gov (United States)

    Anastácio, Amandine; Rodriguez-Wallberg, Kenny A; Chardonnet, Solenne; Pionneau, Cédric; Fédérici, Christian; Almeida Santos, Teresa; Poirot, Catherine

    2017-12-01

    emphasize proteins with different expression profiles between the three follicular stages. Supplementary western blot analysis (using new biological replicates) was performed to confirm the expression variations of three proteins during follicle development in vitro. It was found that 609 out of 1401 identified proteins were common to the three follicle developmental stages investigated. Some proteins were identified uniquely at one stage: 71 of the 775 identified proteins in SF, 181 of 1092 in SMR and 192 of 1100 in AF. Additional qualitative and quantitative analysis highlighted 44 biological processes over-represented in our samples compared to the Mus musculus gene database. In particular, it was possible to identify proteins implicated in the cell cycle, calcium ion binding and glycolysis, with specific expressions and abundance, throughout in vitro follicle development. Data are available via ProteomeXchange with identifier PXD006227. The proteome analyses described in this study were performed after in vitro development. Despite fractionation of the samples before LC-MS/MS, proteomic approaches are not exhaustive, thus proteins that are not identified in a group are not necessarily absent from that group, although they are likely to be less abundant. This study allowed a general view of proteins implicated in follicle development in vitro and it represents the most complete catalog of the whole follicle proteome available so far. Not only were well known proteins of the oocyte identified but also proteins that are probably expressed only in granulosa cells. This study was supported by the Portuguese Foundation for Science and Technology, FCT (PhD fellowship SFRH/BD/65299/2009 to A.A.), the Swedish Childhood Cancer Foundation (PR 2014-0144 to K.A.R-.W.) and Stockholm County Council to K.A.R-.W. The authors of the study have no conflict of interest to report. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human

  5. Study of protein and metabolic profile of sugarcane workers

    Energy Technology Data Exchange (ETDEWEB)

    Polachini, G.M.; Tajara, E.H. [Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), SP (Brazil); Santos, U.P. [Universidade de Sao Paulo (USP), SP (Brazil); Zeri, A.C.M.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil)

    2012-07-01

    Full text: The National Alcohol Program (Proalcool) is a successful Brazilian renewable fuel initiative aiming to reduce the country's oil dependence. Producing ethanol from sugar cane, the program has shown positive results although accompanied by potential damage. The environmental impact mainly derives from the particulate matter emissions due to sugarcane burning, which is potentially harmful to human health. The physical activity of sugarcane workers is repetitive and exhaustive and is carried out in presence of dust, smoke and soot. The efforts by the sugarcane workers during the labor process result in increased risks of nervous, respiratory and cardiovascular system diseases and also in premature death. The aim of the present study was to investigate the effect of occupational stress on protein and metabolic profile of sugarcane workers. Forty serum samples were analyzed by 1-DE and LC MS/MS proteomic shotgun strategy and nuclear magnetic resonance (NMR). A set of proteins was found to be altered in workers after crops when compared with controls. The analysis of NMR spectra by Chenomx also showed differences in the expression of metabolites. For example, lactate displayed higher levels in control subjects than in sugarcane workers, and vice versa for the acetate. The concentrations of the two metabolites were lower after the crop, except in the case of acetate, which remained uniform in the control subjects before and after the crop. The present findings can have important application for rational designs of preventive measures and early disease detection in sugarcane workers. (author)

  6. Study of protein and metabolic profile of sugarcane workers

    International Nuclear Information System (INIS)

    Polachini, G.M.; Tajara, E.H.; Santos, U.P.; Zeri, A.C.M.; Paes Leme, A.F.

    2012-01-01

    Full text: The National Alcohol Program (Proalcool) is a successful Brazilian renewable fuel initiative aiming to reduce the country's oil dependence. Producing ethanol from sugar cane, the program has shown positive results although accompanied by potential damage. The environmental impact mainly derives from the particulate matter emissions due to sugarcane burning, which is potentially harmful to human health. The physical activity of sugarcane workers is repetitive and exhaustive and is carried out in presence of dust, smoke and soot. The efforts by the sugarcane workers during the labor process result in increased risks of nervous, respiratory and cardiovascular system diseases and also in premature death. The aim of the present study was to investigate the effect of occupational stress on protein and metabolic profile of sugarcane workers. Forty serum samples were analyzed by 1-DE and LC MS/MS proteomic shotgun strategy and nuclear magnetic resonance (NMR). A set of proteins was found to be altered in workers after crops when compared with controls. The analysis of NMR spectra by Chenomx also showed differences in the expression of metabolites. For example, lactate displayed higher levels in control subjects than in sugarcane workers, and vice versa for the acetate. The concentrations of the two metabolites were lower after the crop, except in the case of acetate, which remained uniform in the control subjects before and after the crop. The present findings can have important application for rational designs of preventive measures and early disease detection in sugarcane workers. (author)

  7. Strain-dependent profile of misfolded prion protein aggregates.

    Science.gov (United States)

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-02-15

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

  8. Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

    OpenAIRE

    Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

    2011-01-01

    Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, w...

  9. Effect of the cooking method (grilling, roasting, frying and sous-vide) on the oxidation of thiols, tryptophan, alkaline amino acids and protein cross-linking in jerky chicken

    OpenAIRE

    Silva, Fábio A. P.; Ferreira, Valquíria C. S.; Madruga, Marta S.; Estévez, Mario

    2016-01-01

    Broiler breast (pectoralis major) meat was submitted to salting with NaCl + NaNO3 followed by a drying process to produce jerky-type chicken. The final product (raw broiler charqui) was desalted and then cooked using grilled, roasted, fried and sous-vide techniques. Sous-vide cooked samples showed lowest results of moisture loss compared to roasted and fried ones. Fatty acid profile suffered minor changes after cooking of broiler charqui. Regarding to protein oxidation, tryptophan fluorescenc...

  10. Reproducibility of mass spectrometry based protein profiles for diagnosis of breast cancer across clinical studies

    DEFF Research Database (Denmark)

    Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E

    2008-01-01

    Serum protein profiling by mass spectrometry has achieved attention as a promising technology in oncoproteomics. We performed a systematic review of published reports on protein profiling as a diagnostic tool for breast cancer. The MEDLINE, EMBASE, and COCHRANE databases were searched for original...... studies reporting discriminatory protein peaks for breast cancer as either protein identity or as m/ z values in the period from January 1995 to October 2006. To address the important aspect of reproducibility of mass spectrometry data across different clinical studies, we compared the published lists...... of potential discriminatory peaks with those peaks detected in an original MALDI MS protein profiling study performed by our own research group. A total of 20 protein/peptide profiling studies were eligible for inclusion in the systematic review. Only 3 reports included information on protein identity...

  11. Low molecular weight thiols and thioredoxins are important players in Hg(II) resistance in Thermus thermophilus HB27.

    Science.gov (United States)

    Norambuena, J; Wang, Y; Hanson, T; Boyd, J M; Barkay, T

    2017-11-17

    Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low molecular weight and protein thiols may be important cell components used in Hg resistance. To date, the role of low molecular weight thiols in Hg-detoxification remains understudied. The mercury resistance ( mer ) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low molecular weight thiol metabolism. This mer operon encodes for an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low molecular weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in presence of either a thiol oxidizing agent or a thiol alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus spp. mer operons. The presence of a thiol related gene was also detected in some alphaprotobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II) buffering agents while Hg is reduced by MerA. Importance The survival of microorganisms in presence of toxic metals is central to life's sustainability. The affinity of thiol groups to toxic heavy metals drives microbe-metal interactions and modulate metal toxicity. Mercury detoxification ( mer ) genes likely originated early in microbial evolution among geothermal environments. Little is

  12. Serum Protein Profile Study of Clinical Samples Using High Performance Liquid Chromatography-Laser Induced Fluorescence

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Ukendt, Sujatha; Rai, Lavanya

    2009-01-01

    The serum protein profiles of normal subjects, patients diagnosed with cervical cancer, and oral cancer were recorded using High Performance Liquid Chromatography combined with Laser Induced Fluorescence detection (HPLC-LIF). Serum protein profiles of the above three classes were tested for estab...

  13. Protein Profile study of clinical samples using Laser Induced Fluorescence as the detection method

    DEFF Research Database (Denmark)

    Karemore, Gopal Raghunath; Raja, Sujatha N.; Rai, Lavanya

    2009-01-01

      Protein profiles of tissue homogenates were recorded using HPLC separation and LIF detection method. The samples were collected from volunteers with clinically normal or cervical cancer conditions. It is shown that the protein profile can be classified as belonging to malignant or normal state ...

  14. Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways.

    Science.gov (United States)

    Nagy, Péter

    2013-05-01

    Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol-disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. This review is focused on the kinetics and mechanisms of thiol-disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery.

  15. Classification of protein profiles using fuzzy clustering techniques

    DEFF Research Database (Denmark)

    Karemore, Gopal; Mullick, Jhinuk B.; Sujatha, R.

    2010-01-01

     Present  study  has  brought  out  a  comparison  of PCA  and  fuzzy  clustering  techniques  in  classifying  protein profiles  (chromatogram)  of  homogenates  of  different  tissue origins:  Ovarian,  Cervix,  Oral  cancers,  which  were  acquired using HPLC–LIF (High Performance Liquid...... Chromatography- Laser   Induced   Fluorescence)   method   developed   in   our laboratory. Study includes 11 chromatogram spectra each from oral,  cervical,  ovarian  cancers  as  well  as  healthy  volunteers. Generally  multivariate  analysis  like  PCA  demands  clear  data that   is   devoid   of   day......   PCA   mapping   in   classifying   various cancers from healthy spectra with classification rate up to 95 % from  60%.  Methods  are  validated  using  various  clustering indexes   and   shows   promising   improvement   in   developing optical pathology like HPLC-LIF for early detection of various...

  16. Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

    DEFF Research Database (Denmark)

    Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

    2010-01-01

    Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

  17. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  18. Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier.

    Science.gov (United States)

    Hu, Bo; Zhao, Yang; Zhu, Hai-Zhou; Yu, Shu-Hong

    2011-04-26

    Thiol-containing biomolecules show strong affinity with noble metal nanostructures and could not only stably protect them but also control the self-assembly process of these special nanostructures. A highly selective and sensitive chromogenic detection method has been designed for the low and high molecular weight thiol-containing biomolecules, including cysteine, glutathione, dithiothreitol, and bovine serum albumin, using a new type of carbonaceous nanospheres loaded with silver nanoparticles (Ag NPs) as carrier. This strategy relies upon the place-exchange process between the reporter dyes on the surface of Ag NPs and the thiol groups of thiol-containing biomolecules. The concentration of biomolecules can be determined by monitoring with the fluorescence intensity of reporter dyes dispersed in solution. This new chromogenic assay method could selectively detect these biomolecules in the presence of various other amino acids and monosaccharides and even sensitively detect the thiol-containing biomolecules with different molecular weight, even including proteins.

  19. Protein abundance profiling of the Escherichia coli cytosol

    DEFF Research Database (Denmark)

    Ishihama, Y.; Schmidt, T.; Rappsilber, J.

    2008-01-01

    sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI...... approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal...

  20. Effect of soy protein on serum lipid profile and some lipid ...

    African Journals Online (AJOL)

    The effect of soy protein on serum lipid profile and some lipid metabolizing enzymes in rats fed with cholesterol diets was examined in this study. Rats were subjected to feeding trial over a period of six weeks on formulated diets containing: 20% soy protein with 0% cholesterol (group A), 20% soy protein with 5% cholesterol ...

  1. Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

    Science.gov (United States)

    Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

    2012-04-06

    Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

  2. Selenocysteine in thiol/disulfide-like exchange reactions.

    Science.gov (United States)

    Hondal, Robert J; Marino, Stefano M; Gladyshev, Vadim N

    2013-05-01

    Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

  3. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Science.gov (United States)

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  4. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    Energy Technology Data Exchange (ETDEWEB)

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  5. Role of thiols in cellular response to radiation and drugs. Symposium: thiols

    International Nuclear Information System (INIS)

    Biaglow, J.E.; Varnes, M.E.; Clark, E.P.; Epp, E.R.

    1983-01-01

    Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme. A GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells

  6. Evaluation and Control of Thiol-ene/Thiol-epoxy Hybrid Networks

    OpenAIRE

    Carioscia, Jacquelyn A.; Stansbury, Jeffrey W.; Bowman, Christopher N.

    2007-01-01

    The development of thiol-ene/thiol-epoxy hybrid networks offers the advantage of tailorable polymerization kinetics while producing a highly crosslinked, high Tg polymer that has significantly reduced shrinkage stress. Stoichiometric mixtures of pentaerythritol tetra(3-mercaptopropionate) (PETMP)/triallyl-1,3,5-triazine-2,4,6-trione (TATATO) (thiol-ene, mixture 1) and PETMP/bisphenol a diglycidyl ether (BADGE) (thiol-epoxy, mixture 2) were prepared and hybrid mixtures of 75/25, 50/50, 25/75, ...

  7. Integration of the thiol redox status with cytokine response to physical training in professional basketball players.

    Science.gov (United States)

    Zembron-Lacny, A; Slowinska-Lisowska, M; Ziemba, A

    2010-01-01

    The present study was designed to evaluate the plasma markers of reactive oxygen species (ROS) activity and cytokines, and their relationship with thiol redox status of basketball players during training. Sixteen professional players of the Polish Basketball Extraleague participated in the study. The study was performed during the preparatory period and the play-off round. Markers of ROS activity (lipid peroxidation TBARS, protein carbonylation PC) and reduced glutathione (GSH) demonstrated regularity over time, i.e. TBARS, PC and GSH were elevated at the beginning and decreased at the end of training periods. Oxidized glutathione (GSSG) was not affected by exercise training. Thiol redox status (GSH(total)-2GSSG/GSSG) correlated with TBARS and PC in both training periods. The level of interleukin-6 (IL-6) was increased and positively correlated with thiol redox (r=0.423) in the preparatory period, whereas tumor necrosis factor alpha (TNFalpha) was increased and inversely correlated with thiol redox (r= 0.509) in the play-off round. The present study showed significant shifts in markers of ROS activity, thiol redox status and inflammatory mediators (IL-6, TNFalpha) following professional sport training as well as correlation between changes in thiol redox and cytokine response.

  8. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    Directory of Open Access Journals (Sweden)

    Florian Heinke

    2012-01-01

    Full Text Available Diabetes insipidus (DI is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI. NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

  9. Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

    Science.gov (United States)

    Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

    2011-01-01

    Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H2O2. Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H2O2. The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H2O2 and other cellular proteins plays a secondary role. PMID:21282621

  10. Analysis of Proximate and Protein Profile of Kefir from Fermented Goat and Cow Milk

    Directory of Open Access Journals (Sweden)

    Erwin Hidayat

    2015-09-01

    Full Text Available This research aims to analyze the characteristics of proximate and protein profile in kefir from fermented goat milk and cow milk with different concentration of kefir grains. The research design was true experimental with Completely Randomized Design (CRD of 3 repetitions. The research procedures consisted of kefir production, proximate analysis and protein profile characterization. Proximate assay result was analyzed by using LSD, whereas the protein profile was analyzed by descriptive qualitative method. Based on the analysis of kefir proximate levels, the kefir grain (5% showed the highest proximate level of both kefirs from goat milk and cow milk. The analysis of protein profile of cow milk kefir showed 75 kDa of protein ribbon, while the goat milk kefir showed 48 kDa, 60 kDa and 75 kDa. Therefore it can be concluded that the proximate level of goat and cow milk kefir with different concentration of kefir grains showed significant differences in the nutrition content as well as its protein profiles.Tujuan dari penelitian ini adalah menganalisis karakteristik proksimat dan profil protein pada kefir hasil fermentasi susu kambing dan susu sapi dengan konsentrasi biji kefir yang berbeda-beda. Penelitian ini adalah eksperimen murni, dengan Rancangan Acak Lengkap (RAL 3 kali ulangan. Prosedur penelitian meliputi pembuatan kefir, analisis proksimat dan profil protein. Data hasil proksimat dianalisi uji BNT, sedangkan profil protein dianalisis deskriptif kualitatif. Berdasarkan analisis kadar proksimat kefir, kefir grains 5% menunjukan kadar proksimat paling tinggi baik pada kefir susu kambing dan susu sapi. Sedangkan analisis profil protein kefir susu sapi menunjukan pita protein 75 kDa, pada kefir susu kambing yaitu 48 kDa, 60 kDa dan 75 kDa. Simpulan dari penelitian ini bahwa kadar proksimat kefir susu kambing dan susu sapi dengan konsentrasi kefir grains yang berbeda menunjukan perbedaan kandungan yang berbeda secara signifikan dengan

  11. Serum protein profile of Malaria patients through SDS-PAGE method ...

    African Journals Online (AJOL)

    Serum protein profile of Malaria patients through SDS-PAGE method. ... reliable method in the diagnosis of antibodies produced against Plasmodium spps. ... of malaria patients may be undertaken for study to develop possible future vaccine.

  12. Thiol/Disulfide system plays a crucial role in redox protection in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum.

    Directory of Open Access Journals (Sweden)

    Javiera Norambuena

    Full Text Available Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. Leptospirillum ferriphilum DSM14647, an acidophilic bacterium that uses Fe(2+ as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from L. ferriphilum are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR, periplasmic thiol oxidation system (DsbA/DsbB and a c-type cytochrome maturation system (DsbD/DsbE. Upon exposure of L. ferriphilum to reactive oxygen species (ROS-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of L. ferriphilum favoring the survival of this microorganism under extreme environmental oxidative conditions.

  13. Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago sativa).

    Science.gov (United States)

    Zhang, Jing Jing; Xu, Jiang Yan; Lu, Feng Fan; Jin, She Feng; Yang, Hong

    2017-10-16

    Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich nonprotein compounds and play primary and multiple roles in cellular redox homeostasis, enzyme activities, and xenobiotics detoxification. This study focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environment. Using high-throughput RNA-sequencing, a set of ATZ-responsive thiols-related protein genes highly up-regulated and differentially expressed in alfalfa was identified. Most of the differentially expressed genes (DEGs) were involved in regulation of biotic and abiotic stress responses. By analyzing the genes involved in thiols-mediated redox homeostasis, we found that many of them were thiols-synthetic enzymes such as γ-glutamylcysteine synthase (γECS), homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we further characterized a group of ATZ-thiols conjugates, which are the detoxified forms of ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite detected by MS. Several other ATZ-conjugates were also examined as ATZ-detoxified metabolites. Such results were validated by characterizing their analogs in rice. Our data showed that some conjugates under ATZ stress were detected in both plants, indicating that some detoxified mechanisms and pathways can be shared by the two plant species. Overall, these results indicate that LMW thiols play critical roles in detoxification of ATZ in the plants.

  14. Investigations of thiol-modified phenol derivatives for the use in thiol-ene photopolymerizations.

    Science.gov (United States)

    Reinelt, Sebastian; Tabatabai, Monir; Fischer, Urs Karl; Moszner, Norbert; Utterodt, Andreas; Ritter, Helmut

    2014-01-01

    Thiol-ene photopolymerizations gain a growing interest in academic research. Coatings and dental restoratives are interesting applications for thiol-ene photopolymerizations due to their unique features. In most studies the relative flexible and hydrophilic ester derivative, namely pentaerythritoltetra(3-mercaptopropionate) (PETMP), is investigated as the thiol component. Thus, in the present study we are encouraged to investigate the performance of more hydrophobic ester-free thiol-modified bis- and trisphenol derivatives in thiol-ene photopolymerizations. For this, six different thiol-modified bis- and trisphenol derivatives exhibiting four to six thiol groups are synthesized via the radical addition of thioacetic acid to suitable allyl-modified precursors and subsequent hydrolysis. Compared to PETMP better flexural strength and modulus of elasticity are achievable in thiol-ene photopolymerizations employing 1,3,5-triallyl-1,3,5-triazine-2,4,6-trione (TATATO) as the ene derivative. Especially, after storage in water, the flexural strength and modulus of elasticity is twice as high compared to the PETMP reference system.

  15. Evaluation and Control of Thiol-ene/Thiol-epoxy Hybrid Networks.

    Science.gov (United States)

    Carioscia, Jacquelyn A; Stansbury, Jeffrey W; Bowman, Christopher N

    2007-03-08

    The development of thiol-ene/thiol-epoxy hybrid networks offers the advantage of tailorable polymerization kinetics while producing a highly crosslinked, high T(g) polymer that has significantly reduced shrinkage stress. Stoichiometric mixtures of pentaerythritol tetra(3-mercaptopropionate) (PETMP)/triallyl-1,3,5-triazine-2,4,6-trione (TATATO) (thiol-ene, mixture 1) and PETMP/bisphenol a diglycidyl ether (BADGE) (thiol-epoxy, mixture 2) were prepared and hybrid mixtures of 75/25, 50/50, 25/75, and 10/90 w/w of mixtures 1 and 2 were polymerized using a combination of both radical and anionic initiation. The light exposure timing and the relative initiation conditions of the two types were used to control the order and relative rates of the radical and anionic polymerizations. The 50/50 w/w thiol-ene/thiol-epoxy hybrid material exhibited a final stress of only 0.2 MPa, which is 90 % lower than the stress developed in a control dimethacrylate resin. Kinetic analysis indicates composition affects network development in thiol-ene/thiol-epoxy hybrid networks and produces materials with robust mechanical properties.

  16. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Science.gov (United States)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  17. Formation of Underbrushes on thiolated Poly (ethylene glycol) PEG monolayers by Oligoethylene glycol (OEG) terminated Alkane Thiols on Gold

    DEFF Research Database (Denmark)

    Lokanathan, Arcot R.

    2011-01-01

    Adding underbrushes of oligoethylene glycol (OEG) to monolayers of long chain PEG molecules on a surface is one of the strategies [1] in designing a suitable platform for antifouling purpose, where it is possible to have high graft density and molecular conformational freedom[4] simultaneously......, there by maximal retention of activity of covalently immobilised antifouling enzyme [2] on PEG surfaces along with resistance to protein adsorption[3]. Here we present some our studies on the addition of OEG thiol molecules over a self assembled monolayer of PEG thiol on gold. The kinetics of addition of OEG thiol...

  18. Hierarchical partitioning of metazoan protein conservation profiles provides new functional insights.

    Directory of Open Access Journals (Sweden)

    Jonathan Witztum

    Full Text Available The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles. We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO analysis tool, we explore functional enrichment of the "universal proteins", those with homologues in all 17 other species, and of the "non-universal proteins". A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the "tree of life" (TOL consistent, as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the "life style" of the related clades. Most previous approaches for studying function and conservation are "bottom up", studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is "top down". We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life.

  19. glue protein profiles in the nasuta–albomicans complex

    Indian Academy of Sciences (India)

    . Manasagangotri ... Further, quantitative analysis also shows lack of correlation between the chromosomal ... involving these two races followed by karyotypic screening of hybrid .... The molecular masses of the variable protein fractions were ...

  20. The Reducing Capacity of Thioredoxin on Oxidized Thiols in Boiled Wort

    DEFF Research Database (Denmark)

    Murmann, Anne N.; Hägglund, Per; Svensson, Birte

    2017-01-01

    system was also capable of increasing the free thiol concentration, although with lower efficiency to 187 and 170 μM, respectively. The presence of sulfite, an important antioxidant in beer secreted by the yeast during fermentation, was found to inactivate thioredoxin by sulfitolysis. Reduction......Free thiol-containing proteins are suggested to work as antioxidants in beer, but the majority of thiols in wort are present in their oxidized form as disulfides and are therefore not active as antioxidants. Thioredoxin, a disulfide-reducing protein, is released into the wort from some yeast...... and fluorescence detection of thiol-derivatives. When boiled wort was incubated with all components of the thioredoxin system at pH 7.0 and 25 °C for 60 min under anaerobic conditions, the free thiol concentration increased from 25 to 224 μM. At pH values similar to wort (pH 5.7) and beer (pH 4.5), the thioredoxin...

  1. Thiol-disulfide exchange in peptides derived from human growth hormone.

    Science.gov (United States)

    Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M; Topp, Elizabeth M

    2014-04-01

    Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  2. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

    Science.gov (United States)

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-01

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

  3. A structurally driven analysis of thiol reactivity in mammalian albumins.

    Science.gov (United States)

    Spiga, Ottavia; Summa, Domenico; Cirri, Simone; Bernini, Andrea; Venditti, Vincenzo; De Chiara, Matteo; Priora, Raffaella; Frosali, Simona; Margaritis, Antonios; Di Giuseppe, Danila; Di Simplicio, Paolo; Niccolai, Neri

    2011-04-01

    Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence-specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental and predicted albumin structures reveal that thiolation rates are influenced by hydrogen bonding pattern and stability of the acceptor C34 sulphur atom with donor groups of nearby residues. Atom depth evolution of albumin C34 thiol groups has been monitored during Molecular Dynamic trajectories. The most reactive albumins appeared also the ones presenting the C34 sulphur atom on the protein surface with the highest accessibility. High C34 sulphur atom reactivity in rat and porcine albumins seems to be determined by the presence of additional positively charged amino acid residues favoring both the C34 S⁻ form and the approach of DTNB. Copyright © 2011 Wiley Periodicals, Inc.

  4. Correlations between RNA and protein expression profiles in 23 human cell lines

    Directory of Open Access Journals (Sweden)

    Pontén Fredrik

    2009-08-01

    Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  5. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    OpenAIRE

    Feng, Yi; Tian, Zhong-Min; Wan, Ming-Xi; Zheng, Zhao-Bin

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.

  6. Optimization of translation profiles enhances protein expression and solubility.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  7. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  8. Buffalo milk: proteins electrophoretic profile and somatic cell count

    Directory of Open Access Journals (Sweden)

    S. Mattii

    2011-03-01

    Full Text Available Water buffalo milk differs from the cow’s milk for greater fat and protein content, very important features in cheese making. Proteins, casein and whey-proteins in particular, are the most important factors determining cheese yield. Several previous research discussed the rule of SCC in cow milk production (Varisco, 1999 and the close relationship existing between cow’s milk cheese yield and somatic cell count (Barbano, 2000. In particular the inverse correlation between cheese yields and somatic cells’content have been demonstrated. In Italy the regulation in force DPR 54/97 acknowledges what expressed in EEC 46/92 Directive (Tripodi, 1999 without fixing the limit threshold of somatic cells for buffalo’s milk....

  9. Variation in the protein profiles in the gamma-irradiated chick pea (Cicer arietinum L.) seeds

    International Nuclear Information System (INIS)

    Farook, S.A.F.; Nizam, Jafar

    1978-01-01

    Water soluble seed proteins from the control as well as gamma ray treated material from the M 2 generation of Chick pea (Cicer arietinum L.) were separated by disc electrophoresis using 7.5 percent poly acrylamide gels. Average Rf values and percentage of similarity values were calculated. The comparisons of number and Rf values of protein bands were made to elucidate the differences in the treated material. Differences obtained in the seed protein profiles of the treated material suggest the presence of the qualitative variation in the proteins. Attempts were made to correlate the variation in the protein bands with the morphological changes in the mutants. (author)

  10. Analysis on Protein Profile and Amino Acid of Edible Bird's Nest (Collocalia Fuchiphaga) From Painan

    OpenAIRE

    Elfita, Lina

    2014-01-01

    This study was aimed to analyze protein profile and amino acid composition of bird nest from Painan, Pesisir Selatan Distric, West Sumatra. Protein analysis was performed by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE), meanwhile High Performance Liquid Chromatography (HPLC) was used for analysis of amino acid. Analysis on water extract of bird nest by SDS-PAGE showed six bands which correspond to molecular protein which had molecular weight of 147.2; 142.6; 133.4; 73...

  11. Design and application of natural product derived probes for activity based protein profiling

    OpenAIRE

    Battenberg, Oliver Alexander

    2015-01-01

    The identification of new antibacterial protein targets by activity based protein profiling (ABPP) is an important approach to face the increasing emergence of resistant bacteria. The scope of this work focuses on three new strategies for the labeling of antibacterial protein-targets with natural product derived ABPP-probes: A.) Evaluation of the intrinsic photo-reactivity of α-pyrones and pyrimidones for use as photo-crosslinkers. B.) Synthesis of a benzophenone-tag that combines photo-cross...

  12. Changes in the protein profile of Habanero pepper (Capsicum ...

    African Journals Online (AJOL)

    2012-06-12

    Jun 12, 2012 ... (Capsicum chinense J.) somatic embryos during ... molecular weights to those reported for storage proteins in other .... were isolated by cutting with a razor blade. ... electrophoresis (SDS-PAGE; 5% stacking gel [pH 6.8], 15% running ... Developmental stages correspond to C) globular, D) heart-shaped, ...

  13. Synthesis of soybean oil-based thiol oligomers.

    Science.gov (United States)

    Wu, Jennifer F; Fernando, Shashi; Weerasinghe, Dimuthu; Chen, Zhigang; Webster, Dean C

    2011-08-22

    Industrial grade soybean oil (SBO) and thiols were reacted to generate thiol-functionalized oligomers via a thermal, free radical initiated thiol-ene reaction between the SBO double bond moieties and the thiol functional groups. The effect of the reaction conditions, including thiol concentration, catalyst loading level, reaction time, and atmosphere, on the molecular weight and the conversion to the resultant soy-thiols were examined in a combinatorial high-throughput fashion using parallel synthesis, combinatorial FTIR, and rapid gel permeation chromatography (GPC). High thiol functionality and concentration, high thermal free radical catalyst concentration, long reaction time, and the use of a nitrogen reaction atmosphere were found to favor fast consumption of the SBO, and produced high molecular weight products. The thiol conversion during the reaction was inversely affected by a high thiol concentration, but was favored by a long reaction time and an air reaction atmosphere. These experimental observations were explained by the initial low affinity of the SBO and thiol, and the improved affinity between the generated soy-thiol oligomers and unreacted SBO during the reaction. The synthesized soy-thiol oligomers can be used for renewable thiol-ene UV curable materials and high molecular solids and thiourethane thermal cure materials. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    Institute of Scientific and Technical Information of China (English)

    Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

  15. Protein profile of Chlamydophila abortus isolates from Kerala, India

    Directory of Open Access Journals (Sweden)

    Binu K Mani

    Full Text Available Chlamydiae are of microbiological interest because of their mode of interaction with eukaryotic host cells and their specialized life cycle with unique features of parasitism. Reports regarding prevalence of infections of Chlamydophila abortus, the causative organism for chlamydial abortions in livestock, was the basis of the study. Two isolates, one each from cattle and goat abortion along with a reference isolate, were used for characterization with Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE. Elementary bodies infected Mc Coy cells, harvested from bottle cultures were disrupted by Teflon coated magnetic pellet. Urografin-76 diluted with Tris-Potassium hydrochloride was used for purification of Elementary bodies of Chlamydophila abortus organism. On protein estimation of Elementary bodies by Biuret method, all the three isolates revealed protein concentration between 500-1000 mg/100ml, which were sufficient for electrophoresis. Ten percent of resolving gel and five percent of stacking gel of polyacrylamide in which 10g of processed isolate samples along with standard protein marker and Mc Coy cell protein (control were electrophoresed. Using Alpha Imager Gel Documentation System, the protein bands were analyzed. Twelve bands each for local bovine isolate and reference isolate were noticed while only 10 bands were there in the caprine isolate. Additional bands of 148 kDa and 135 kDa were present in bovine isolate, compared to the reference isolate, while 152 kDa and 137 kDa bands were unique for caprine isolate. [Vet. World 2011; 4(10.000: 470-472

  16. Novel thermal curing of cycloaliphatic resins by thiol-epoxy click process with several multifunctional thiols

    OpenAIRE

    Guzman, Dailyn; Mateu, Blai; Fernández Francos, Xavier; Ramis Juan, Xavier; Serra Albet, Àngels

    2017-01-01

    Novel thermosets were prepared by the base-catalysed reaction between a cycloaliphatic resin (ECC) and various thiol crosslinkers. 4-(N,N-Dimethylaminopyridine) (DMAP) was used as base catalyst for the thiol–epoxy reaction. A commercial tetrathiol (PETMP) and three different thiols synthesized by us, 6SH-SQ, 3SH-EU and 3SH-ISO, were tested. 6SH-SQ and 3SH-EU were prepared from vinyl or allyl compounds from renewable resources such as squalene and eugenol, respectively. Thiol 3SH-ISO was prepa...

  17. Analysis of the protein profiles of the antibiotic- resistant Salmonella ...

    African Journals Online (AJOL)

    Owner

    2005-05-18

    May 18, 2005 ... ice-cold 100% acetone and air-dried. The dried whole cell proteins and other samples (flagillin, CFUS) for 2DE were digested (100oC,. 5 min) in 4 µl of 10% SDS and dissolved in 100 µl of urea sample buffer containing 8 M urea, 4% Triton X-100, 20 mM dithiothreitol,. 2% ampholyte (pH 3.5~10) and traces ...

  18. Fabrication and bonding of thiol-ene-based microfluidic devices

    DEFF Research Database (Denmark)

    Sikanen, Tiina M; Lafleur, Josiane P.; Moilanen, Maria-Elisa

    2013-01-01

    In this work, the bonding strength of microchips fabricated by thiol-ene free-radical polymerization was characterized in detail by varying the monomeric thiol/allyl composition from the stoichiometric ratio (1:1) up to 100% excess of thiol (2:1) or allyl (1:2) functional groups. Four different...... properties for each application. Here, a capillary electrophoresis separation is performed to demonstrate the attractive properties of stoichiometric thiol-ene microchips....

  19. Transsulfuration pathway thiols and methylated arginines: the Hunter Community Study.

    Directory of Open Access Journals (Sweden)

    Arduino A Mangoni

    Full Text Available Serum homocysteine, when studied singly, has been reported to be positively associated both with the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine [ADMA, via inhibition of dimethylarginine dimethylaminohydrolase (DDAH activity] and with symmetric dimethylarginine (SDMA. We investigated combined associations between transsulfuration pathway thiols, including homocysteine, and serum ADMA and SDMA concentrations at population level.Data on clinical and demographic characteristics, medication exposure, C-reactive protein, serum ADMA and SDMA (LC-MS/MS, and thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione, and cysteinylglycine; capillary electrophoresis were collected from a sample of the Hunter Community Study on human ageing [n = 498, median age (IQR = 64 (60-70 years].REGRESSION ANALYSIS SHOWED THAT: a age (P = 0.001, gender (P = 0.03, lower estimated glomerular filtration rate (eGFR, P = 0.08, body mass index (P = 0.008, treatment with beta-blockers (P = 0.03, homocysteine (P = 0.02, and glutamylcysteine (P = 0.003 were independently associated with higher ADMA concentrations; and b age (P = 0.001, absence of diabetes (P = 0.001, lower body mass index (P = 0.01, lower eGFR (P<0.001, cysteine (P = 0.007, and glutamylcysteine (P < 0.001 were independently associated with higher SDMA concentrations. No significant associations were observed between methylated arginines and either glutathione or taurine concentrations.After adjusting for clinical, demographic, biochemical, and pharmacological confounders the combined assessment of transsulfuration pathway thiols shows that glutamylcysteine has the strongest and positive independent associations with ADMA and SDMA. Whether this reflects a direct effect of glutamylcysteine on DDAH activity (for ADMA and/or cationic amino acid transport requires further investigations.

  20. Involvement of thiol-based mechanisms in plant development.

    Science.gov (United States)

    Rouhier, Nicolas; Cerveau, Delphine; Couturier, Jérémy; Reichheld, Jean-Philippe; Rey, Pascal

    2015-08-01

    Increasing knowledge has been recently gained regarding the redox regulation of plant developmental stages. The current state of knowledge concerning the involvement of glutathione, glutaredoxins and thioredoxins in plant development is reviewed. The control of the thiol redox status is mainly ensured by glutathione (GSH), a cysteine-containing tripeptide and by reductases sharing redox-active cysteines, glutaredoxins (GRXs) and thioredoxins (TRXs). Indeed, thiol groups present in many regulatory proteins and metabolic enzymes are prone to oxidation, ultimately leading to post-translational modifications such as disulfide bond formation or glutathionylation. This review focuses on the involvement of GSH, GRXs and TRXs in plant development. Recent studies showed that the proper functioning of root and shoot apical meristems depends on glutathione content and redox status, which regulate, among others, cell cycle and hormone-related processes. A critical role of GRXs in the formation of floral organs has been uncovered, likely through the redox regulation of TGA transcription factor activity. TRXs fulfill many functions in plant development via the regulation of embryo formation, the control of cell-to-cell communication, the mobilization of seed reserves, the biogenesis of chloroplastic structures, the metabolism of carbon and the maintenance of cell redox homeostasis. This review also highlights the tight relationships between thiols, hormones and carbon metabolism, allowing a proper development of plants in relation with the varying environment and the energy availability. GSH, GRXs and TRXs play key roles during the whole plant developmental cycle via their antioxidant functions and the redox-regulation of signaling pathways. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Berle Magnus

    2011-05-01

    Full Text Available Abstract Background The mechanisms behind formation and filling of intracranial arachnoid cysts (AC are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1 Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2 Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF and plasma. Methods AC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM initiated detection and sequence analysis. Results We did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC. In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that

  2. A Search for Interstellar Monohydric Thiols

    Energy Technology Data Exchange (ETDEWEB)

    Gorai, Prasanta; Das, Ankan; Das, Amaresh; Chakrabarti, Sandip K. [Indian Centre for Space Physics, 43 Chalantika, Garia Station Rd., Kolkata, 700084 (India); Sivaraman, Bhalamurugan [Atomic Molecular and Optical Physics Division, Physical Research Laboratory, Ahmedabad, 380009 (India); Etim, Emmanuel E., E-mail: ankan.das@gmail.com [Indian Institute of Science Bangalore, 560012 (India)

    2017-02-10

    It has been pointed out by various astronomers that a very interesting relationship exists between interstellar alcohols and the corresponding thiols (sulfur analog of alcohols) as far as the spectroscopic properties and chemical abundances are concerned. Monohydric alcohols such as methanol and ethanol are widely observed and 1-propanol was recently claimed to have been seen in Orion KL. Among the monohydric thiols, methanethiol (chemical analog of methanol) has been firmly detected in Orion KL and Sgr B2(N2) and ethanethiol (chemical analog of ethanol) has been observed in Sgr B2(N2), though the confirmation of this detection is yet to come. It is very likely that higher order thiols could be observed in these regions. In this paper, we study the formation of monohydric alcohols and their thiol analogs. Based on our quantum chemical calculation and chemical modeling, we find that the Tg conformer of 1-propanethiol is a good candidate of astronomical interest. We present various spectroscopically relevant parameters of this molecule to assist in its future detection in the interstellar medium.

  3. [The Hypo Ionic Protein Profile (HIPP). Laboratory analytical evaluation in Complementary and Alternative Medicine].

    Science.gov (United States)

    Berth, M; Stalpaert, M; Bosmans, E

    2008-01-01

    The hypo ionic protein profile (HIPP) is a test based on the reticulo-endothelial index of Sandor. We evaluated the analytical performance of this test by comparing the obtained data in the HIPP to the concentration of some frequently measured specific serum proteins. The alfa euglobulin zone mainly comprises of ceruloplasmin, complement factor 3, apolipoprotein B and haptoglobin. The beta and gamma euglobulin zone reflect the concentration of the immunoglobulins. Since these proteins cannot be distinguished from each other, the diagnostic value of the HIPP will be limited. The HIPP is an outdated and aspecific assay for protein measurements.

  4. Systematic Characterisation of Cellular Localisation and Expression Profiles of Proteins Containing MHC Ligands

    DEFF Research Database (Denmark)

    Juncker, Agnieszka; Larsen, Mette Voldby; Weinhold, Nils

    2009-01-01

    Background: Presentation of peptides on Major Histocompatibility Complex (MHC) molecules is the cornerstone in immune system activation and increased knowledge of the characteristics of MHC ligands and their source proteins is highly desirable. Methodology/Principal Finding: In the present large......-scale study, we used a large data set of proteins containing experimentally identified MHC class I or II ligands and examined the proteins according to their expression profiles at the mRNA level and their Gene Ontology (GO) classification within the cellular component ontology. Proteins encoded by highly...

  5. Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

    Science.gov (United States)

    Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

    2011-01-01

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  6. Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

    International Nuclear Information System (INIS)

    Hameed, A.; Iqbal, N.; Shah, T.M.

    2012-01-01

    A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

  7. 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

  8. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

    DEFF Research Database (Denmark)

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affe...

  9. Thiol Redox Transitions in Cell Signaling: a Lesson from N-Acetylcysteine

    Directory of Open Access Journals (Sweden)

    Tiziana Parasassi

    2010-01-01

    Full Text Available The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.

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

    Science.gov (United States)

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

    2014-11-27

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

  11. Oxidation of lipid and protein in horse mackerel (Trachurus trachurus) mince and washed minces during processing and storage

    DEFF Research Database (Denmark)

    Eymard, Sylvie; Baron, Caroline; Jacobsen, Charlotte

    2009-01-01

    : M1, M2 and M3, with one, two and three washing steps, respectively. The different products were characterised (i.e. lipid content, protein, water, iron, fatty acid profile and tocopherol content) and analysed for protein and lipid oxidation in order to investigate the impact of the washing steps...... was followed by determination of protein solubility, protein thiol groups and protein carbonyl groups using colorimetric methods as well as western blotting for protein carbonyl groups. Lipid and protein oxidation markers indicated that both lipid and protein oxidation took place during processing...

  12. Analysis of protein profiles in diabetic rat blood plasma that induced by alloxan

    Science.gov (United States)

    Hidayati, Dewi; Abdulgani, Nurlita; Setiyawan, Hengki; Trisnawati, Indah; Ashuri, Nova Maulidina; Sa'adah, Noor Nailis

    2017-06-01

    Proteomics is the study to identify the proteins involved in physiological metabolic pathway. The protein profiles of blood plasma from alloxan-induced diabetic rats has investigated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Data were analyzed descriptively based on variations of the type and intensity of the protein. There were identified the similarity of protein variant between diabetic and control rats included ankyrin (200kDa), IgG (150kDa), nephrin (136 kDa), IDE (112 kDA), albumin (66 kDa), prealbumin (55 kDA), CICP (43 kDa), ApoA-V (39 kDa), GAPDH (35 kDa), C-RP (27,1 kDa), leptin (16 kDa) and apelin (13 kDa). However, the apelin profile at diabetic rats shows the higher intensity than control.

  13. Protein and Amino Acid Profile of Filial Etawah Crossbred and Castrated Filial Boer Crossbred Goat Meat

    Directory of Open Access Journals (Sweden)

    Hari Purnomo

    2012-03-01

    Full Text Available The aim of this study was to know the protein content and amino acid profile of filial Etawah and castrated Boer goat meat. The results were expected to be used as information about protein content and amino acid composition of filial Etawah and filial castrated Boer goat meat and  as a reference for further experiment about different livestock. The material of the research were loin meat, front  and back thigh of filial Etawah and filial castrated Boer goat meat. Data were analysed with t-test. The results showed that castrated filial Boer goat meat had significantly higher protein content  and 7 essensial amino acids namely lysine, leucine, arginine, phenylalanine, isoleucine, valine and histidine compared to the one from filial Etawah goat meat. Key words: protein, amino acid profiles, goat  meat

  14. HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features.

    Science.gov (United States)

    Zaman, Rianon; Chowdhury, Shahana Yasmin; Rashid, Mahmood A; Sharma, Alok; Dehzangi, Abdollah; Shatabda, Swakkhar

    2017-01-01

    DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM) as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

  15. HMMBinder: DNA-Binding Protein Prediction Using HMM Profile Based Features

    Directory of Open Access Journals (Sweden)

    Rianon Zaman

    2017-01-01

    Full Text Available DNA-binding proteins often play important role in various processes within the cell. Over the last decade, a wide range of classification algorithms and feature extraction techniques have been used to solve this problem. In this paper, we propose a novel DNA-binding protein prediction method called HMMBinder. HMMBinder uses monogram and bigram features extracted from the HMM profiles of the protein sequences. To the best of our knowledge, this is the first application of HMM profile based features for the DNA-binding protein prediction problem. We applied Support Vector Machines (SVM as a classification technique in HMMBinder. Our method was tested on standard benchmark datasets. We experimentally show that our method outperforms the state-of-the-art methods found in the literature.

  16. ANALISIS PROFIL PROTEIN DARAH ANAK KAMBING PERANAKAN ETAWAH DENGAN PEMBERIAN PAKAN SUBSTITUSI SUSU SAPI

    Directory of Open Access Journals (Sweden)

    Teguh Wicaksono

    2017-08-01

    Full Text Available The objective of this study is to determine the protein profile of pre-weaning kids fed with cow's milk as a substitute for dam’s milk. The materials used were 18 Etawah Descendant (PE kids born the twin at the age of 5-13 days from 3-4-year-old dams. This experimental design was a completely randomized design with three treatments with six replications per treatment, namely the control (T0 fed 100% goat’s milk, treatment 1 (T1 fed 50% goat’s milk and 50% cow’s milk, treatment 2 (T2 fed 100% cow’s milk. The protein profile serum was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE method, 12,5% of the resolving gel and 3% of the stacking gel were used. The protein profile of the 5-14 days old PE kids were 19 protein bands with the molecular weight ranging from 15-160 kDa. The kids fed with 100% goat milk (T0 and those substituted by 50% cow's milk (T1, it was produced 19 protein bands with molecular weights ranging from 15 kDa to 155 kDa, while those fed with 100 % cow's milk (T2, it was produced 17 protein bands with molecular weights ranging from 13 kDa to 160 kDa. It can be concluded that the dam's milk substitute using cow's milk at the 50% level does not affect the blood protein profile of goat kids, while the 100% substitute produces the different number and types of protein

  17. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    Science.gov (United States)

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-10-23

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro.

  18. Density functional study of a typical thiol tethered on a gold surface: ruptures under normal or parallel stretch

    International Nuclear Information System (INIS)

    Wang, Guan M; Sandberg, William C; Kenny, Steven D

    2006-01-01

    The mechanical and dynamical properties of a model Au(111)/thiol surface system were investigated by using localized atomic-type orbital density functional theory in the local density approximation. Relaxing the system gives a configuration where the sulfur atom forms covalent bonds to two adjacent gold atoms as the lowest energy structure. Investigations based on ab initio molecular dynamics simulations at 300, 350 and 370 K show that this tethering system is stable. The rupture behaviour between the thiol and the surface was studied by displacing the free end of the thiol. Calculated energy profiles show a process of multiple successive ruptures that account for experimental observations. The process features successive ruptures of the two Au-S bonds followed by the extraction of one S-bonded Au atom from the surface. The force required to rupture the thiol from the surface was found to be dependent on the direction in which the thiol was displaced, with values comparable with AFM measurements. These results aid the understanding of failure dynamics of Au(111)-thiol-tethered biosurfaces in microfluidic devices where fluidic shear and normal forces are of concern

  19. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

    Science.gov (United States)

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

    2016-06-20

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/.

  20. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    Science.gov (United States)

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

    Science.gov (United States)

    Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

    2012-04-01

    Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

  2. A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

    Directory of Open Access Journals (Sweden)

    T.R. Sutton

    2018-06-01

    Full Text Available Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed

  3. Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

    Science.gov (United States)

    Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

    2015-10-01

    Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

  4. Oriented Immobilization of His-Tagged Protein on a Redox Active Thiol Derivative of DPTA-Cu(II Layer Deposited on a Gold Electrode—The Base of Electrochemical Biosensors

    Directory of Open Access Journals (Sweden)

    Hanna Radecka

    2013-09-01

    Full Text Available This paper concerns the development of an electrochemical biosensor for the determination of Aβ16–23' and Aβ1–40 peptides. The His-tagged V and VC1 domains of Receptor for Advanced Glycation end Products (RAGE immobilized on a gold electrode surface were used as analytically active molecules. The immobilization of His6–RAGE domains consists of: (i formation of a mixed layer of N-acetylcysteamine (NAC and the thiol derivative of pentetic acid (DPTA; (ii complexation of Cu(II by DPTA; (iii oriented immobilization of His6–RAGE domains via coordination bonds between Cu(II sites from DPTA–Cu(II complex and imidazole nitrogen atoms of a histidine tag. Each modification step was controlled by cyclic voltammetry (CV, Osteryoung square-wave voltammetry (OSWV, and atomic force microscopy (AFM. The applicability of the proposed biosensor was tested in the presence of human plasma, which had no influence on its performance. The detection limits for Aβ1–40 determination were 1.06 nM and 0.80 nM, in the presence of buffer and human plasma, respectively. These values reach the concentration level of Aβ1–40 which is relevant for determination of its soluble form in human plasma, as well as in brain. This indicates the promising future application of biosensor presented for early diagnosis of neurodegenerative diseases.

  5. Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

    Science.gov (United States)

    Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

    2011-10-01

    Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

  6. Surface modification of cyclomatrix polyphosphazene microsphere by thiol-ene chemistry and lectin recognition

    International Nuclear Information System (INIS)

    Chen, Chen; Zhu, Xue-yan; Gao, Qiao-ling; Fang, Fei; Huang, Xiao-jun

    2016-01-01

    glucosyl microspheres. Thus, the thiol-ene modified polyphosphazene microspheres displayed chemical flexibility in post-functionalization. These microspheres can be potentially applicated in enzyme immobilization, protein adsorption and chromatographic separation.

  7. Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

    Science.gov (United States)

    Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

    2016-01-01

    Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

  8. Surface modification of cyclomatrix polyphosphazene microsphere by thiol-ene chemistry and lectin recognition

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chen; Zhu, Xue-yan; Gao, Qiao-ling; Fang, Fei; Huang, Xiao-jun, E-mail: hxjzxh@zju.edu.cn

    2016-11-30

    glucosyl microspheres. Thus, the thiol-ene modified polyphosphazene microspheres displayed chemical flexibility in post-functionalization. These microspheres can be potentially applicated in enzyme immobilization, protein adsorption and chromatographic separation.

  9. Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions.

    NARCIS (Netherlands)

    Tuomainen, M.H.; Nunan, N.; Lehesranta, S.J.; Tervahauta, A.I.; Hassinen, V.H.; Schat, H.; Koistinen, K.M.; Auriola, S.; McNicol, J.; Karenlampi, S.O.

    2006-01-01

    Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyper-accumulation, we used proteomic profiling to identify differences in protein intensities among three T caerulescens

  10. Protein profiles of field isolates ofBacillus anthracis from different endemic areas of Indonesia

    Directory of Open Access Journals (Sweden)

    M Bhakti Poerwadikarta

    1998-03-01

    Full Text Available Sonicated cell-free extract proteins of 14 field isolates ofBacillus anthracis from six different endemic areas of Indonesia were analyzed by the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE methods . The protein profiles of each field isolate tested demonstrated slightly different at the protein bands with molecular weights of 18, 37, 52, 65 and 70 kDa, and varied between the field isolates and vaccine strains. The variation could provide clues to the source of anthrax transmission whether it was originated from similar strain or not.

  11. Operation of trans-thylakoid thiol-metabolizing pathways in photosynthesis

    Directory of Open Access Journals (Sweden)

    Mohamed eKaramoko

    2013-11-01

    Full Text Available Thiol oxidation to disulfides and the reverse reaction, i.e. disulfide reduction to free thiols, are under the control of catalysts in vivo. Enzymatically assisted thiol-disulfide chemistry is required for the biogenesis of all energy-transducing membrane systems. However, until recently, this had only been demonstrated for the bacterial plasma membrane. Long considered to be vacant, the thylakoid lumen has now moved to the forefront of photosynthesis research with the realization that its proteome is far more complicated than initially anticipated. Several lumenal proteins are known to be disulfide bonded in Arabidopsis, highlighting the importance of sulfhydryl oxidation in the thylakoid lumen. While disulfide reduction in the plastid stroma is known to activate several enzymatic activities, it appears that it is the reverse reaction, i.e. thiol oxidation that is required for the activity of several lumen-resident proteins. This paradigm for redox regulation in the thylakoid lumen has opened a new frontier for research in the field of photosynthesis. Of particular significance in this context is the discovery of trans-thylakoid redox pathways controlling disulfide bond formation and reduction, which are required for photosynthesis.

  12. Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

    Science.gov (United States)

    Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

    2009-05-01

    To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

  13. Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

    Science.gov (United States)

    Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

    2017-03-01

    Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

  14. Effects of Holder pasteurization on the protein profile of human milk.

    Science.gov (United States)

    Peila, Chiara; Coscia, Alessandra; Bertino, Enrico; Cavaletto, Maria; Spertino, Stefano; Icardi, Sara; Tortone, Claudia; Visser, Gerard H A; Gazzolo, Diego

    2016-04-07

    The most widespread method for the treatment of donor milk is the Holder pasteurization (HoP). The available literature data show that HoP may cause degradation of some bioactive components. The aim of this study was to determine the effect of HoP on the protein profile of human milk (HM) using a GeLC-MS method, a proteomic approach and a promising technique able to offer a qualitative HM protein profile. HM samples were collected by standardized methods from 20 mothers carrying both preterm and term newborns. A aliquot of each sample was immediately frozen at -80 °C, whilst another one was Holder pasteurized and then frozen. All samples were then analyzed by GeLC-MS. The protein bands of interest were excised from the gel, digested with trypsin and identified by nano-HPLC-MS/MS analysis. The protein profile before and after HoP showed qualitative differences only in 6 samples out of 20, while in the remaining 14 no detectable differences were found. The differences interested only colostrums and transitional milk samples and regarded the decrease of the electrophoretic bands corresponding to alpha and beta-casein, tenascin, lactoferrin and immunoglobulin. In the majority of samples, HoP did not cause any modification, thereby preserving the biological activity of HM proteins.

  15. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    Science.gov (United States)

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  16. Major urinary protein (MUP) profiles show dynamic changes rather than individual 'barcode' signatures.

    Science.gov (United States)

    Thoß, M; Luzynski, K C; Ante, M; Miller, I; Penn, D J

    2015-06-30

    House mice ( Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This 'barcode hypothesis' requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent ('major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous ('minor') bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis ('dynamic changes' hypothesis).

  17. Spectroscopic Characterization of Extracellular Polymeric Substances from Escherichia coli and Serratia marcescens: Suppression using Sub-Inhibitory Concentrations of Bismuth Thiols

    Energy Technology Data Exchange (ETDEWEB)

    Badireddy, Appala R.; Korpol, Bhoom Reddy; Chellam, Shankararaman; Gassman, Paul L.; Engelhard, Mark H.; Lea, Alan S.; Rosso, Kevin M.

    2008-10-21

    Free and capsular EPS produced by Escherichia coli and Serratia marcescens were characterized in detail using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Auger electron spectroscopy (AES). Total EPS production decreased upon treatment with sub-inhibitory concentrations of lipophilic bismuth thiols (bismuth dimercaptopropanol, BisBAL; bismuth ethanedithiol, BisEDT; and bismuth pyrithione, BisPYR), BisBAL being most effective. Bismuth thiols also influenced acetylation and carboxylation of polysaccharides in EPS from S. marcescens. Extensive homology between EPS samples in the presence and absence of bismuth was observed with proteins, polysaccharides, and nucleic acids varying predominantly only in the total amount expressed. Second derivative analysis of the amide I region of FTIR spectra revealed decreases in protein secondary structures in the presence of bismuth thiols. Hence, anti-fouling properties of bismuth thiols appear to originate in their ability to suppress O-acetylation and protein secondary structures in addition to total EPS secretion.

  18. PROFIL PROTEIN HIPOFISA SAPI PERAH PERANAKAN FRIES HOLLAND (PFH BETINA FASE FOLIKULER DAN LUTEA

    Directory of Open Access Journals (Sweden)

    Nurul Isnaini

    2012-04-01

    Full Text Available ABSTRAK   Penelitian dilakukan dengan tujuan untuk mengetahui profil protein hipofisa sapi perah PFH betina fase folikuler dan fase luteal dengan menggunakan metode SDS-PAGE. Hasil penelitian ini diharapkan dapat digunakan sebagai acuan untuk melaksanakan pengujian jenis protein tertentu yang terdapat dalam hipofisa sapi perah PFH.Materi yang digunakan dalam penelitian ini adalah hipofisa sapi perah PFH betina Fase Folikuler dan Fase Luteal. Sampel hipofisa didapatkan dari Rumah Potong Hewan (RPH Singosari Malang. Metode penelitian yang digunakan dalam penelitian ini adalah metode observasi. Dimana sampel hipofisa sapi perah PFH fase folikuler dan fase luteal dilakukan isolasi protein, dan ditentukan Berat Molekul (BM proteinnya. Data yang diperoleh dianalisis dengan menggunakan analisis diskriptif. Hasil penelitian menunjukkan bahwa profil protein hipofisa fase folikuler dan fase luteal sapi perah PFH memiliki perbedaan. Pada hipofisa fase folikuler memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 80,6; 71,2; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pada hipofisa fase luteal memiliki 12 pita protein, yaitu 163; 112,3; 101,3; 85,8; 71,2; 60,3; 53,3; 45,2; 39,9; 32,4; 29,8; dan 24,8 kDa. Pita protein yang membedakan adalah pita dengan berat molekul 80,6 kDa hanya terdapat pada hipofisa fase folikuler, dan pita dengan berat molekul 60,3 kDa hanya terdapat pada hipofisa fase luteal. Kesimpulan yang diperoleh dari penelitian ini adalah terdapat perbedaan profil protein yang dihasilkan pada hipofisa fase folikuler dengan hipofisa fase luteal. Saran yang diberikan adalah perlu dilakukan penelitian lanjutan yaitu uji immunoblothing atau westernblothing (WB untuk memastikan keberadaan  protein-protein tertentu.   Kata kunci: Hipofisa, folikuler, luteal, berat molekul protein. HIPOPHISIS PROTEIN PROFILE OF CROSSBREED FRIESH HOLLAND DAIRY CATTLE IN FOLLICULAR AND LUTEAL PHASE   ABSTRACT   The aim of this research was to identification of

  19. Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density.

    Science.gov (United States)

    Byström, Sanna; Eklund, Martin; Hong, Mun-Gwan; Fredolini, Claudia; Eriksson, Mikael; Czene, Kamila; Hall, Per; Schwenk, Jochen M; Gabrielson, Marike

    2018-02-14

    Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

  20. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

    Science.gov (United States)

    Meinicke, Peter

    2009-09-02

    Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

  1. Protein profiling as early detection biomarkers for TiO2 nanoparticle toxicity in Daphnia magna.

    Science.gov (United States)

    Sá-Pereira, Paula; Diniz, Mário S; Moita, Liliana; Pinheiro, Teresa; Mendonça, Elsa; Paixão, Susana M; Picado, Ana

    2018-05-01

    The mode of action for nanoparticle (NP) toxicity in aquatic organisms is not yet fully understood. In this work, a strategy other than toxicity testing was applied to Daphnia magna exposed to TiO 2 -NPs: the use of nuclear microscopy and the assessment of protein profile. D. magna is a keystone species broadly used as a model system in ecotoxicology. Titanium (Ti) was found in the D. magna digestive tract, mainly in the gut. The penetration of Ti into the epithelial region was greater at higher exposure levels and also observed in eggs in the brood pouch. The protein profile of individuals exposed to different concentrations showed that 2.8 and 5.6 mg/L TiO 2 -NP concentrations induced an over-expression of the majority of proteins, in particular proteins with molecular weight of ∼120, 85 and 15 kDa, while 11.2 mg/L TiO 2 -NP had an inhibitory effect on protein expression. The Matrix-assisted laser desorption ionization with tandem time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis of these proteins consistently identified them as vitellogenin (Vtg)-like proteins, associated with enzymes involved in redox balance. These results indicate that Vtg-like proteins are up-regulated in D. magna exposed to TiO 2 -NPs. Vitellogenesis is associated with the reproduction system, suggesting that TiO 2 -NP exposure can impair reproduction by affecting this process. The precise mode of action of TiO 2 -NPs is still unclear and the results from this study are a first attempt to identify specific proteins as potential markers of TiO 2 -NP toxicity in D. magna, providing useful information for future research.

  2. Serum protein profiling and proteomics in autistic spectrum disorder using magnetic bead-assisted mass spectrometry.

    Science.gov (United States)

    Taurines, Regina; Dudley, Edward; Conner, Alexander C; Grassl, Julia; Jans, Thomas; Guderian, Frank; Mehler-Wex, Claudia; Warnke, Andreas; Gerlach, Manfred; Thome, Johannes

    2010-04-01

    The pathophysiology of autistic spectrum disorder (ASD) is not fully understood and there are no diagnostic or predictive biomarkers. Proteomic profiling has been used in the past for biomarker research in several non-psychiatric and psychiatric disorders and could provide new insights, potentially presenting a useful tool for generating such biomarkers in autism. Serum protein pre-fractionation with C8-magnetic beads and protein profiling by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-ToF-MS) were used to identify possible differences in protein profiles in patients and controls. Serum was obtained from 16 patients (aged 8-18) and age-matched controls. Three peaks in the MALDI-ToF-MS significantly differentiated the ASD sample from the control group. Sub-grouping the ASD patients into children with and without comorbid Attention Deficit and Hyperactivity Disorder, ADHD (ASD/ADHD+ patients, n = 9; ASD/ADHD- patients, n = 7), one peak distinguished the ASD/ADHD+ patients from controls and ASD/ADHD- patients. Our results suggest that altered protein levels in peripheral blood of patients with ASD might represent useful biomarkers for this devastating psychiatric disorder.

  3. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  4. Physicochemical, sensory attributes and protein profile by SDS-PAGE of beef sausage substituted with texturized vegetable protein

    Directory of Open Access Journals (Sweden)

    Hidayat, B.T.,

    2017-08-01

    Full Text Available The effect of texturized vegetable protein (TVP on the quality of beef sausages was investigated in this research. Several formulations which replaced by beef meat with TVP ranging from 10-20% w/w were investigated for their physical, chemical, sensory properties and also protein profile by SDS-PAGE. The addition of TVP concentration significantly influences physicochemical characteristics e.g. water, fat content, the color parameter (L and b value, WHC, Texture (Hardness and cooking yield (P<0.05. The protein profile also influenced by the addition of TVP in beef sausage formula. Higher substitution of meat with TVP will increase the water and will decrease fat content significantly (P<0.05. The highest water content is 40% TVP (64.02 ± 1.15% where the lowest water content is control (61.29 ± 1.88%. The highest fat content is control (12.16 ± 1.87% where the highest fat content is (8.53 ± 2.09%. For the physicochemical properties, e.g. L* and b* value, WHC and Cooking yield will increase during the substitution of meat with TVP in sausage products (P<0.05. The hardness will decrease during the substitution of meat with TVP in sausage products (P<0.05. Sensory results indicated that sensory attributed of beef sausage showed good acceptance until 30% of TVP substitution.

  5. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

    Science.gov (United States)

    Ullal, Adeeti V; Peterson, Vanessa; Agasti, Sarit S; Tuang, Suan; Juric, Dejan; Castro, Cesar M; Weissleder, Ralph

    2014-01-15

    Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

  6. SDS-Page Seed Storage Protein Profiles in Chili Peppers (Capsicum L.

    Directory of Open Access Journals (Sweden)

    Owk ANIEL KUMAR

    2010-09-01

    Full Text Available Seed protein banding patterns (SDS-PAGE were studied from eighteen genotypes of chili pepper (Capsicum L. A total of 21 protein polypeptide bands with molecular weight ranging from 18.6 to 72.0 kD were recorded. Among the genotypes CA18, CA21 and CA27 represented maximum number of protein bands (12. Band no. (11 and (5,12 are exclusive to C. annuum L. and C. frutescens L. genotypes respectively. Average percent similarity was highest (100% between CA2 and CA8 genotypes and the UPGMA dendrogram represented low genetic diversity. The study revealed that considerable intra and inter-specific differences were found in the genotypes. The variability of protein profiles in the genotypes suggested that these selected genotypes can be a good source for crop improvement through hybridization programs.

  7. The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols.

    Science.gov (United States)

    Turell, Lucía; Steglich, Martina; Alvarez, Beatriz

    2018-03-22

    Nitroalkene fatty acids can be formed in vivo and administered exogenously. They exert pleiotropic signaling actions with cytoprotective and antiinflammatory effects. The presence of the potent electron withdrawing nitro group confers electrophilicity to the adjacent β-carbon. Thiols (precisely, thiolates) are strong nucleophiles and can react with nitroalkene fatty acids through reversible Michael addition reactions. In addition, nitroalkene fatty acids can undergo several other processes including metabolic oxidation, reduction, esterification, nitric oxide release and partition into hydrophobic compartments. The signaling actions of nitroalkenes are mainly mediated by reactions with critical thiols in regulatory proteins. Thus, the thio-Michael addition reaction provides a framework for understanding the molecular basis of the biological effects of nitroalkene fatty acids at the crossroads of thiol signaling and electrophilic lipid signaling. In this review, we describe the reactions of nitroalkene fatty acids in biological contexts. We focus on the Michael addition-elimination reaction with thiols and its mechanism, and extrapolate kinetic and thermodynamic considerations to in vivo settings. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Hybrid Organic/Inorganic Thiol-ene-Based Photopolymerized Networks

    OpenAIRE

    Schreck, Kathleen M.; Leung, Diana; Bowman, Christopher N.

    2011-01-01

    The thiol-ene reaction serves as a more oxygen tolerant alternative to traditional (meth)acrylate chemistry for forming photopolymerized networks with numerous desirable attributes including energy absorption, optical clarity, and reduced shrinkage stress. However, when utilizing commercially available monomers, many thiol-ene networks also exhibit decreases in properties such as glass transition temperature (Tg) and crosslink density. In this study, hybrid organic/inorganic thiol-ene resins ...

  9. Fabrication and bonding of thiol-ene-based microfluidic devices

    International Nuclear Information System (INIS)

    Sikanen, Tiina M; Moilanen, Maria-Elisa; Lafleur, Josiane P; Zhuang, Guisheng; Jensen, Thomas G; Kutter, Jörg P

    2013-01-01

    In this work, the bonding strength of microchips fabricated by thiol-ene free-radical polymerization was characterized in detail by varying the monomeric thiol/allyl composition from the stoichiometric ratio (1:1) up to 100% excess of thiol (2:1) or allyl (1:2) functional groups. Four different thiol-ene to thiol-ene bonding combinations were tested by bonding: (i) two stoichiometric layers, (ii) two layers bearing complementary excess of thiols and allyls, (iii) two layers both bearing excess of thiols, or (iv) two layers both bearing excess of allyls. The results showed that the stiffness of the cross-linked polymer plays the most crucial role regarding the bonding strength. The most rigid polymer layers were obtained by using the stoichiometric composition or an excess of allyls, and thus, the bonding combinations (i) and (iv) withstood the highest pressures (up to the cut-off value of 6 bar). On the other hand, excess of thiol monomers yielded more elastic polymer layers and thus decreased the pressure tolerance for bonding combinations (ii) and (iii). By using monomers with more thiol groups (e.g. tetrathiol versus trithiol), a higher cross-linking ratio, and thus, greater stiffness was obtained. Surface characterization by infrared spectroscopy confirmed that the changes in the monomeric thiol/allyl composition were also reflected in the surface chemistry. The flexibility of being able to bond different types of thiol-enes together allows for tuning of the surface chemistry to yield the desired properties for each application. Here, a capillary electrophoresis separation is performed to demonstrate the attractive properties of stoichiometric thiol-ene microchips. (technical note)

  10. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    Energy Technology Data Exchange (ETDEWEB)

    Higa, A.M.; Noronha, M.D.N. [Universidade do Estado do Amazonas (UEA), Manaus, AM (Brazil). Rede Proteomica do Amazonas (Proteam). Lab. de Genomica e Proteomica; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L. [Universidade Federal do Amazonas (UFAM), Manaus, AM (Brazil). Pos-Graduacao em Biotecnologia

    2008-07-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with {approx} 60, 70 and 80 kDa were detected in gel acidic region with pI {approx} 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI {approx} 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with {approx} 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI {approx} 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course.

  11. 1-D and 2-D electrophoresis protein profiles of the scorpion venom from Brotheas amazonicus

    International Nuclear Information System (INIS)

    Higa, A.M.; Noronha, M.D.N.; Rocha-Oliveira, F.; Lopez-Lozano, J.L.L.

    2008-01-01

    Full text: Introduction: Scorpions venoms show specific neurotoxins to insect or mammals. These toxins are very important molecular tools to development of news drugs or bioinsecticides. Brotheas amazonicus scorpion is an endemic specie in Amazonian Rain Forest, but your venom do not show toxicity in humans. Information about biological specific activity on insect of this venom is not known yet. Objectives: Molecular protein toxins profiles of the venom from Brotheas amazonicus scorpion by 1-D and 2-D electrophoresis methods to detected toxins with potential biotech applications. Results: Several spots 'families' with ∼ 60, 70 and 80 kDa were detected in gel acidic region with pI ∼ 4,5 - 6 range, in the same region 1-D zimography showed proteolytic activity on gelatin and fibrinogen and proteolytic activity was inhibited by PMSF, suggesting scorpion serine proteinases. 50 kDa proteins were detected with pI ∼ 6,5 - 7 range. In 23 - 50 kDa gel acid region were observed some proteins. In 23 - 14 kDa gel acidic region were detected proteins with pI 4 - 7 range. 1-D Tris-tricine gel showed proteins with ∼ 7 kDa, suggesting scorpion neurotoxins. In gel basic region only 14 kDa proteins were observed with pI ∼ 9 - 10 range. Conclusion: Molecular profile of the scorpion venom from B. amazonicus showed proteins with high and low molecular masses, mainly with acidic pI. Proteolytic activity suggest serine proteinases with high molecular masses and 7 kDa proteins in B. amazonicus venom suggest scorpion neurotoxins. Purification and molecular characterization of these toxins are in course

  12. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  13. Transcriptional profiling of protein expression related genes of Pichia pastoris under simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Feng Qi

    Full Text Available The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG were substantially changed compared with normal gravity (NG control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes and protein transportation or secretion (82.2% of 45 genes were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.

  14. Soy Germ Protein With or Without-Zn Improve Plasma Lipid Profile in Metabolic Syndrome Women

    Directory of Open Access Journals (Sweden)

    SIWI PRAMATAMA MARS WIJAYANTI

    2012-03-01

    Full Text Available The aim of this research was to determine the effect of soy germ protein on lipid profile of metabolic syndrome (MetS patients. Respondents were 30 women with criteria, i.e. blood glucose level > normal, body mass index > 25 kg/m2, hypertriglyceridemia, low cholesterol-HDL level, 40-65 years old, living in Purwokerto, and signed the informed consent. The project was approved by the ethics committee of the Medical Faculty from Gadjah Mada University-Yogyakarta. Respondents were divided into three randomly chosen groups consisting of ten women each. The first, second, and third groups were treated, respectively, with milk enriched soy germ protein plus Zn, milk enriched soy germ protein (without Zn, and placebo for two months. Blood samples were taken at baseline, one and two months after observation. Two months after observation the groups consuming milk enriched with soy germ protein, both with or without Zn, had their level of cholesterol-total decrease from 215.8 to 180.2 mg/dl (P = 0.03, triglyceride from 240.2 to 162.5 mg/dl (P = 0.02, and LDL from 154.01 to 93.85 mg/dl (P = 0.03. In contrast, HDL increased from 38.91 to 49.49 mg/dl (P = 0.0008. In conclusion, soy germ protein can improve lipid profile, thus it can inhibit atherosclerosis incident.

  15. Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

    Directory of Open Access Journals (Sweden)

    Marian Kacerovsky

    Full Text Available OBJECTIVE: This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. METHODS: A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. RESULTS: The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. CONCLUSIONS: The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

  16. Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

    Science.gov (United States)

    Kacerovsky, Marian; Celec, Peter; Vlkova, Barbora; Skogstrand, Kristin; Hougaard, David M; Cobo, Teresa; Jacobsson, Bo

    2013-01-01

    This study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology. A retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology. The presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1. The presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

  17. Evaluation of a type 1 diabetes serum cohort by SELDI-TOF MS protein profiling

    DEFF Research Database (Denmark)

    Albrethsen, J.; Kaas, A.; Schonle, E.

    2009-01-01

    Proteomics analysis of serum from patients with type 1 diabetes (T1D) may lead to novel biomarkers for prediction of disease and for patient monitoring. However, the serum proteome is highly sensitive to sample processing and before proteomics biomarker research serum cohorts should preferably...... be examined for potential bias between sample groups. S ELDI-TOF MS protein profiling was used for preliminary evaluation of a biological-bank with 766 serum samples from 270 patients with T1D, collected at 18 different paediatric centers representing 15 countries in Europe and Japan over 2 years (2000......-2002). Samples collected 1 (n = 270), 6 (n = 248), and 12 (n = 248) months after T1D diagnosis were grouped across centers and compared. The serum protein profiles varied with collection site and day of analysis; however, markers of sample processing were not systematically different between samples collected...

  18. Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

    Science.gov (United States)

    Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

    2017-10-01

    While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Prioritization of candidate disease genes by topological similarity between disease and protein diffusion profiles.

    Science.gov (United States)

    Zhu, Jie; Qin, Yufang; Liu, Taigang; Wang, Jun; Zheng, Xiaoqi

    2013-01-01

    Identification of gene-phenotype relationships is a fundamental challenge in human health clinic. Based on the observation that genes causing the same or similar phenotypes tend to correlate with each other in the protein-protein interaction network, a lot of network-based approaches were proposed based on different underlying models. A recent comparative study showed that diffusion-based methods achieve the state-of-the-art predictive performance. In this paper, a new diffusion-based method was proposed to prioritize candidate disease genes. Diffusion profile of a disease was defined as the stationary distribution of candidate genes given a random walk with restart where similarities between phenotypes are incorporated. Then, candidate disease genes are prioritized by comparing their diffusion profiles with that of the disease. Finally, the effectiveness of our method was demonstrated through the leave-one-out cross-validation against control genes from artificial linkage intervals and randomly chosen genes. Comparative study showed that our method achieves improved performance compared to some classical diffusion-based methods. To further illustrate our method, we used our algorithm to predict new causing genes of 16 multifactorial diseases including Prostate cancer and Alzheimer's disease, and the top predictions were in good consistent with literature reports. Our study indicates that integration of multiple information sources, especially the phenotype similarity profile data, and introduction of global similarity measure between disease and gene diffusion profiles are helpful for prioritizing candidate disease genes. Programs and data are available upon request.

  20. Plasma Protein Profiles Differ Between Women Diagnosed with Cervical Intraepithelial Neoplasia (CIN 1 and 3

    Directory of Open Access Journals (Sweden)

    Edward E. Partridge

    2006-01-01

    Full Text Available Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR-human papillomavirus (HPV are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI time of flight (TOF mass spectrometry (MS protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3 from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases and 28 women with CIN1 (controls. Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge (m-z values, an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions.

  1. Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

    Directory of Open Access Journals (Sweden)

    Rotem Gura Sadovsky

    2017-03-01

    Full Text Available The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists.

  2. Thiol X Click Foldamers for Polymer Affinity

    Science.gov (United States)

    2016-06-24

    polymers   e. Invention  of  a  novel,  robust,  and  ambient   polymerization ...efficiently   polymerized   to   moderate  sized   polymers  capable  of  forming  >>1012  sequence  distinct   polymers ... polymerization  of  nucleobase  appended   thiol-­‐ene  monomers.    Naturally,   the  average   composition  of  the  

  3. Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab.

    Directory of Open Access Journals (Sweden)

    Kenta Mukaihara

    Full Text Available Giant cell tumors of bone (GCTB are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the

  4. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

    Science.gov (United States)

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

    2016-02-18

    Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

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

    Science.gov (United States)

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

    2018-03-13

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

  6. Dietary live yeast alters metabolic profiles, protein biosynthesis and thermal stress tolerance of Drosophila melanogaster.

    Science.gov (United States)

    Colinet, Hervé; Renault, David

    2014-04-01

    The impact of nutritional factors on insect's life-history traits such as reproduction and lifespan has been excessively examined; however, nutritional determinant of insect's thermal tolerance has not received a lot of attention. Dietary live yeast represents a prominent source of proteins and amino acids for laboratory-reared drosophilids. In this study, Drosophila melanogaster adults were fed on diets supplemented or not with live yeast. We hypothesized that manipulating nutritional conditions through live yeast supplementation would translate into altered physiology and stress tolerance. We verified how live yeast supplementation affected body mass characteristics, total lipids and proteins, metabolic profiles and cold tolerance (acute and chronic stress). Females fed with live yeast had increased body mass and contained more lipids and proteins. Using GC/MS profiling, we found distinct metabolic fingerprints according to nutritional conditions. Metabolite pathway enrichment analysis corroborated that live yeast supplementation was associated with amino acid and protein biosyntheses. The cold assays revealed that the presence of dietary live yeast greatly promoted cold tolerance. Hence, this study conclusively demonstrates a significant interaction between nutritional conditions and thermal tolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

    Science.gov (United States)

    Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

    2016-11-01

    usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

  8. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

    Science.gov (United States)

    Sinclair, John; Timms, John F

    2011-08-01

    Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus

    DEFF Research Database (Denmark)

    Sebastiana, Mónica; Martins, Joana; Figueiredo, Andreia

    2017-01-01

    in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus...... to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered...... in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root...

  10. Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

    DEFF Research Database (Denmark)

    Sultan, Abida; Andersen, Birgit; Svensson, Birte

    2016-01-01

    Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant...... xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC–MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface......-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation...

  11. Proteomic Profiling Comparing the Effects of Different Heat Treatments on Camel (Camelus dromedarius) Milk Whey Proteins.

    Science.gov (United States)

    Benabdelkamel, Hicham; Masood, Afshan; Alanazi, Ibrahim O; Alzahrani, Dunia A; Alrabiah, Deema K; AlYahya, Sami A; Alfadda, Assim A

    2017-03-28

    Camel milk is consumed in the Middle East because of its high nutritional value. Traditional heating methods and the duration of heating affect the protein content and nutritional quality of the milk. We examined the denaturation of whey proteins in camel milk by assessing the effects of temperature on the whey protein profile at room temperature (RT), moderate heating at 63 °C, and at 98 °C, for 1 h. The qualitative and quantitative variations in the whey proteins before and after heat treatments were determined using quantitative 2D-difference in gel electrophoresis (DIGE)-mass spectrometry. Qualitative gel image analysis revealed a similar spot distribution between samples at RT and those heated at 63 °C, while the spot distribution between RT and samples heated at 98 °C differed. One hundred sixteen protein spots were determined to be significantly different ( p protein spots were decreased in common in both the heat-treated samples and an additional 25 spots were further decreased in the 98 °C sample. The proteins with decreased abundance included serum albumin, lactadherin, fibrinogen β and γ chain, lactotransferrin, active receptor type-2A, arginase-1, glutathione peroxidase-1 and, thiopurine S, etc. Eight protein spots were increased in common to both the samples when compared to RT and included α-lactalbumin, a glycosylation-dependent cell adhesion molecule. Whey proteins present in camel milk were less affected by heating at 63 °C than at 98 °C. This experimental study showed that denaturation increased significantly as the temperature increased from 63 to 98 °C.

  12. Protein Profile in Corpus Luteum during Pregnancy in Korean Native Cows

    Directory of Open Access Journals (Sweden)

    H. J. Chung

    2012-11-01

    Full Text Available Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5 and pregnant cows (n = 6; until d-90. A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each. Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4 was compared with that in CL of cyclic cows at d 6 to 13 (n = 5. Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE, and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS. Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.

  13. A novel strategy for global analysis of the dynamic thiol redox proteome.

    Science.gov (United States)

    Martínez-Acedo, Pablo; Núñez, Estefanía; Gómez, Francisco J Sánchez; Moreno, Margoth; Ramos, Elena; Izquierdo-Álvarez, Alicia; Miró-Casas, Elisabet; Mesa, Raquel; Rodriguez, Patricia; Martínez-Ruiz, Antonio; Dorado, David Garcia; Lamas, Santiago; Vázquez, Jesús

    2012-09-01

    Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.

  14. Profile of total protein, albumin, globulin and albumin globulin ratio in bulls

    Directory of Open Access Journals (Sweden)

    Ida Zahidah Irfan

    2014-06-01

    Full Text Available Determination of serum total protein concentration and main fractions (albumin and globulin can be used as an important diagnostic tool in clinical biochemistry. Several factors can affect the concentration of total protein, albumin, globulin and albumin globulin ratio (A/G. The aim of this study is to obtain serum protein profiles, albumin, globulin and A/G ratio based on breed, age and BCS (body condition score. Blood samples from 160 bulls were collected. Blood chemistry were analyzed by photometer principle using a commercial kit. There were significant (P<0.001 breed variation on total protein, albumin, globulin and albumin globulin ratio. Significant age differences were observed on total protein and albumin concentration (P<0.001, while globulin concentration and A/G ratio were also significant (P<0.05. Amongs groups of BCS, significant difference was verified only in the albumin concentration (P<0.05. The concentration of total proteins, albumins and globulins in the serum of the bulls are higher than standard values for cattle, while A/G ratio is lower.

  15. Insights into the immune manipulation mechanisms of pollen allergens by protein domain profiling.

    Science.gov (United States)

    Patel, Seema; Rani, Aruna; Goyal, Arun

    2017-10-01

    Plant pollens are airborne allergens, as their inhalation causes immune activation, leading to rhinitis, conjunctivitis, sinusitis and oral allergy syndrome. A myriad of pollen proteins belonging to profilin, expansin, polygalacturonase, glucan endoglucosidase, pectin esterase, and lipid transfer protein class have been identified. In the present in silico study, the protein domains of fifteen pollen sequences were extracted from the UniProt database and submitted to the interactive web tool SMART (Simple Modular Architecture Research Tool), for finding the protein domain profiles. Analysis of the data based on custom-made scripts revealed the conservation of pathogenic domains such as OmpH, PROF, PreSET, Bet_v_1, Cpl-7 and GAS2. Further, the retention of critical domains like CHASE2, Galanin, Dak2, DALR_1, HAMP, PWI, EFh, Excalibur, CT, PbH1, HELICc, and Kelch in pollen proteins, much like cockroach allergens and lethal viruses (such as HIV, HCV, Ebola, Dengue and Zika) was observed. Based on the shared motifs in proteins of taxonomicall-ydispersed organisms, it can be hypothesized that allergens and pathogens manipulate the human immune system in a similar manner. Allergens, being inanimate, cannot replicate in human body, and are neutralized by immune system. But, when the allergens are unremitting, the immune system becomes persistently hyper-sensitized, creating an inflammatory milieu. This study is expected to contribute to the understanding of pollen allergenicity and pathogenicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Role of endogenous thiols in protection

    Science.gov (United States)

    Vos, O.

    Aminothiols represent the most important group of radioprotective compounds. The most effective compounds administered at an optimal dose and time before irradiation are able to provide a protection in mice with a dose reduction factor (DRF) of about 2-2.5. The working mechanism can partly be explained as a scavenging process of radicals induced in water and partly as a chemical repair process of injured DNA. The endogenous aminothiol which has far-out the highest intracellular concentration is glutathione (GSH). The importance of intracellular GSH in determining cellular radiosensitivity has been shown by irradiating cells that had very low GSH levels. Such cells appear to have a high radiosensitivity, especially in hypoxic conditions. On the other hand, it has been demonstrated that induction of a high GSH level (100-200% above the normal level) provides only a small protection. In vitro experiments with DNA indicate that thiols with a high positive charge condense in the vicinity of DNA and are effective protectors, whereas thiols with a negative charge are kep away from it and are poor protectors. In comparison with the most effective exogenous aminothiols like cysteamine and WR1065, GSH is not an effective radioprotector. Putative explanations for this relatively poor protective ability of GSH are presented.

  17. Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

    Directory of Open Access Journals (Sweden)

    Haddy K S Fye

    Full Text Available Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose

  18. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences

    Directory of Open Access Journals (Sweden)

    Meinicke Peter

    2009-09-01

    Full Text Available Abstract Background Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Description Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. Conclusion For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

  19. Chitosan scaffold modified with D-(+) raffinose and enriched with thiol-modified gelatin for improved osteoblast adhesion

    International Nuclear Information System (INIS)

    Galli, C; Parisi, L; Smerieri, A; Lumetti, S; Manfredi, E; Macaluso, G M; Elviri, L; Bianchera, A; Bettini, R; Lagonegro, P

    2016-01-01

    The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml −1 BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties. (paper)

  20. Electrophoretic protein profiles of mid-sized copepod Calanoides patagoniensis steadily fed bloom-forming diatoms

    Directory of Open Access Journals (Sweden)

    Victor M Aguilera

    2015-09-01

    Full Text Available Recent field and experimental evidence collected in the southern upwelling region off Concepción (36°5'S, 73°3'W showed an abrupt reduction (<72 h in the egg production rates (EPR of copepods when they were fed steadily and solely with the local bloom-forming diatom Thalassiosira rotula. Because diatoms were biochemically similar to dinoflagellate Prorocentrum minimum, a diet which supported higher reproductive outcomes, the fecundity reduction observed in copepod females fed with the diatom may have obeyed to post-ingestive processes, giving rise to resources reallocation. This hypothesis was tested by comparing feeding (clearance and ingestion rates, reproduction (EPR and hatching success and the structure of protein profiles (i.e., number and intensity of electrophoretic bands of copepods (adults and eggs incubated during 96 h with the two food conditions. The structure of protein profiles included molecular sizes that were calculated from the relative mobility of protein standards against the logarithm of their molecular sizes. After assessing the experimental conditions, feeding decreased over time for those females fed with T. rotula, while reproduction was higher in females fed with P. minimum. Electrophoretic profiles resulted similar mostly at a banding region of 100 to 89-kDa, while they showed partial differences around the region of 56-kDa band, especially in those females fed and eggs produced with T. rotula. Due to reproductive volume was impacted while larvae viability, a physiological processes with specific and high nutritional requirements, was independent on food type; post-ingestive processes, such as expression of stress-related proteins deviating resources to metabolic processes others than reproduction, are discussed under framework of nutritional-toxic mechanisms mediating copepod-diatoms relationships in productive upwelling areas.

  1. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

    Directory of Open Access Journals (Sweden)

    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  2. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    International Nuclear Information System (INIS)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi

    2014-01-01

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au 8 -clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au 8 -cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au 8 -cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au 8 -cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au 8 -cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au 8 -cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot

  3. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi, E-mail: shuyi@nhri.org.tw

    2014-11-07

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au{sub 8}-clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au{sub 8}-cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au{sub 8}-cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au{sub 8}-cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au{sub 8}-cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au{sub 8}-cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot.

  4. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

  5. Profiles

    International Nuclear Information System (INIS)

    2004-01-01

    Profiles is a synthetic overview of more than 100 national energy markets in the world, providing insightful facts and key energy statistics. A Profile is structured around 6 main items and completed by key statistics: Ministries, public agencies, energy policy are concerned; main companies in the oil, gas, electricity and coal sectors, status, shareholders; reserve, production, imports and exports, electricity and refining capacities; deregulation of prices, subsidies, taxes; consumption trends by sector, energy market shares; main energy projects, production and consumption prospects. Statistical Profiles are present in about 3 pages the main data and indicators on oil, gas, coal and electricity. (A.L.B.)

  6. Discovery of protein profiles for differentiated thyroid cancer using SELDI TOF MS

    International Nuclear Information System (INIS)

    Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young

    2003-01-01

    Low sensitivity of diagnostic whole body iodine scintigraphy and intermediate range of serum thyroglobulin (Tg) with or without anti-Tg antibody make it difficult to select the patients with differentiated thyroid cancer who need further treatment. Surfaced Enhanced Laser Desorption /Ionization - Time of Flight - Mass Spectrometry (SELDI TOF MS) is a useful method to evaluate cancer proteome, biomarkers and patterns of biomarkers. In this preliminary study, we evaluated and developed protein profiles for the discrimination between patients with differentiated thyroid cancer and non-cancer controls using SELDI technology. Serum samples from 10 healthy controls and from 14 patients with papillary thyroid cancer before thyroidectomy were analyzed by SELDI MS. Multiple protein peaks detected were analyzed by the computer software to develop a classifier for separating cancer patients form controls. The classifier was then challenged to 24 serum samples to determine the validity and accuracy of the classification system. All patients with papillary thyroid cancer had no other concomitant cancer or thyroiditis. Their serum Tg concentration was 55.8 (1.5 - 249.7) and 2 patients had extra-thyroidal extension. According to the SELDI analysis, protein peaks at 3696 Da, 4178 Da, and 8149 Da were more prominent in cancer patients than controls in various degrees. Among those, protein peak at 4178 Da was determined as classifier by computer software, and the sensitivity, specificity and accuracy for discrimination of cancer patients from controls was 92.9% (13/14), 90% (9/10) and 91.7% respectively. This preliminary study suggests that serum protein profiles of differentiated thyroid cancer can be used for differentiation between cancer patients and non-cancer controls. And further clinical studies in various test sets will offer useful information in selecting patients who require treatment

  7. Discovery of protein profiles for differentiated thyroid cancer using SELDI TOF MS

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Joon Kee; Lee, Myung Hoon; Joh, Chul Woo; Yoon, Seok Nam; Soh, Eui Young [College of Medicine, Univ. of Ajou, Suwon (Korea, Republic of)

    2003-07-01

    Low sensitivity of diagnostic whole body iodine scintigraphy and intermediate range of serum thyroglobulin (Tg) with or without anti-Tg antibody make it difficult to select the patients with differentiated thyroid cancer who need further treatment. Surfaced Enhanced Laser Desorption /Ionization - Time of Flight - Mass Spectrometry (SELDI TOF MS) is a useful method to evaluate cancer proteome, biomarkers and patterns of biomarkers. In this preliminary study, we evaluated and developed protein profiles for the discrimination between patients with differentiated thyroid cancer and non-cancer controls using SELDI technology. Serum samples from 10 healthy controls and from 14 patients with papillary thyroid cancer before thyroidectomy were analyzed by SELDI MS. Multiple protein peaks detected were analyzed by the computer software to develop a classifier for separating cancer patients form controls. The classifier was then challenged to 24 serum samples to determine the validity and accuracy of the classification system. All patients with papillary thyroid cancer had no other concomitant cancer or thyroiditis. Their serum Tg concentration was 55.8 (1.5 - 249.7) and 2 patients had extra-thyroidal extension. According to the SELDI analysis, protein peaks at 3696 Da, 4178 Da, and 8149 Da were more prominent in cancer patients than controls in various degrees. Among those, protein peak at 4178 Da was determined as classifier by computer software, and the sensitivity, specificity and accuracy for discrimination of cancer patients from controls was 92.9% (13/14), 90% (9/10) and 91.7% respectively. This preliminary study suggests that serum protein profiles of differentiated thyroid cancer can be used for differentiation between cancer patients and non-cancer controls. And further clinical studies in various test sets will offer useful information in selecting patients who require treatment.

  8. Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

    Science.gov (United States)

    Raghav, Sunil Kumar; Deplancke, Bart

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

  9. Predicting adverse drug reaction profiles by integrating protein interaction networks with drug structures.

    Science.gov (United States)

    Huang, Liang-Chin; Wu, Xiaogang; Chen, Jake Y

    2013-01-01

    The prediction of adverse drug reactions (ADRs) has become increasingly important, due to the rising concern on serious ADRs that can cause drugs to fail to reach or stay in the market. We proposed a framework for predicting ADR profiles by integrating protein-protein interaction (PPI) networks with drug structures. We compared ADR prediction performances over 18 ADR categories through four feature groups-only drug targets, drug targets with PPI networks, drug structures, and drug targets with PPI networks plus drug structures. The results showed that the integration of PPI networks and drug structures can significantly improve the ADR prediction performance. The median AUC values for the four groups were 0.59, 0.61, 0.65, and 0.70. We used the protein features in the best two models, "Cardiac disorders" (median-AUC: 0.82) and "Psychiatric disorders" (median-AUC: 0.76), to build ADR-specific PPI networks with literature supports. For validation, we examined 30 drugs withdrawn from the U.S. market to see if our approach can predict their ADR profiles and explain why they were withdrawn. Except for three drugs having ADRs in the categories we did not predict, 25 out of 27 withdrawn drugs (92.6%) having severe ADRs were successfully predicted by our approach. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Variance decomposition of protein profiles from antibody arrays using a longitudinal twin model

    Directory of Open Access Journals (Sweden)

    Kato Bernet S

    2011-11-01

    Full Text Available Abstract Background The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental components. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components. Results Using antibody suspension bead arrays and antibodies from the Human Protein Atlas, we study unfractionated serum from a longitudinal study on 154 twins. In this study, we provide a detailed description of how the variation in a molecular phenotype in terms of protein profile can be decomposed into familial i.e. genetic and common environmental; individual environmental, short-term biological and experimental components. The results show that across 69 antibodies analyzed in the study, the median proportion of the total variation explained by familial sources is 12% (IQR 1-22%, and the median proportion of the total variation attributable to experimental sources is 63% (IQR 53-72%. Conclusion The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discoveries with exploratory, high-throughput and multiplexed methods.

  11. Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

    Science.gov (United States)

    Yu, Xiaomin; Yuan, Fengjie; Fu, Xujun; Zhu, Danhua

    2016-04-01

    Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement. Copyright © 2015. Published by Elsevier Ltd.

  12. Protein profile analysis of Malaysian snake venoms by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    J Vejayan

    2010-01-01

    Full Text Available Snake venoms comprise a highly complex mixture of proteins, which requires for their characterization the use of versatile two-dimensional electrophoresis techniques. In the present study, venoms obtained from eight snakes (Ophiophagus hannah, Naja kaouthia, Naja sumatrana, Bungarus fasciatus, Trimeresurus sumatranus, Tropidolaemus wagleri, Enhydrina schistosa and Calloselasma rhodostoma commonly found in Malaysia were separated based on two independent properties, isoelectric point (pI and molecular weight (MW. Many differences in snake venoms at the inter-family, inter-subfamily, inter-genus and inter-species levels were revealed. Notably, proteins from individuals of the Viperidae family - Trimeresurus sumatranus, Tropidolaemus wagleri and Calloselasma rhodostoma - were found to be numerous and scattered by the two-dimensional gel electrophoresis (2DE specifically in regions between 37 and 100 kDa compared to the Elapidae venom proteins. The latter were clustered at the basic and lower molecular mass region (less than 20 kDa. Trains of spots were commonly observed, indicating that these proteins may be derived from post-translational modifications. Ophiophagus hannah (Elapidae revealed a great amount of protein spots in the higher molecular mass range when compared to Enhydrina schistosa, Naja kaouthia, Naja sumatrana and Bungarus fasciatus. Overall 2DE showed large differences in the venom profile of each species, which might be employed as an ancillary tool to the identification of venomous snake species.

  13. Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers.

    Science.gov (United States)

    Thelin, Eric Peter; Just, David; Frostell, Arvid; Häggmark-Månberg, Anna; Risling, Mårten; Svensson, Mikael; Nilsson, Peter; Bellander, Bo-Michael

    2018-03-15

    The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Sprague Dawley rats (n=73) received a 3mm deep controlled cortical impact ("severe injury"). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  14. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: Subcellular distribution, chemical forms and thiol pools

    International Nuclear Information System (INIS)

    Weng Bosen; Xie Xiangyu; Weiss, Dominik J.; Liu Jingchun; Lu Haoliang; Yan Chongling

    2012-01-01

    Highlights: ► Cadmium tolerance mechanisms of Kandelia obovata was investigated systematacially. ► Thiol pool can play roles in cadmium detoxification mechanisms. ► Increasing cadmium treatment strength caused proportional increase of cadmium uptake. ► More than half of cadmium was localized in cell walls, and lowest in membranes. ► Sodium chloride and acetic acid extractable fractions were dominant. - Abstract: In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  15. Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

    Directory of Open Access Journals (Sweden)

    Yinliang Yang

    2013-10-01

    Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

  16. Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor

    Science.gov (United States)

    Diehl, Katharine L.; Kolesnichenko, Igor V.; Robotham, Scott A.; Bachman, J. Logan; Zhong, Ye; Brodbelt, Jennifer S.; Anslyn, Eric V.

    2016-10-01

    The coupling and decoupling of molecular units is a fundamental undertaking of organic chemistry. Herein we report the use of a very simple conjugate acceptor, derived from Meldrum's acid, for the sequential ‘clicking’ together of an amine and a thiol in aqueous conditions at neutral pH. Subsequently, this linkage can be ‘declicked’ by a chemical trigger to release the original amine and thiol undisturbed. The reactivity differs from that of other crosslinking agents because the selectivity for sequential functionalization derives from an altering of the electrophilicity of the conjugate acceptor on the addition of the amine. We describe the use of the procedure to modify proteins, create multicomponent libraries and synthesize oligomers, all of which can be declicked to their starting components in a controlled fashion when desired. Owing to the mild reaction conditions and ease of use in a variety of applications, the method is predicted to have wide utility.

  17. Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

    Science.gov (United States)

    M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2016-11-01

    The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

    Science.gov (United States)

    Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

    2017-12-01

    It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

  19. Methionine sulfoxide profiling of milk proteins to assess the influence of lipids on protein oxidation in milk.

    Science.gov (United States)

    Wüst, Johannes; Pischetsrieder, Monika

    2016-06-15

    Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC-ESI-MS/MS-sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk.

  20. Identification of Protein Pupylation Sites Using Bi-Profile Bayes Feature Extraction and Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Xiaowei Zhao

    2013-01-01

    Full Text Available Pupylation, one of the most important posttranslational modifications of proteins, typically takes place when prokaryotic ubiquitin-like protein (Pup is attached to specific lysine residues on a target protein. Identification of pupylation substrates and their corresponding sites will facilitate the understanding of the molecular mechanism of pupylation. Comparing with the labor-intensive and time-consuming experiment approaches, computational prediction of pupylation sites is much desirable for their convenience and fast speed. In this study, a new bioinformatics tool named EnsemblePup was developed that used an ensemble of support vector machine classifiers to predict pupylation sites. The highlight of EnsemblePup was to utilize the Bi-profile Bayes feature extraction as the encoding scheme. The performance of EnsemblePup was measured with a sensitivity of 79.49%, a specificity of 82.35%, an accuracy of 85.43%, and a Matthews correlation coefficient of 0.617 using the 5-fold cross validation on the training dataset. When compared with other existing methods on a benchmark dataset, the EnsemblePup provided better predictive performance, with a sensitivity of 80.00%, a specificity of 83.33%, an accuracy of 82.00%, and a Matthews correlation coefficient of 0.629. The experimental results suggested that EnsemblePup presented here might be useful to identify and annotate potential pupylation sites in proteins of interest. A web server for predicting pupylation sites was developed.

  1. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    Science.gov (United States)

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob

    2005-01-01

    for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI...

  3. Thiol derivatization of Xanthan gum and its evaluation as a mucoadhesive polymer.

    Science.gov (United States)

    Bhatia, Meenakshi; Ahuja, Munish; Mehta, Heena

    2015-10-20

    Thiol-derivatization of xanthan gum polysaccharide was carried out by esterification with mercaptopropionic acid and thioglycolic acid. Thiol-derivatization was confirmed by Fourier-transformed infra-red spectroscopy. Xanthan-mercaptopropionic acid conjugate and xanthan-thioglycolic acid conjugate were found to possess 432.68mM and 465.02mM of thiol groups as determined by Ellman's method respectively. Comparative evaluation of mucoadhesive property of metronidazole loaded buccal pellets of xanthan and thiolated xanthan gum using chicken buccal pouch membrane revealed higher ex vivo bioadhesion time of thiolated xanthan gum as compared to xanthan gum. Improved mucoadhesive property of thiolated xanthan gum over the xanthan gum can be attributed to the formation of disulfide bond between mucus and thiolated xanthan gum. In vitro release study conducted using phosphate buffer (pH 6.8) revealed a sustained release profile of metronidazole from thiolated xanthan pellets as compared to xanthan pellets. In conclusion, thiolation of xanthan improves its mucoadhesive property and sustained the release of metronidazole over a prolonged period. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Thiol-linked alkylation of RNA to assess expression dynamics.

    Science.gov (United States)

    Herzog, Veronika A; Reichholf, Brian; Neumann, Tobias; Rescheneder, Philipp; Bhat, Pooja; Burkard, Thomas R; Wlotzka, Wiebke; von Haeseler, Arndt; Zuber, Johannes; Ameres, Stefan L

    2017-12-01

    Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s 4 U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N 6 -methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

  5. Genetic characteristic of swamp buffalo (Bubalus bubalis) from Pampangan, South Sumatra based on blood protein profile

    Science.gov (United States)

    Windusari, Yuanita; Hanum, Laila; Wahyudi, Rizki

    2017-11-01

    Swamp buffalo (Bubalus bubalis) is an endemic species and one of the genetic wealth of South Sumatra with a distribution area in the district of Pampangan (OganIlir and OganOganIlir). Suspected inbreeding causes decreased phenotypic properties. Inbreeding among various swamp buffalo is certainly not only lower the qualities but also genotypes and phenotypes. It is of interest to determine kinship variants swamp buffaloes from Pampangan through the analysis of a blood protein profile. Blood protein profile of four variants swamps buffalo was studied by using five electrophoresis system i.e. pre-albumin (Palb), albumin (Alb), ceruloplasmin (Cp), transferrin (Tf) and transferrin post (Ptf). In this paper, it is obtained that there was no significant differences among the four variants of the buffaloes were used as a sample. Prealbumin has two alleles (Palb1 and Palb2), albumin has three alleles (Alba, AlbB, AlbC), ceruloplasmin has one allele (BPA), post-transferrin has one allele (PTFA) with an allele frequency 1.0000 at any time transferrin has two alleles (TFA and TFB) with the allele frequency of 0.7500 and 1.0000. Characteristics prealbumin (Palb), albumin (Alb), ceruloplasmin (Cp), and post-transferrin (P-tf) is monomorphic, while transferrin is polymorphic average heterozygosity values all loci (H) 0.1286. Based on average heterozygosity, the swamp buffalo (Bubalusbubalis) from Pampangan has low genetic variation and closest genetic relationship.

  6. Micropropagation and protein profile analysis by SDS-PAGE of Gracilaria changii (Rhodophyta, Solieriaceae

    Directory of Open Access Journals (Sweden)

    Lin Wei Jong

    2015-05-01

    Full Text Available Gracilaria changii seaweed is primarily important as a source of agar with wide applications in food industries. The high demand of agar led to gradual depletion of G. changii in natural resources. Establishment of in vitro culture of G. changii has an important role and allowing G. changii explants to grow optimally under controlled conditions to provide constant, continuous and sufficient seedlings supply for Gracilaria farming. This study focused on micropropagation culture of G. changii in which different exogenous factors influencing seaweed growth were investigated: strength of chosen medium Provasoli’s enriched seawater (PES, types and concentration of fertilizers/biostimulant, supplementation of plant growth regulators and seawater salinity. The results were presented in daily growth rate of explants and data analysis was carried out using one-way ANOVA. The results demonstrated high growth rate of G. changii in 25% of PES supplemented with 5 mg L−1 AMPEP, and seawater salinity range between 30 and 40 ppt, respectively. Protein profiles of tissue-cultured and farm cultivated G. changii were produced by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. The results demonstrated no remarkable difference in the protein profiles and indicated the suitability of the culture condition for the growth of G. changii.

  7. Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature.

    Science.gov (United States)

    Wölter, M; Röwer, C; Koy, C; Rath, W; Pecks, U; Glocker, M O

    2016-10-21

    Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III 0 . Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III 1 , and apolipoprotein C-III 2 , which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics. Identification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired. It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement protein composition (IUGR signature) which can be determined just ahead of delivery and at date of delivery, respectively using a minimal invasive blood sampling approach. With this manuscript we describe the use of a mass spectrometric profiling method of 30

  8. Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13

    KAUST Repository

    Narasimhan, Kothandaraman

    2013-02-01

    Objectives: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. Methods: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Results: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Conclusions: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies. © 2013 John Wiley & Sons, Ltd.

  9. Surface functionalized thiol-ene waveguides for fluorescence biosensing in microfluidic devices

    DEFF Research Database (Denmark)

    Feidenhans'l, Nikolaj Agentoft; Lafleur, Josiane P.; Jensen, Thomas Glasdam

    2013-01-01

    -ene waveguides were fabricated from 40% excess thiol thiol-ene to ensure the presence of thiol functional groups at the surface of the waveguide. Biotin alkyne was photografted at specific locations using a photomask, directly at the interface between the microfluidic channel and the thiol-ene waveguide prior...

  10. Profil Protein Trypanosoma evansi dari Daerah Geografis Berbeda di Indonesia Tahun 2012-2014 dengan Sodium Dodecil Sulphate Polyacrylamide Gel Electrophoresis (TRYPANOSOMA EVANSI PROTEIN PROFILE OF DIFFERENT GEOGRAPHICAL AREAS ORIGIN IN INDONESIA

    Directory of Open Access Journals (Sweden)

    Fitrine Ekawasti

    2018-01-01

    Full Text Available Surra outbreak in 2012 has led to more than 1,700 animals have died in the province of East Nusa Tenggara (NTT Indonesia. Surra case sporadically continues throughout the year in various areas, especially Kalimantan, Banten as well as other areas. Some reports reveal differences in protein profiles among multiple isolates of T. evansi. Therefore the purpose of this research were to find out the protein profile of each isolate T. evansi in Indonesia and the possible biological differences among them. Eleven isolates originating from the province of East Nusa Tenggara, South Kalimantan and Central Kalimantan, Banten, Lampung and Bengkulu has been isolated and purified Using DEAE. Trypanosoma isolate were frezeethawing repeatedly to obtain soluble protein. Furthermore, soluble protein is treated with heating or without heating and then each was run on SDS PAGE with Coomassie Blue staining. The protein profiles of all isolates were compared each other. The results showed that eleven isolates of T. evansi in Indonesia has a very diverse protein profile. Then for the purposes of development of diagnostic kit can be used whole lysate cell (WCL as stock antigen in serological test process.

  11. Phylogenetic profiles of all membrane transport proteins of the malaria parasite highlight new drug targets

    Directory of Open Access Journals (Sweden)

    January Weiner 3rd

    2016-08-01

    Full Text Available In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date.

  12. SVM-PB-Pred: SVM based protein block prediction method using sequence profiles and secondary structures.

    Science.gov (United States)

    Suresh, V; Parthasarathy, S

    2014-01-01

    We developed a support vector machine based web server called SVM-PB-Pred, to predict the Protein Block for any given amino acid sequence. The input features of SVM-PB-Pred include i) sequence profiles (PSSM) and ii) actual secondary structures (SS) from DSSP method or predicted secondary structures from NPS@ and GOR4 methods. There were three combined input features PSSM+SS(DSSP), PSSM+SS(NPS@) and PSSM+SS(GOR4) used to test and train the SVM models. Similarly, four datasets RS90, DB433, LI1264 and SP1577 were used to develop the SVM models. These four SVM models developed were tested using three different benchmarking tests namely; (i) self consistency, (ii) seven fold cross validation test and (iii) independent case test. The maximum possible prediction accuracy of ~70% was observed in self consistency test for the SVM models of both LI1264 and SP1577 datasets, where PSSM+SS(DSSP) input features was used to test. The prediction accuracies were reduced to ~53% for PSSM+SS(NPS@) and ~43% for PSSM+SS(GOR4) in independent case test, for the SVM models of above two same datasets. Using our method, it is possible to predict the protein block letters for any query protein sequence with ~53% accuracy, when the SP1577 dataset and predicted secondary structure from NPS@ server were used. The SVM-PB-Pred server can be freely accessed through http://bioinfo.bdu.ac.in/~svmpbpred.

  13. Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

    Science.gov (United States)

    Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

    2018-01-24

    Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

  14. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Directory of Open Access Journals (Sweden)

    Muhammad Ibrahim

    Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  15. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Science.gov (United States)

    Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

    2012-01-01

    Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  16. Modification of the mitochondrial sulfonylurea receptor by thiol reagents.

    Science.gov (United States)

    Szewczyk, A; Wójcik, G; Lobanov, N A; Nalecz, M J

    1999-08-19

    The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor. Copyright 1999 Academic Press.

  17. Facially amphiphilic thiol capped gold and silver nanoparticles

    Indian Academy of Sciences (India)

    Abstract. A series of bile acid-derived facially amphiphilic thiols have been used to cap sliver and gold nanoparticles. The self-assembling properties of these steroid-capped nanoparticles have been investigated and reported in this article.

  18. Sex- and Tissue-Specific Expression Profiles of Odorant Binding Protein and Chemosensory Protein Genes in Bradysia odoriphaga (Diptera: Sciaridae

    Directory of Open Access Journals (Sweden)

    Yunhe Zhao

    2018-04-01

    Full Text Available Bradysia odoriphaga is an agricultural pest insect affecting the production of Chinese chive and other liliaceous vegetables in China, and it is significantly attracted by sex pheromones and the volatiles derived from host plants. Despite verification of this chemosensory behavior, however, it is still unknown how B. odoriphaga recognizes these volatile compounds on the molecular level. Many of odorant binding proteins (OBPs and chemosensory proteins (CSPs play crucial roles in olfactory perception. Here, we identified 49 OBP and 5 CSP genes from the antennae and body transcriptomes of female and male adults of B. odoriphaga, respectively. Sequence alignment and phylogenetic analysis among Dipteran OBPs and CSPs were analyzed. The sex- and tissue-specific expression profiles of 54 putative chemosensory genes among different tissues were investigated by quantitative real-time PCR (qRT-PCR. qRT-PCR analysis results suggested that 22 OBP and 3 CSP genes were enriched in the antennae, indicating they might be essential for detection of general odorants and pheromones. Among these antennae-enriched genes, nine OBPs (BodoOBP2/4/6/8/12/13/20/28/33 were enriched in the male antennae and may play crucial roles in the detection of sex pheromones. Moreover, some OBP and CSP genes were enriched in non-antennae tissues, such as in the legs (BodoOBP3/9/19/21/34/35/38/39/45 and BodoCSP1, wings (BodoOBP17/30/32/37/44, abdomens and thoraxes (BodoOBP29/36, and heads (BodoOBP14/23/31 and BodoCSP2, suggesting that these genes might be involved in olfactory, gustatory, or other physiological processes. Our findings provide a starting point to facilitate functional research of these chemosensory genes in B. odoriphaga at the molecular level.

  19. Reversible inactivation of CO dehydrogenase with thiol compounds

    Energy Technology Data Exchange (ETDEWEB)

    Kreß, Oliver [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Gnida, Manuel [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Pelzmann, Astrid M. [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Marx, Christian [Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena (Germany); Meyer-Klaucke, Wolfram [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Meyer, Ortwin, E-mail: Ortwin.Meyer@uni-bayreuth.de [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany)

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  20. 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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  2. Simultaneous determination of albumin and low-molecular-mass thiols in plasma by HPLC with UV detection.

    Science.gov (United States)

    Borowczyk, Kamila; Wyszczelska-Rokiel, Monika; Kubalczyk, Paweł; Głowacki, Rafał

    2015-02-15

    In this paper, we describe a simple and robust HPLC based method for determination of total low- and high-molecular-mass thiols, protein S-linked thiols and reduced albumin in plasma. The method is based on derivatization of analytes with 2-chloro-1-methylquinolinium tetrafluoroborate, separation and quantification by reversed-phase liquid chromatography followed by UV detection. Disulfides were converted to their thiol counterparts by reductive cleavage with tris(2-carboxyethyl)phosphine. Linearity in detector response for total thiols was observed over the range of 1-40 μmol L(-1) for Hcy and glutathione (GSH), 5-100 μmol L(-1) for Cys-Gly, 20-300 μmol L(-1) for Cys and 3.1-37.5 μmol L(-1) (0.2-2.4gL(-1)) for human serum albumin (HSA). For the protein S-bound forms these values were as follows: 0.5-30 μmol L(-1) for Hcy and GSH, 2.5-60 μmol L(-1) for Cys-Gly and 5-200 μmol L(-1) for Cys. The LOQs for total HSA, Cys, Hcy, Cys-Gly and GSH were 0.5, 0.2, 0.4, 0.3 and 0.4 μmol L(-1), respectively. The estimated validation parameters for all analytes are more than sufficient to allow the analytical method to be used for monitoring of the total and protein bound thiols as well as redox status of HSA in plasma. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Dinitrosyl iron complexes with thiol-containing ligands as a "working form" of endogenous nitric oxide.

    Science.gov (United States)

    Vanin, Anatoly F

    2016-04-01

    The material presented herein is an overview of the results obtained by our research team during the many years' study of biological activities and occurrence of dinitrosyl iron complexes (DNIC) with thiol-containing ligands in human and animal organisms. With regard to their dose dependence and vast diversity of biological activities, DNIC are similar to the system of endogenous NO, one of the most universal regulators of biological processes. The role of biologically active components in DNIC is played by their iron-dinitrosyl fragments, [Fe(NO)2], endowed with the ability to generate neutral NO molecules and nitrosonium ions (NO(+)). Their release is effected by heme-and thiol-containing proteins, which fulfill the function of biological targets and acceptors of NO and NO(+). Beneficial regulatory effects of DNIC on physiological and metabolic processes are numerous and diverse and include, among other things, lowering of arterial pressure and accelerated healing of skin wounds. In the course of fast decomposition of their Fe(NO)2 fragments (e.g., in the presence of iron chelators), DNIC produce adverse (cytotoxic) effects, which can best be exemplified by their ability to suppress the development of experimental endometriosis in animals. In animal tissues, DNIC with thiol-containing ligands are predominantly represented by the binuclear form, which, contrary to mononuclear DNIC detectable by the 2.03 signal, is EPR-silent. The ample body of evidence on biological activities and occurrence of DNIC gained so far clearly demonstrates that in human and animal organisms DNIC with thiol-containing ligands represent a "working form" of the system of endogenous NO responsible for its accumulation and stabilization in animal tissues as well as its further transfer to its biological targets. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Seasonal variations in the amino acid profile and protein nutritional value of Saccharina latissima cultivated in a commercial IMTA system

    DEFF Research Database (Denmark)

    Silva Marinho, Goncalo; Holdt, Susan Løvstad; Angelidaki, Irini

    2015-01-01

    . Aspartic and glutamic acids dominated the amino acid profile, accounting for up to 49 % of the total. Greatest seasonal differences in amino acid composition occurred in July, with leucine contributing most (22.7–26.7 %) of the observed differences. A maximal essential amino acid (EAA) score of 68......Seaweeds have potential for the provision of biomass for food and feed supplements. The demand is increasing especially for proteins as ingredients; however, the amino acid profile is essential for evaluation of the nutritional value of proteins. The year-round protein concentration and amino acid.......9 % (based on WHO/FAO/UNU requirements) was achieved in November 2013. The presence of epiphytes in July to November changed neither the amino acid content nor the EAA score. S. latissima is comparable with wheat as a protein ingredient for fish feed and appears to be a suitable protein/amino acid source...

  5. Changes in the serum protein profile during radiotherapy to the upper respiratory and gastro-intestinal tracts

    International Nuclear Information System (INIS)

    David, M.; Lobera, A.; Legrand, E.

    1984-01-01

    Patients with a cancer of the upper airways of upper gastro-intestinal tract present a state of malnutrition as a result of the disease itself and, more importantly, as a result of its localisation. Loco-regional radiotherapy often leads to an aggravation, of this state. The protein profile, consisting of nine serum proteins, was determined each week in 54 patients with cancer of the upper respirato-gastro-intestinal tract receiving radiotherapy. During the course of radiotherapy, the already altered nutritional state of these patients deteriorated further, as shown by a regular and significant downturn in the weight curve. The weekly monitoring of the protein profile showed a gradual and significant decrease in the levels of nutritional proteins (prealbumin, retinol binding protein, transferrin) and immunoglobulins (IgM, IgA) and a small variation in the levels of inflammatory proteins (haptoglobin, orosomucoid, C3 complement fraction, alpha 1 -antitrypsin). The protein profile, established on the basis of carefully selected proteins, can provide useful information in the monitoring of a patient's nutritional state [fr

  6. Changes in the serum protein profile during radiotherapy to the upper respiratory and gastro-intestinal tracts

    Energy Technology Data Exchange (ETDEWEB)

    David, M; Lobera, A; Legrand, E [Fondation Bergorie, Bordeaux (France)

    1984-01-01

    Patients with a cancer of the upper airways on upper gastro-intestinal tract present a state of malnutrition as a result of the disease itself and, more importantly, as a result of its localisation. Loco-regional radiotherapy often leads to an aggravation, of this state. The protein profile, consisting of nine serum proteins, was determined each week in 54 patients with cancer of the upper respirato-gastro-intestinal tract receiving radiotherapy. During the course of radiotherapy, the already altered nutritional state of these patients deteriorated further, as shown by a regular and significant downturn in the weight curve. The weekly monitoring of the protein profile showed a gradual and significant decrease in the levels of nutritional proteins (prealbumin, retinol binding protein, transferrin) and immunoglobulins (IgM, IgA) and a small variation in the levels of inflammatory proteins (haptoglobin, orosomucoid, C3 complement fraction, alpha/sub 1/-antitrypsin). The protein profile, established on the basis of carefully selected proteins, can provide useful information in the monitoring of a patient's nutritional state.

  7. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    Science.gov (United States)

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

  8. Metallophilic interactions in polymeric group 11 thiols

    Science.gov (United States)

    Kolari, Kalle; Sahamies, Joona; Kalenius, Elina; Novikov, Alexander S.; Kukushkin, Vadim Yu.; Haukka, Matti

    2016-10-01

    Three polymeric group 11 transition metal polymers featuring metallophilic interactions were obtained directly via self-assembly of metal ions and 4-pyridinethiol ligands. In the cationic [Cu2(S-pyH)4]n2+ with [ZnCl4]n2- counterion (1) and in the neutral [Ag(S-py) (S-pyH)]n (2) 4-pyridinethiol (S-pyH) and its deprotonated form (S-py) are coordinated through the sulfur atom. Both ligands are acting as bridging ligands linking the metal centers together. In the solid state, the gold(I) polymer [Au(S-pyH)2]Cl (3) consists of the repeating cationic [Au(S-pyH)2]+ units held together by aurophilic interactions. Compound 1 is a zig-zag chain, whereas the metal chains in the structures of 2 and 3 are linear. The protonation level of the thiol ligand had an impact on the crystallization of polymers. Both nature of the metal center and reaction conditions affected the polymerization. QTAIM analysis confirmed direct metal-metal contacts only in polymers 1 and 3. In polymer 2, no theoretical evidence of argentophilic contacts was obtained even though the AgṡṡṡAg distance was found to be less than sum of the Bondi's van der Waals radius of silver.

  9. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    Science.gov (United States)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  10. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Directory of Open Access Journals (Sweden)

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  11. Studies on alterations of the 86-rubidium efflux from rat pancreatic islets caused by thiol and thiol oxidants

    International Nuclear Information System (INIS)

    Wahl, M.A.

    1983-01-01

    The following findings were revealed by this study: 1) Oxidation-reduction (redox) of the intracellular system of glutathione influences the potassium efflux by way of an increase in the 86-rubidium efflux brought about by the oxidation of intracellular thiols. 2) The 86-rubidium efflux is not subject to change by oxidation of extracellular thiols located in the membrane, nor can it in any way be influenced by reduced glutathione of exogenous origin. 3) The potassium efflux from rat pancreatic islets, being generally known to trigger the electric activities of the beta-cell, is controlled by the oxidation-reduction of intracellular thiols rather than by that of extracellular thiols. (TRV) [de

  12. Investigation of thiol-disulphide balance in patients with acute urticaria and chronic spontaneous urticaria.

    Science.gov (United States)

    Akbas, Ayse; Kilinc, Fadime; Sener, Sertac; Aktaş, Akın; Baran, Pervin; Ergin, Merve

    2017-09-01

    Thiol-disulphide balance plays a major role in health and diseases. This balance may be disrupted by various diseases. We aimed to determine status of the effect of thiol-disulphide balance in urticaria. We aimed to investigate the thiol-disulphide balance in patients with acute urticaria (AUP) and chronic spontaneous urticaria (CSU). Study included 53 AUP and 47 healthy controls plus 57 patients with chronic spontaneous urticaria (CSUP) and 57 healthy controls. Levels of native thiols, disulphides and total thiols were evaluated in plasma using a new and automated spectrophotometric method. Ratios of disulphides/total thiols, disulphides/native thiols and native thiols/total thiols were calculated. For AU, there was no statistical difference compared to control group in levels of native thiols, disulphides and total thiols. For CSU, however, there was an increase in levels of native thiols, disulphides and total thiols and the ratio of thiol/disulphide in favour of disulphide. Thiol-disulphide balance was not affected by AU but shifted towards to disulphide in CSU indicating the presence of oxidative stress (OS).

  13. PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

    Science.gov (United States)

    Paul, Sandip; Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V; Chattopadhyay, Sujay

    2015-12-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. PanCoreGen – profiling, detecting, annotating protein-coding genes in microbial genomes

    Science.gov (United States)

    Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V.

    2015-01-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen – a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars – Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. PMID:26456591

  15. Thiol-yne/thiol-epoxy hybrid crosslinked materials based on propargyl modified hyperbranched poly(ethyleneimine) and diglycidylether of bisphenol A resins

    OpenAIRE

    Acebo Gorostiza, Cristina; Fernández Francos, Xavier; Ramis Juan, Xavier; Serra Albet, Àngels

    2016-01-01

    A novel curing methodology based on the combination of thiol-yne and thiol-epoxy click reactions has been developed. The curing process consists of a first photoinitiated thiol-yne reaction, followed by a thermal thiol-epoxy process. As alkyne substrate a new propargyl terminated hyperbranched poly(ethyleneimine) (PEIyne) has been synthesized from the reaction of commercial poly(ethylenimine) (PEI) and glycidyl propargyl ether. The evolution of the curing of different mixtures of PEIyne and d...

  16. Postprandial lipemia detects the effect of soy protein on cardiovascular disease risk compared with the fasting lipid profile.

    Science.gov (United States)

    Santo, Antonio S; Santo, Ariana M; Browne, Richard W; Burton, Harold; Leddy, John J; Horvath, Steven M; Horvath, Peter J

    2010-12-01

    Studies examining the effect of soy protein on cardiovascular disease (CVD) risk factors have not taken advantage of the postprandial state as an adjunct to the fasting lipid profile. The American Heart Association has acknowledged the efficacy of soy protein in reducing CVD risk factors to be limited. We hypothesized that the postprandial state would be more sensitive to any favorable changes associated with consuming soy protein compared with the fasting lipid profile. Furthermore, the presence of isoflavones in soy would enhance this effect. Thirty sedentary males aged 18-30 years were randomly assigned to milk protein (Milk), isoflavone-poor soy (Soy-), or isoflavone-rich soy (Soy+). Usual diets were supplemented with 25 g/day of protein for 28 days. Serum samples were collected before and after supplementation in a fasted state and postprandially at 30, 60, 120, 240, and 360 min after a high-fat, 1,000 kcal shake. Triacylglycerol (TAG), total cholesterol, non-esterified fatty acids, apolipoproteins B-100 and A-I and glucose concentrations were quantified. Fasting concentrations were not different after any protein supplementation. Postprandial TAG and TAG AUC increased after Soy-consumption supporting the postprandial state as a more sensitive indicator of soy ingestion effects on CVD risk factors compared with the fasting lipid profile. Furthermore, the absence of isoflavones in soy protein may have deleterious consequences on purported cardio-protective effects.

  17. Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

    Science.gov (United States)

    Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

    2015-01-01

    Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

  18. MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

    DEFF Research Database (Denmark)

    Reinert, Line; Shi, B; Nandi, S

    2006-01-01

    MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification...... enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up......-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis....

  19. Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with Coccidiosis

    Science.gov (United States)

    Gilbert, Elizabeth R.; Cox, Chasity M.; Williams, Patricia M.; McElroy, Audrey P.; Dalloul, Rami A.; Ray, W. Keith; Barri, Adriana; Emmerson, Derek A.; Wong, Eric A.; Webb, Kenneth E.

    2011-01-01

    Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at PEimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods. PMID:21297942

  20. Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

    Science.gov (United States)

    Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

    2017-08-08

    Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

  1. A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins

    Directory of Open Access Journals (Sweden)

    Mizuguchi Kenji

    2008-09-01

    Full Text Available Abstract Background Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. Results A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. Conclusion The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the α-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in

  2. Profile of Students' Creative Thinking Skills on Quantitative Project-Based Protein Testing using Local Materials

    Directory of Open Access Journals (Sweden)

    D. K. Sari

    2017-04-01

    Full Text Available The purpose of this study is to obtain a profile of students’ creative thinking skills on quantitative project-based protein testing using local materials. Implementation of the research is using quasi-experimental method pre-test post-test control group design with 40 students involved in Biochemistry lab. The research instrument is pre-test and post-test using creative thinking skills in the form of description and students’ questionnaire. The analysis was performed with SPSS 22.0 program to see the significance normality, U Mann-Whitney test for nonparametric statistics, N-Gain score, and the percentage of student responses to the practicum performed. The research result shows that the pretest rate in the experimental group is 8.25 while in the control group is 6.90. After attending a project-based practicum with local materials, the experimental group obtained the mean of posttest is 37.55 while in control class is 11.18. The students’ improvement on creative thinking skills can be seen from the average of N-Gain in the experimental class with 0.32 (medium category and in the control category with 0.05 (low category. The experimental and control class have different creative thinking skills significantly different fluency, flexibility, novelty, and detail. It can be concluded that quantitative project-based protein testing using local materials can improve students’ creative thinking skills. 71% of total students feel that quantitative project-based protein testing using local materials make them more creative in doing a practicum in the laboratory.

  3. Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

    Directory of Open Access Journals (Sweden)

    Kuballa Anna V

    2007-10-01

    Full Text Available Abstract Background Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992 previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path

  4. Redox Reactivity of Cerium Oxide Nanoparticles Induces the Formation of Disulfide Bridges in Thiol-Containing Biomolecules.

    Science.gov (United States)

    Rollin-Genetet, Françoise; Seidel, Caroline; Artells, Ester; Auffan, Mélanie; Thiéry, Alain; Vidaud, Claude

    2015-12-21

    The redox state of disulfide bonds is implicated in many redox control systems, such as the cysteine-cystine couple. Among proteins, ubiquitous cysteine-rich metallothioneins possess thiolate metal binding groups susceptible to metal exchange in detoxification processes. CeO2 NPs are commonly used in various industrial applications due to their redox properties. These redox properties that enable dual oxidation states (Ce(IV)/Ce(III)) to exist at their surface may act as oxidants for biomolecules. The interaction among metallothioneins, cysteine, and CeO2 NPs was investigated through various biophysical approaches to shed light on the potential effects of the Ce(4+)/Ce(3+) redox system on the thiol groups of these biomolecules. The possible reaction mechanisms include the formation of a disulfide bridge/Ce(III) complex resulting from the interaction between Ce(IV) and the thiol groups, leading to metal unloading from the MTs, depending on their metal content and cluster type. The formation of stable Ce(3+) disulfide complexes has been demonstrated via their fluorescence properties. This work provides the first evidence of thiol concentration-dependent catalytic oxidation mechanisms between pristine CeO2 NPs and thiol-containing biomolecules.

  5. Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

    Science.gov (United States)

    2014-03-10

    leishmaniasis.56 Pre-exposure of PROFILING OF SAND FLY SALIVARY PROTEINS 935 murine cells to L. intermedia salivary sonicates resulted in decreased IP-10...Thompson JD, Higgins DG, 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7...Brodskyn C, Barral A, de Oliveira CI, 2010. Immunity to Lutzomyia intermedia saliva modulates the inflammatory environ- ment induced by Leishmania

  6. Structural Basis of a Thiol-Disulfide Oxidoreductase in the Hedgehog-Forming Actinobacterium Corynebacterium matruchotii.

    Science.gov (United States)

    Luong, Truc Thanh; Tirgar, Reyhaneh; Reardon-Robinson, Melissa E; Joachimiak, Andrzej; Osipiuk, Jerzy; Ton-That, Hung

    2018-05-01

    The actinobacterium Corynebacterium matruchotii has been implicated in nucleation of oral microbial consortia leading to biofilm formation. Due to the lack of genetic tools, little is known about basic cellular processes, including protein secretion and folding, in this organism. We report here a survey of the C. matruchotii genome, which encodes a large number of exported proteins containing paired cysteine residues, and identified an oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA (MdbA Cd ). Crystallization studies uncovered that the 1.2-Å resolution structure of C. matruchotii MdbA (MdbA Cm ) possesses two conserved features found in actinobacterial MdbA enzymes, a thioredoxin-like fold and an extended α-helical domain. By reconstituting the disulfide bond-forming machine in vitro , we demonstrated that MdbA Cm catalyzes disulfide bond formation within the actinobacterial pilin FimA. A new gene deletion method supported that mdbA is essential in C. matruchotii Remarkably, heterologous expression of MdbA Cm in the C. diphtheriae Δ mdbA mutant rescued its known defects in cell growth and morphology, toxin production, and pilus assembly, and this thiol-disulfide oxidoreductase activity required the catalytic motif CXXC. Altogether, the results suggest that MdbA Cm is a major thiol-disulfide oxidoreductase, which likely mediates posttranslocational protein folding in C. matruchotii by a mechanism that is conserved in Actinobacteria IMPORTANCE The actinobacterium Corynebacterium matruchotii has been implicated in the development of oral biofilms or dental plaque; however, little is known about the basic cellular processes in this organism. We report here a high-resolution structure of a C. matruchotii oxidoreductase that is highly homologous to the Corynebacterium diphtheriae thiol-disulfide oxidoreductase MdbA. By biochemical analysis, we demonstrated that C. matruchotii MdbA catalyzes disulfide

  7. Dynamic thiol/disulfide homeostasis and effects of smoking on homeostasis parameters in patients with psoriasis.

    Science.gov (United States)

    Emre, Selma; Demirseren, Duriye Deniz; Alisik, Murat; Aktas, Akin; Neselioglu, Salim; Erel, Ozcan

    2017-12-01

    Recently, increased reactive oxygen species (ROS), reduced antioxidant capacity, and oxidative stress have been suggested in the pathogenesis of psoriasis. The aim of this study to evaluate the thiol/disulfide homeostasis in patients with psoriasis. Ninety patients with psoriasis who did not receive any systemic treatment in the last six  months were included in the study. Seventy-six age and gender-matched healthy volunteers served as control group. Thiol/disulfide homeostasis was measured in venous blood samples obtained from patient and control groups. Native thiol and total thiol levels were significantly higher in patients than in control group. When thiol/disulfide hemostasis parameters and clinical and demographic characteristics were compared, a negative correlation was detected between native thiol and total thiol with age. The levels of total thiols had also negative correlation with PASI and duration of the disease. When we divided the patients into smokers and non-smokers, native thiol and total thiol levels were significantly higher in smokers than in controls, whereas native thiol and total thiol levels were comparable in non-smoker patients and controls. Thiol/disulfide balance shifted towards thiol in psoriasis patients and this may be responsible for increased keratinocyte proliferation in the pathogenesis of psoriasis.

  8. Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

    Science.gov (United States)

    da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

    2017-08-09

    Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

  9. Protection by thiols against poisoning by radiomimetic agents. Chapter 8

    International Nuclear Information System (INIS)

    Bacq, Z.M.

    1975-01-01

    A review is presented of reports of studies aimed at detecting a protective effect of thiols against radiomimetic alkylating agents such as those used in cancer therapy (nitrogen mustards (HN2), sarcolysine, busulfan, etc.). Protection by thiols against alkylating agents has been observed in mammals, plant cells, bacteria, isolated mammalian cells and in model systems. The lack of correlation between the protective power of various thiols against radiomimetic agents and ionizing radiations indicates that different mechanisms are involved. Studies have been made of the toxicity of the protector and the competition factor, increased excretion of detoxication products of alkylating agents, decreased alkylation of DNA and RNA both in vivo and in vitro, the protection of hematopoietic tissues, tumours and the adrenal cortex, and the modification of the effects of nitrosoalkylamines, carbon tetrachloride and fungistatics by thiols. The restriction of DNA alkylation by the competitive removal of radiomimetic agents is thought to account for the protective effect of thiols against radiomimetic agents. (U.K.)

  10. Effect of Ramadan fasting on serum heat shock protein 70 and serum lipid profile.

    Science.gov (United States)

    Zare, A; Hajhashemi, M; Hassan, Z M; Zarrin, S; Pourpak, Z; Moin, M; Salarilak, S; Masudi, S; Shahabi, S

    2011-07-01

    Ramadan, the holy month for the Islamic world, is a period every year when food and fluid intake is restricted to the pre-sunrise and post-sunset hours. The aim of this study was to evaluate the effect of Ramadan fasting on the serum concentration of heat shock protein 70 (HSP70) and serum lipid profile in healthy men. A total of 32 male volunteers with a mean age of 28.5 (range 23-37) years were selected for the study. Blood samples were obtained one day prior to Ramadan and on the 3rd and 25th days of fasting. Serum HSP70, triglyceride (TG), cholesterol (Chol), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), LDL/HDL and Chol/HDL ratios were investigated. It was observed that the mean concentrations of serum HSP70 and HDL on the 25th day of Ramadan were significantly higher than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels on the 3rd day of Ramadan was significantly higher than those recorded one day before Ramadan. Mean concentrations of serum TG, Chol, LDL, and LDL/HDL and Chol/HDL ratios on the 25th day of Ramadan were significantly lower than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels found on the 3rd day of Ramadan were also significantly lower than those recorded one day before Ramadan. Ramadan fasting increases serum HSP70 and improves serum lipid profile.

  11. Reproducibility of mass spectrometry based protein profiles for diagnosis of ovarian cancer across clinical studies: A systematic review

    DEFF Research Database (Denmark)

    Callesen, AK; Mogensen, O; Jensen, AK

    2012-01-01

    of published discriminatory peaks to peaks found in an original MALDI MS protein profiling study was made to address the key question of reproducibility across studies. An overlap was found despite substantial heterogeneity between studies relating to study design, biological material, pre-analytical treatment...

  12. Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Godiksen, Helene; Morzel, M.; Hyldig, Grethe

    2009-01-01

    Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days oil ice were obtained by SDS-PAGE. The link between protein hand intensities and firmness of trout fillets was examined through a correlation...

  13. Urinary protein profiles in ketorolac-associated acute kidney injury in patients undergoing orthopedic day surgery

    Directory of Open Access Journals (Sweden)

    Mariano F

    2017-09-01

    Full Text Available Filippo Mariano,1 Chiara Cogno,1 Fulvia Giaretta,2,3 Ilaria Deambrosis,2,3 Simona Pozza,4 Maurizio Berardino,5 Giuseppe Massazza,6 Luigi Biancone1,3 1Department of General and Specialist Medicine, Nephrology, Dialysis and Transplantation Unit, City of Health and Science, CTO Hospital, Turin, 2Department of General and Specialist Medicine, Laboratory of Nephrology and Immunopathology, City of Health and Science, Molinette Hospital, Turin, 3Department of Medical Sciences, University of Turin, Turin, 4Department of Radiology and Radiotherapy, CTO Radiology, City of Health and Science, CTO Hospital, Turin, 5Department of Anesthesiology and Intensive Care, Anesthesiology and Intensive Care 5, City of Health and Science, CTO Hospital, Turin, 6Department of Orthopedics and Traumatology, Week Hospital Unit, City of Health and Science, CTO Hospital, and University of Turin, Turin, Italy Background: Parenteral administration of ketorolac is very effective in controlling postoperative pain for orthopedic surgery. Ketorolac can induce clinically relevant renal alterations in elderly patients, whereas its short course is considered safe for young adults with normal preoperative renal function. In this study, of a cohort of young adults undergoing elective orthopedic day surgery, we sought cases complicated by readmission due to acute kidney injury (AKI.Patients and methods: Among 1397 young adults, aged 18–32 years who were admitted to undergo orthopedic day surgery from 2013 to 2015, four patients (0.29%, three males/one female treated in postprocedure with ketorolac (from 60 to 90 mg/day for 1–2 days were readmitted for suspected severe AKI. We evaluated functional outcome, urinary protein profiles and kidney biopsy (1 patient.Results: After day surgery discharge, they experienced gastrointestinal disturbances, flank pain and fever. Readmitted on post-surgery days 3–4, they presented with oliguric AKI (creatinine range 158.4–466.4 µmol/L and

  14. Integrated transcriptomics and proteomics analysis of storage protein composition in developing barley grain to improve nutritional profile

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Dionisio, Giuseppe; Renaut, Jenny

    2012-01-01

    The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe......-regimes caused significant differences in both quantity and quality of the storage proteins transcripts. Principal Component Analysis of the amino acid (AA) profiles also indicated dissimilarity in individual AA percentages, correlated to hordein content. The abundance values of proteins of interest confirmed...

  15. Protein content and electrophoretic profile of fat body and ovary extracts from workers of Melipona quadrifasciata anthidioides (Hymenoptera, Meliponini

    Directory of Open Access Journals (Sweden)

    Vagner T. Paes de Oliveira

    Full Text Available Workers of Melipona quadrifasciata anthidioides (Lepeletier, 1836 develop their ovaries and lay eggs, therefore the production of vitellogenin is expected. In electrophoretic profiles only fat body extracts from nurse workers and ovary extracts from newly-emerged workers show protein with molecular mass similar to vitellogenin. However, an increase in the protein content was detected in forager fat body. This increase was attributed to storage of vitellogenin or other proteins in the previous phase and not discharged into the hemolymph or to an effect of the increased titre of juvenile hormone in this phase of worker life over the fat body functioning.

  16. Analysis of whey protein hydrolysates: peptide profile and ACE inhibitory activity

    Directory of Open Access Journals (Sweden)

    Marialice Pinto Coelho Silvestre

    2012-12-01

    Full Text Available The aim of this study was to prepare enzymatic hydrolysates from whey protein concentrate with a nutritionally adequate peptide profile and the ability to inhibit angiotensin-converting enzyme (ACE activity. The effects of the type of enzyme used (pancreatin or papain, the enzyme:substrate ratio (E:S ratio=0.5:100, 1:100, 2:100 and 3:100 and the use of ultrafiltration (UF were investigated. The fractionation of peptides was performed by size-exclusion-HPLC, and the quantification of the components of the chromatographic fractions was carried out by a rapid Corrected Fraction Area method. The ACE inhibitory activity (ACE-IA was determined by Reverse Phase-HPLC. All parameters tested affected both the peptide profile and the ACE-IA. The best peptide profile was achieved for the hydrolysates obtained with papain, whereas pancreatin was more advantageous in terms of ACE-IA. The beneficial effect of using a lower E:S ratio on the peptide profile and ACE-IA was observed for both enzymes depending on the conditions used to prepare the hydrolysates. The beneficial effect of not using UF on the peptide profile was observed in some cases for pancreatin and papain. However, the absence of UF yielded greater ACE-IA only when using papain.O objetivo deste estudo foi preparar hidrolisados enzimáticos do concentrado proteico do soro de leite com perfil peptídico nutricionalmente adequado e com capacidade para inibir a atividade da enzima conversora da angiotensina (ECA. Os efeitos do tipo de enzima usado (pancreatina ou papaína, da relação enzima:substrato (E:S=0,5:100, 1:100, 2:100 e 3:100 e do uso da ultrafiltração (UF foram investigados. O fracionamento dos peptídeos foi feito por CLAE de exclusão molecular e a quantificação dos componentes das frações cromatográficas foi realizada pelo método da Área Corrigida da Fração. A atividade inibitória da ECA (AI-ECA foi determinada por CLAE de fase reversa. Todos os parâmetros testados afetaram

  17. Cytokine profile after oral food challenge in infants with food protein-induced enterocolitis syndrome.

    Science.gov (United States)

    Kimura, Mitsuaki; Ito, Yasunori; Shimomura, Masaki; Morishita, Hideaki; Meguro, Takaaki; Adachi, Yuichi; Seto, Shiro

    2017-07-01

    Although food protein-induced enterocolitis syndrome (FPIES) is supposed to be caused by inflammation, the role of cytokines has not yet been clarified. To elucidate the role of cytokines in the development of symptoms and abnormal laboratory findings at an oral food challenge (OFC), changes in serum cytokine levels were analyzed for 6 OFCs in 4 patients with FPIES. The result of OFC was judged positive if any gastrointestinal (GI) symptoms (vomiting, diarrhea, or bloody stool) were induced. Among 11 cytokines profiled, serum levels of interleukin (IL)-2, IL-5, and IL-8 were clearly increased in all 4 positive OFCs in which elevations of the serum level of C-reactive protein (CRP) and peripheral blood neutrophilia were also seen. The level of serum IL-10 also rose in 2 positive OFCs. Remarkable increases in the serum level of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-6, and IL-12 were observed in a positive OFC where the serum level of CRP rose markedly (6.75 mg/dL). The serum levels of IL-5 were also elevated in 2 negative OFCs. No apparent specific correlations were found between cytokines and GI symptoms. These results suggest that IL-2 and IL-8 are involved in the antigen-specific immune responses in most patients with FPIES. Further studies are needed to elucidate the significance of these cytokine in the pathogenesis of FPIES. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  18. Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

    Science.gov (United States)

    Kobayashi, Tetsuo; Yokoyama, Tomoko; Ito, Satoshi; Kobayashi, Daisuke; Yamagata, Akira; Okada, Moe; Oofusa, Ken; Narita, Ichiei; Murasawa, Akira; Nakazono, Kiyoshi; Yoshie, Hiromasa

    2014-11-01

    Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

  19. The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling

    Directory of Open Access Journals (Sweden)

    Johana A. Luna Coronell

    2018-02-01

    Full Text Available Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology. Keywords: Autoantibody tumor biomarker, Cancer immunology, Colorectal cancer, Immunomics, Protein microarray

  20. Highly tailorable thiol-ene based emulsion-templated monoliths

    DEFF Research Database (Denmark)

    Lafleur, J. P.; Kutter, J. P.

    2014-01-01

    The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off-stoichiometr......The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off......-stoichiometry thiolene chemistry. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single step. Variations in the monomer stoichiometric ratios and/or amount of porogen used allow for the creation of extremely varied polymer morphologies, from foam-like materials to dense networks...

  1. Resistivity of thiol-modified gold thin films

    International Nuclear Information System (INIS)

    Correa-Puerta, Jonathan; Del Campo, Valeria; Henríquez, Ricardo; Häberle, Patricio

    2014-01-01

    In this work, we study the effect of thiol self assembled monolayers on the electrical resistivity of metallic thin films. The analysis is based on the Fuchs–Sondheimer–Lucas theory and on electrical transport measurements. We determined resistivity change due to dodecanethiol adsorption on gold thin films. For this purpose, we controlled the deposition and annealing temperatures of the films to change the surface topography and to diminish the effect of electron grain boundary scattering. Results show that the electrical response to the absorption of thiols strongly depends on the initial topography of the surface. - Highlights: • We study the effect of self assembled monolayers on the resistivity of thin films. • Fuchs–Sondheimer theory reproduces the resistivity increase due to thiol deposition. • We determined resistivity change due to dodecanethiol deposition on gold thin films. • The electrical response strongly depends on the substrate surface topography

  2. Resistivity of thiol-modified gold thin films

    Energy Technology Data Exchange (ETDEWEB)

    Correa-Puerta, Jonathan [Instituto de Física, Pontificia Universidad Católica de Valparaíso, Av. Universidad 330, Curauma, Valparaíso (Chile); Del Campo, Valeria [Departamento de Física, Universidad Técnica Federico Santa María, Av. España 1680, Valparaiso 2390123 (Chile); Henríquez, Ricardo, E-mail: ricardo.henriquez@usm.cl [Departamento de Física, Universidad Técnica Federico Santa María, Av. España 1680, Valparaiso 2390123 (Chile); Häberle, Patricio [Departamento de Física, Universidad Técnica Federico Santa María, Av. España 1680, Valparaiso 2390123 (Chile)

    2014-11-03

    In this work, we study the effect of thiol self assembled monolayers on the electrical resistivity of metallic thin films. The analysis is based on the Fuchs–Sondheimer–Lucas theory and on electrical transport measurements. We determined resistivity change due to dodecanethiol adsorption on gold thin films. For this purpose, we controlled the deposition and annealing temperatures of the films to change the surface topography and to diminish the effect of electron grain boundary scattering. Results show that the electrical response to the absorption of thiols strongly depends on the initial topography of the surface. - Highlights: • We study the effect of self assembled monolayers on the resistivity of thin films. • Fuchs–Sondheimer theory reproduces the resistivity increase due to thiol deposition. • We determined resistivity change due to dodecanethiol deposition on gold thin films. • The electrical response strongly depends on the substrate surface topography.

  3. Sensitive determination of thiols in wine samples by a stable isotope-coded derivatization reagent d0/d4-acridone-10-ethyl-N-maleimide coupled with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis.

    Science.gov (United States)

    Lv, Zhengxian; You, Jinmao; Lu, Shuaimin; Sun, Weidi; Ji, Zhongyin; Sun, Zhiwei; Song, Cuihua; Chen, Guang; Li, Guoliang; Hu, Na; Zhou, Wu; Suo, Yourui

    2017-03-31

    As the key aroma compounds, varietal thiols are the crucial odorants responsible for the flavor of wines. Quantitative analysis of thiols can provide crucial information for the aroma profiles of different wine styles. In this study, a rapid and sensitive method for the simultaneous determination of six thiols in wine using d 0 /d 4 -acridone-10-ethyl-N-maleimide (d 0 /d 4 -AENM) as stable isotope-coded derivatization reagent (SICD) by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. Quantification of thiols was performed by using d 4 -AENM labeled thiols as the internal standards (IS), followed by stable isotope dilution HPLC-ESI-MS/MS analysis. The AENM derivatization combined with multiple reactions monitoring (MRM) not only allowed trace analysis of thiols due to the extremely high sensitivity, but also efficiently corrected the matrix effects during HPLC-MS/MS and the fluctuation in MS/MS signal intensity due to instrument. The obtained internal standard calibration curves for six thiols were linear over the range of 25-10,000pmol/L (R 2 ≥0.9961). Detection limits (LODs) for most of analytes were below 6.3pmol/L. The proposed method was successfully applied for the simultaneous determination of six kinds of thiols in wine samples with precisions ≤3.5% and recoveries ≥78.1%. In conclusion, the developed method is expected to be a promising tool for detection of trace thiols in wine and also in other complex matrix. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

    Directory of Open Access Journals (Sweden)

    Nicholas T. Ingolia

    2014-09-01

    Full Text Available Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs. Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

  5. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    Science.gov (United States)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  6. Comparative study of heparin-binding proteins profile of Murrah buffalo (Bubalus bubalis semen

    Directory of Open Access Journals (Sweden)

    S. S. Ramteke

    2014-09-01

    Full Text Available Aim: The experiment was conducted to study the total seminal plasma protein (TSPP and heparin-binding proteins (HBPs in relation to initial semen quality of buffalo bull. Materials and Methods: Semen from two Murrah buffalo bulls (bull no. 605 and 790 with mass motility of ≥3+ were used for the study and categorized into three groups (Group I- Mass motility 3+, Group II- Mass motility 4+ and Group III- Mass motility 5+. Seminal plasma from semen was separated by centrifugation. HBPs was isolated and purified from heparin-agarose affinity column by modified elution buffer. TSPP and isolated HBPs concentration was estimated by Lowry’s method. The purified HBPs were resolved on Sodium dodecyl sulfate polyacrylamide gel electrophoresis to check the protein profile of two bulls. Results: The mean values of TSPP concentrations in bull no. 605 and 790 in Group I, II and III were 30.64±0.12, 31.66±0.09, 32.53±0.19 and 28.51±0.09, 29.49±0.15, 30.45±0.17 mg/mL, respectively. The mean values of HBPs concentrations in bull no. 605 and 790 in Group I, II and III were 3.11±0.07, 3.32±0.06, 3.46±0.08 and 2.51±0.08, 2.91±0.05, 3.10±0.03 mg/mL, respectively. Both the values of TSPP and HBPs were significantly higher (p<0.01 in bull no. 605 when compared to 790 in all the three groups. 31 kDa HBP was more intensely present in bull no. 605, thus may indicate its superiority over bull no. 790 in relation to fertility potential. Conclusion: TSPP and HBPs shows variation in concentration with respect to initial semen quality. Furthermore, presence of fertility related 31 kDa HBPs in one of the bull may be an indication of high fertility of a bull. In future, in-vivo and in-vitro correlative study on larger basis is needed for the establishment of fertility-related HBPs in semen which might establish criteria for selection of buffalo bull with high fertility potential.

  7. Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1 in Maize

    Directory of Open Access Journals (Sweden)

    Jin Yu

    2014-05-01

    Full Text Available Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L. and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1; which did not carry a gametophytic factor and W22 (Ga1, a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1 and W22 (Ga1. A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1 and W22 (Ga1 whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1 and W22 (Ga1. However, in pollen, 2 proteins were identified only in the W22 (ga1 and 12 proteins only in the W22 (Ga1 whereas 10 proteins were confirmed from the both of W22 (ga1 and W22 (Ga1. In contrary, 10 proteins were appeared only in the pistil of W22 (ga1 and 7 proteins from W22 (Ga1 while 3 proteins confirmed in the both of W22 (ga1 and W22 (Ga1. Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

  8. Molecular profiling of signalling proteins for effects induced by the anti-cancer compound GSAO with 400 antibodies

    International Nuclear Information System (INIS)

    Cadd, Verity A; Hogg, Philip J; Harris, Adrian L; Feller, Stephan M

    2006-01-01

    GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide) is a hydrophilic derivative of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO). It inhibits angiogenesis and tumour growth in mouse models and may be evaluated in a phase I clinical trial in the near future. Initial experiments have implicated GSAO in perturbing mitochondrial function. Other molecular effects of GSAO in human cells, for example on the phosphorylation of proteins, are still largely unknown. Peripheral white blood cells (PWBC) from healthy volunteers were isolated and used to profile effects of GSAO vs. a control compound, GSCA. Changes in site-specific phosphorylations, other protein modifications and expression levels of many signalling proteins were analysed using more than 400 different antibodies in Western blots. PWBC were initially cultured in low serum conditions, with the aim to reduce basal protein phosphorylation and to increase detection sensitivity. Under these conditions pleiotropic intracellular signalling protein changes were induced by GSAO. Subsequently, PWBC were cultured in 100% donor serum to reflect more closely in vivo conditions. This eliminated detectable GSAO effects on most, but not all signalling proteins analysed. Activation of the MAP kinase Erk2 was still observed and the paxillin homologue Hic-5 still displayed a major shift in protein mobility upon GSAO-treatment. A GSAO induced change in Hic-5 mobility was also found in endothelial cells, which are thought to be the primary target of GSAO in vivo. Serum conditions greatly influence the molecular activity profile of GSAO in vitro. Low serum culture, which is typically used in experiments analysing protein phosphorylation, is not suitable to study GSAO activity in cells. The signalling proteins affected by GSAO under high serum conditions are candidate surrogate markers for GSAO bioactivity in vivo and can be analysed in future clinical trials. GSAO effects on Hic-5 in endothelial cells may

  9. Determination of the separate lipid and protein profile structures derived from the total membrane profile structure or isolated sarcoplasmic reticulum via x-ray and neutron diffraction

    International Nuclear Information System (INIS)

    Herbette, L.; Blasie, J.K.

    1984-01-01

    Sarcoplasmic reticulum (SR) membranes were prepared to contain biosynthetically deuterated SR phospholipids utilizing specific and general phospholipid exchange proteins (PLEP). Functional measurements and freeze fracture on SR dispersions and x-ray diffraction of hydrated oriented membrane multilayers revealed that the exchanged SR membranes were very similar to unexchanged SR membranes. Low resolution (28-A) neutron diffraction studies utilizing SR membranes exchanged with either protonated or perdeuterated SR phospholipids allowed direct determination of the lipid profile within the isolated SR membrane at two different unit cell repeat distances. These lipid profile structures were found to be highly asymmetric regarding the conformation of the fatty acid chain extents and compositional distribution of phospholipid molecules in the inner vs. outer monolayer of the SR membrane bilayer. The relatively high resolution (11-A) electron-density profile from x-ray diffraction was decomposed by utilizing the asymmetry in the number of phospholipid molecules residing in the inner vs. outer monolayer of the SR lipid bilayer as obtained from the neutron diffraction study. To our knowledge, this represents the first direct determination of a lipid bilayer profile structure within an isolated membrane system

  10. Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Smita Rai

    Full Text Available BACKGROUND: In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s and expression profiles of known genes involved in transport and thiol based redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We selected 7 clinical isolates (2 sensitive and 5 resistant in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. CONCLUSIONS/SIGNIFICANCE: Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

  11. Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

    Science.gov (United States)

    Zhang, J; Wang, J; Lee, Y-M; Lim, T-K; Lin, Q; Shen, H-M

    2017-01-01

    Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy. © 2017 Elsevier Inc. All rights reserved.

  12. Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

    Science.gov (United States)

    Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

    2009-04-01

    Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

  13. Effect of Heating Method on Alteration of Protein Molecular Structure in Flaxseed: Relationship with Changes in Protein Subfraction Profile and Digestion in Dairy Cows.

    Science.gov (United States)

    Ahmad Khan, Nazir; Booker, Helen; Yu, Peiqiang

    2015-02-04

    This study evaluated the effect of heating methods on alteration of protein molecular structure in flaxseed (Linum usitatissimum L.) in relation to changes in protein subfraction profile and digestion in dairy cows. Seeds from two flaxseed varieties, sampled from two replicate plots at two locations, were evaluated. The seeds were either maintained in their raw state or heated in an air-draft oven (dry heating) or autoclave (moist heating) for 60 min at 120 °C or by microwave irradiation (MIR) for 5 min. Compared to raw seeds, moist heating decreased (P RUP) content (36.0 ± 5.19 to 46.9 ± 2.72% CP) and intestinal digestibility of RUP (61.0 ± 2.28 to 63.8 ± 2.67% RUP). Dry heating did not alter (P > 0.05) the protein subfraction profile and rumen degradation kinetics, whereas MIR increased (P RUP content from 36.0 ± 5.19 to 40.4 ± 4.67% CP. The MIR and dry heating did not alter (P > 0.05) the amide I to amide II ratio, but moist heating decreased (P RUP (R 2 = 0.71), and intestinal digestibility of RUP (R 2 = 0.72). Overall, heat-induced changes in protein nutritive value and digestion were strongly associated with heat-induced alteration in protein molecular structures.

  14. Effect of thiol group on the curing process of alkaline developable photo-resists

    International Nuclear Information System (INIS)

    Hidetaka Oka; Masaki Ohwa; Hisatoshi Kura

    1999-01-01

    Photosensitivity of a conventional radical photo-initiator in an alkaline developable photoresist is boosted by substitution with a thiol group. Evidence is presented that the thiol group acts via chain transfer mechanism

  15. Effects of D,L-buthionine-S,R-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Astor, M.B.; Hall, E.J.; Biaglow, J.E.; Hartog, B.

    1984-01-01

    The role of glutathione (GSH) and total non-protein thiols (NPSH) in repairing radiation-induced free radical damage incurred under aerated and hypoxic conditions was investigated using Chinese hamster V79 cells cultured in vitro. GSH and NPSH levels were depleted in V79 cells of varying cell densities using the gamma-glutamyl-cysteine-synthetase inhibitor, D,L-Buthionine-S,R-sulfoximine (BSO). A small change in hypoxic cell radiosensitivity could be attributed to the loss of GSH while depletion of thiols to lower levels affected both aerated and hypoxic cell radiosensitivity, resulting in no change in the OER

  16. Arsenic affects the production of glucosinolate, thiol and phytochemical compounds: A comparison of two Brassica cultivars.

    Science.gov (United States)

    Pandey, Chandana; Augustine, Rehna; Panthri, Medha; Zia, Ismat; Bisht, Naveen C; Gupta, Meetu

    2017-02-01

    Arsenic (As), a non-essential metalloid, severely affects the normal functioning of plants, animals and humans. Plants play a crucial role in metabolic, physiological and numerous detoxification mechanisms to cope up with As induced stress. This study aimed to examine the differential response in two Brassica juncea cultivars, Varuna and Pusa Jagannath (PJn) exposed to different doses of As (50, 150, 300 μM) for 48 h duration. Change in morphological traits, concentration of individual as well as total GSL, sulfur related thiol proteins, sulfur content, and phytochemicals were analyzed in both cultivars. Accumulation pattern of As showed dose dependent accumulation in both the cultivars, being more in PJn. Our finding revealed that both cultivars were tolerant at low concentrations of As, while at higher concentration Varuna excelled over PJn. The increased tolerance of Varuna cultivar exposed to 150 and 300 μM concentration of As, correlated with its increased thiol related proteins, sulfur content and phytochemicals, which serves as defence strategy in the plant against oxidative stress. Differential pattern of total as well as individual GSLs content was observed in both Varuna and PJn cultivars. Varuna cultivar showed higher level of total and aliphatic GSLs, which serves as defence compound with other detoxification machineries to combat As stress. Our findings provide foundation for developing metalloid tolerant crops by analyzing the role of different genes involved in GSL mechanism and signaling pathways in different organs of plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Adsorption characteristics of self-assembled thiol and dithiol layer on gold

    International Nuclear Information System (INIS)

    Tlili, A.; Abdelghani, A.; Aguir, K.; Gillet, M.; Jaffrezic-Renault, N.

    2007-01-01

    Monolayers of functional proteins are important in many fields related to pure and applied biochemistry and biophysics. The formation of extended uniform protein monolayers by single- or multiple-step self-chemisorption depends on the quality of the functionalized gold surface. The optical and the electrical properties of the 1-nonanethiol and 1,9-nonanedithiol deposited on gold with the self-assembled technique were investigated. We use cyclic voltammetry and impedance spectroscopy to characterize the insulating properties of the two layers. The analysis of the impedance spectra in terms of equivalent circuit of the gold/electrolyte and gold/SAM/electrolyte interface allows defining the thickness of the two thiols and the percentage of coverage area. Atomic force microscopy, contact angle measurement and Fourier transform infra-red spectroscopy have been used for homogeneity, hydrophobic properties and molecular structure of the formed thiols layer, respectively. The measured thickness with impedance spectroscopy fit well the results found with atomic force microscopy

  18. Small RNA fragments in complex culture media cause alterations in protein profiles of three species of bacteria.

    Science.gov (United States)

    Pavankumar, Asalapuram R; Ayyappasamy, Sudalaiyadum Perumal; Sankaran, Krishnan

    2012-03-01

    Efforts to delineate the basis for variations in protein profiles of different membrane fractions from various bacterial pathogens led to the finding that even the same medium [e.g., Luria Bertani (LB) broth] purchased from different commercial sources generates remarkably dissimilar protein profiles despite similar growth characteristics. Given the pervasive roles small RNAs play in regulating gene expression, we inquired if these source-specific differences due to media arise from disparities in the presence of small RNAs. Indeed, LB media components from two different commercial suppliers contained varying, yet significant, amounts of 10-80 bp small RNAs. Removal of small RNA from LB using RNaseA during media preparation resulted in significant changes in bacterial protein expression profiles. Our studies underscore the fact that seemingly identical growth media can lead to dramatic alterations in protein expression patterns, highlighting the importance of utilizing media free of small RNA during bacteriological studies. Finally, these results raise the intriguing possibility that similar pools of small RNAs in the environment can influence bacterial adaptation.

  19. Thiol-ene and photo-cleavage chemistry for controlled presentation of biomolecules in hydrogels.

    Science.gov (United States)

    Grim, Joseph C; Marozas, Ian A; Anseth, Kristi S

    2015-12-10

    Hydrogels have emerged as promising scaffolds in regenerative medicine for the delivery of biomolecules to promote healing. However, increasing evidence suggests that the context that biomolecules are presented to cells (e.g., as soluble verses tethered signals) can influence their bioactivity. A common approach to deliver biomolecules in hydrogels involves physically entrapping them within the network, such that they diffuse out over time to the surrounding tissues. While simple and versatile, the release profiles in such system are highly dependent on the molecular weight of the entrapped molecule relative to the network structure, and it can be difficult to control the release of two different signals at independent rates. In some cases, supraphysiologically high loadings are used to achieve therapeutic local concentrations, but uncontrolled release can then cause deleterious off-target side effects. In vivo, many growth factors and cytokines are stored in the extracellular matrix (ECM) and released on demand as needed during development, growth, and wound healing. Thus, emerging strategies in biomaterial chemistry have focused on ways to tether or sequester biological signals and engineer these bioactive scaffolds to signal to delivered cells or endogenous cells. While many strategies exist to achieve tethering of peptides, protein, and small molecules, this review focuses on photochemical methods, and their usefulness as a mild reaction that proceeds with fast kinetics in aqueous solutions and at physiological conditions. Photo-click and photo-caging methods are particularly useful because one can direct light to specific regions of the hydrogel to achieve spatial patterning. Recent methods have even demonstrated reversible introduction of biomolecules to mimic the dynamic changes of native ECM, enabling researchers to explore how the spatial and dynamic context of biomolecular signals influences important cell functions. This review will highlight how two

  20. A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

    Science.gov (United States)

    Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

    2003-06-01

    A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

  1. Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

    Science.gov (United States)

    Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam

    2017-01-30

    Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense

  2. Redox regulation of Rac1 by thiol oxidation

    Science.gov (United States)

    Hobbs, G. Aaron; Mitchell, Lauren E.; Arrington, Megan E.; Gunawardena, Harsha P.; DeCristo, Molly J.; Loeser, Richard F.; Chen, Xian; Cox, Adrienne D.; Campbell, Sharon L.

    2016-01-01

    The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys18) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that

  3. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

    2016-01-29

    Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and

  4. Protein Profiles Reveal Diverse Responsive Signaling Pathways in Kernels of Two Maize Inbred Lines with Contrasting Drought Sensitivity

    Directory of Open Access Journals (Sweden)

    Liming Yang

    2014-10-01

    Full Text Available Drought stress is a major factor that contributes to disease susceptibility and yield loss in agricultural crops. To identify drought responsive proteins and explore metabolic pathways involved in maize tolerance to drought stress, two maize lines (B73 and Lo964 with contrasting drought sensitivity were examined. The treatments of drought and well water were applied at 14 days after pollination (DAP, and protein profiles were investigated in developing kernels (35 DAP using iTRAQ (isobaric tags for relative and absolute quantitation. Proteomic analysis showed that 70 and 36 proteins were significantly altered in their expression under drought treatments in B73 and Lo964, respectively. The numbers and levels of differentially expressed proteins were generally higher in the sensitive genotype, B73, implying an increased sensitivity to drought given the function of the observed differentially expressed proteins, such as redox homeostasis, cell rescue/defense, hormone regulation and protein biosynthesis and degradation. Lo964 possessed a more stable status with fewer differentially expressed proteins. However, B73 seems to rapidly initiate signaling pathways in response to drought through adjusting diverse defense pathways. These changes in protein expression allow for the production of a drought stress-responsive network in maize kernels.

  5. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling.

    Science.gov (United States)

    Kandasamy, Saveetha; Loganathan, Karthiba; Muthuraj, Raveendran; Duraisamy, Saravanakumar; Seetharaman, Suresh; Thiruvengadam, Raguchander; Ponnusamy, Balasubramanian; Ramasamy, Samiyappan

    2009-12-24

    Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  6. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    Directory of Open Access Journals (Sweden)

    Thiruvengadam Raguchander

    2009-12-01

    Full Text Available Abstract Background Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.

  7. THE INFLUENCE OF AUTOLYSIS ON THE PROTEIN-PEPTIDE PROFILE OF Bos taurus AND Sus scrofa HEART AND AORTA TISSUES

    Directory of Open Access Journals (Sweden)

    I. M. Chernukha

    2016-01-01

    Full Text Available The article presents the results of autolytic processes impact on the protein-peptide profile of Bos taurus and Sus scrofa cardiac muscle and aorta. The results of tissue-specific protein identification are also presented as well as the effect of autolysis. Apolipoprotein A-1 involved in the formation of high-density lipoproteins, peroxiredoxin-1 involved in the suppression of oxidative stress, galectin-1 induced apoptosis of T-lymphocytes, as well as number of heat shock proteins with molecular weight less than 30 kDa were identified in Sus scrofa aorta tissue. It was discovered that functional proteins with molecular weight less than 30 kDa are retained during the freezing process, but destroyed under the action of autolytic enzymes. This work was supported by the Russian Science Foundation (project No. 16–16–10073.

  8. Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors.

    Science.gov (United States)

    Bacalocostantis, Irene; Mane, Viraj P; Kang, Michael S; Goodley, Addison S; Muro, Silvia; Kofinas, Peter

    2012-05-14

    Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand

  9. Iron and thiols as two major players in carcinogenesis: friends or foes?

    Science.gov (United States)

    Toyokuni, Shinya

    2014-01-01

    Iron is the most abundant metal in the human body and mainly works as a cofactor for proteins such as hemoglobin and various enzymes. No independent life forms on earth can survive without iron. However, excess iron is intimately associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Biomolecules with redox-active sulfhydryl function(s) (thiol compounds) are necessary for the maintenance of mildly reductive cellular environments to counteract oxidative stress, and for the execution of redox reactions for metabolism and detoxification. Involvement of glutathione S-transferase and thioredoxin has long attracted the attention of cancer researchers. Here, I update recent findings on the involvement of iron and thiol compounds during carcinogenesis and in cancer cells. It is now recognized that the cystine/glutamate transporter (antiporter) is intimately associated with ferroptosis, an iron-dependent, non-apoptotic form of cell death, observed in cancer cells, and also with cancer stem cells; the former with transporter blockage but the latter with its stabilization. Excess iron in the presence of oxygen appears the most common known mutagen. Ironically, the persistent activation of antioxidant systems via genetic alterations in Nrf2 and Keap1 also contributes to carcinogenesis. Therefore, it is difficult to conclude the role of iron and thiol compounds as friends or foes, which depends on the quantity/distribution and induction/flexibility, respectively. Avoiding further mutation would be the most helpful strategy for cancer prevention, and myriad of efforts are being made to sort out the weaknesses of cancer cells.

  10. Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

    Science.gov (United States)

    Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

    2017-03-17

    Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

  11. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    Directory of Open Access Journals (Sweden)

    Yi-Ming Chen

    2016-02-01

    Full Text Available Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA-rich sake protein (SP to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Methods: Male ICR mice were divided into three groups (n = 8 per group for four-week treatment: sedentary controls with vehicle (SC, and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP. Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. Results: four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP weight and area. Levels of aspartate aminotransferase (AST, alanine aminotransferase (ALT, creatinine, and uric acid (UA were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. Conclusion: The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training.

  12. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice.

    Science.gov (United States)

    Chen, Yi-Ming; Lin, Che-Li; Wei, Li; Hsu, Yi-Ju; Chen, Kuan-Neng; Huang, Chi-Chang; Kao, Chin-Hsung

    2016-02-20

    Exercise and fitness training programs have attracted the public's attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. In this study, we designed a power exercise training (PET) program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA)-rich sake protein (SP) to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Male ICR mice were divided into three groups (n = 8 per group) for four-week treatment: sedentary controls with vehicle (SC), and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP). Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK) after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP) weight and area. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and uric acid (UA) were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training.

  13. Facially amphiphilic thiol capped gold and silver nanoparticles

    Indian Academy of Sciences (India)

    Wintec

    *For correspondence. Also at the Chemical Biology Unit,. Jawaharlal Nehru Centre for Advanced Scientific Research,. Bangalore 560 064. Facially amphiphilic thiol capped gold and silver nanoparticles. †. SHREEDHAR BHAT a and UDAY MAITRA*. Department of Organic Chemistry, Indian Institute of Science, Bangalore ...

  14. Lignin-Based Materials Through Thiol-Maleimide "Click" Polymerization.

    Science.gov (United States)

    Buono, Pietro; Duval, Antoine; Averous, Luc; Habibi, Youssef

    2017-03-09

    In the present report an environmentally friendly approach to transforming renewable feedstocks into value-added materials is proposed. This transformation pathway was conducted under green conditions, without the use of solvents or catalyst. First, controlled modification of lignin, a major biopolymer present in wood and plants, was achieved by esterification with 11-maleimidoundecylenic acid (11-MUA), a derivative from castor oil that contains maleimide groups, following its transformation into 11-maleimidoundecanoyl chloride (11-MUC). Different degrees of substitution were achieved by using various amounts of the 11-MUC, leading to an efficient conversion of lignin hydroxy groups, as demonstrated by 1 H and 31 P NMR analyses. These fully biobased maleimide-lignin derivatives were subjected to an extremely fast (ca. 1 min) thiol-ene "click" polymerization with thiol-containing linkers. Aliphatic and aromatic thiol linkers bearing two to four thiol groups were used to tune the reactivity and crosslink density. The properties of the resulting materials were evaluated by swelling tests and thermal and mechanical analyses, which showed that varying the degree of functionality of the linker and the linker structure allowed accurate tailoring of the thermal and mechanical properties of the final materials, thus providing interesting perspectives for lignin in functional aromatic polymers. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Hybrid Organic/Inorganic Thiol-ene-Based Photopolymerized Networks.

    Science.gov (United States)

    Schreck, Kathleen M; Leung, Diana; Bowman, Christopher N

    2011-09-15

    The thiol-ene reaction serves as a more oxygen tolerant alternative to traditional (meth)acrylate chemistry for forming photopolymerized networks with numerous desirable attributes including energy absorption, optical clarity, and reduced shrinkage stress. However, when utilizing commercially available monomers, many thiol-ene networks also exhibit decreases in properties such as glass transition temperature (T(g)) and crosslink density. In this study, hybrid organic/inorganic thiol-ene resins incorporating silsesquioxane (SSQ) species into the photopolymerized networks were investigated as a route to improve these properties. Thiol- and ene-functionalized SSQs (SH-SSQ and allyl-SSQ, respectively) were synthesized via alkoxysilane hydrolysis/condensation chemistry, using a photopolymerizable monomer [either pentaerythriol tetrakis(3-mercaptopropionate) (PETMP) or 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO)] as the reaction solvent. The resulting SSQ-containing solutions (SSQ-PETMP and SSQ-TATATO) were characterized, and their incorporation into photopolymerized networks was evaluated.

  16. Capillary electrophoresis in the analysis of biologically important thiols

    Czech Academy of Sciences Publication Activity Database

    Lačná, J.; Kubáň, Petr; Foret, František

    2017-01-01

    Roč. 38, č. 1 (2017), s. 203-222 ISSN 0173-0835 Institutional support: RVO:68081715 Keywords : biological thiols * capillary electrophoresis * clinical applications Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 2.744, year: 2016

  17. Combining RNA-seq and proteomic profiling to identify seminal fluid proteins in the migratory grasshopper Melanoplus sanguinipes (F).

    Science.gov (United States)

    Bonilla, Martha L; Todd, Christopher; Erlandson, Martin; Andres, Jose

    2015-12-22

    Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs' functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies.

  18. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

    Directory of Open Access Journals (Sweden)

    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

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

    DEFF Research Database (Denmark)

    Wang, Ping; Holst, Claus; Astrup, Arne

    2012-01-01

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

  20. Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer

    DEFF Research Database (Denmark)

    Callesen, Anne Kjærgaard; Vach, Werner; Jørgensen, Per E

    2008-01-01

    it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total...... and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity...... and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis....

  1. Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

    Science.gov (United States)

    Calvano, C D; Aresta, A; Iacovone, M; De Benedetto, G E; Zambonin, C G; Battaglia, M; Ditonno, P; Rutigliano, M; Bettocchi, C

    2010-03-11

    Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between

  2. Molecular spectroscopic features of protein in newly developed chickpea: Relationship with protein chemical profile and metabolism in the rumen and intestine of dairy cows

    Science.gov (United States)

    Sun, Baoli; Khan, Nazir Ahmad; Yu, Peiqiang

    2018-05-01

    The first aim of this study was to investigate the nutritional value of crude protein (CP) in CDC [Crop Development Centre (CDC), University of Saskatchewan] chickpea varieties (Frontier kabuli and Corinne desi) in comparison with a CDC barley variety in terms of: 1) CP chemical profile and subfractions; (2) in situ rumen degradation kinetics and intestinal digestibility of CP; 2) metabolizable protein (MP) supply to dairy cows; and (3) protein molecular structure characteristics using advanced molecular spectroscopy. The second aim was to quantify the relationship between protein molecular spectral characteristics and CP subfractions, in situ rumen CP degradation characteristics, intestinal digestibility of CP, and MP supply to dairy cows. Samples (n = 4) of each variety, from two consecutive years were analyzed. Chickpeas had higher (P content (21.71-22.11 vs 12.96% DM), with higher (P content, and any of the measured in situ degradation and molecular spectral characteristics of protein. The content of RUP was positively (r = 0.94, P content of CP (R2 = 0.91) D-fraction (R2 = 0.82), RDP (R2 = 0.77), RUP (R2 = 0.77), TDP (R2 = 0.98), MP (R2 = 0.80), and FMV (R2 = 0.80) can be predicted from amide II peak height. Despite extensive ruminal degradation, chickpea is a good source of MP for dairy cows, and molecular spectroscopy can be used to rapidly characterize feed protein molecular structures and predict their digestibility and nutritive value.

  3. Molecular spectroscopic features of protein in newly developed chickpea: Relationship with protein chemical profile and metabolism in the rumen and intestine of dairy cows.

    Science.gov (United States)

    Sun, Baoli; Khan, Nazir Ahmad; Yu, Peiqiang

    2018-05-05

    The first aim of this study was to investigate the nutritional value of crude protein (CP) in CDC [Crop Development Centre (CDC), University of Saskatchewan] chickpea varieties (Frontier kabuli and Corinne desi) in comparison with a CDC barley variety in terms of: 1) CP chemical profile and subfractions; (2) in situ rumen degradation kinetics and intestinal digestibility of CP; 2) metabolizable protein (MP) supply to dairy cows; and (3) protein molecular structure characteristics using advanced molecular spectroscopy. The second aim was to quantify the relationship between protein molecular spectral characteristics and CP subfractions, in situ rumen CP degradation characteristics, intestinal digestibility of CP, and MP supply to dairy cows. Samples (n=4) of each variety, from two consecutive years were analyzed. Chickpeas had higher (Pmolecular spectral data of chickpeas can be distinguished from the barley. The two chickpeas did not differ in CP content, and any of the measured in situ degradation and molecular spectral characteristics of protein. The content of RUP was positively (r=0.94, Pmolecular spectroscopy can be used to rapidly characterize feed protein molecular structures and predict their digestibility and nutritive value. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

  4. Volatile profile, lipid oxidation and protein oxidation of irradiated ready-to-eat cured turkey meat products

    International Nuclear Information System (INIS)

    Feng, Xi; Ahn, Dong Uk

    2016-01-01

    Irradiation had little effects on the thiobarbituric acid reactive substances (TBARS) values in ready-to-eat (RTE) turkey meat products, while it increased protein oxidation at 4.5 kGy. The volatile profile analyses indicated that the amount of sulfur compounds increased linearly as doses increased in RTE turkey meat products. By correlation analysis, a positive correlation was found between benzene/ benzene derivatives and alcohols with lipid oxidation, while aldehydes, ketones and alkane, alkenes and alkynes were positively correlated with protein oxidation. Principle component analysis showed that irradiated meat samples can be discriminated by two categories of volatile compounds: Strecker degradation products and radiolytic degradation products. The cluster analysis of volatile data demonstrated that low-dose irradiation had minor effects on the volatile profile of turkey sausages (<1.5 kGy). However, as the doses increased, the differences between the irradiated and non-irradiated cured turkey products became significant. - Highlights: • Irradiation had little effects on lipid oxidation of ready-to-eat cured turkey. • 4.5 kGy irradiation increased protein oxidation. • Irradiated samples were isolated due to Strecker/radiolytic degradation products. • 1.5 kGy irradiation had limited effects on the volatile profile of turkey sausages. • Dimethyl disulfide can be used as a potential marker for irradiated meat products.

  5. Detection of lung cancer using plasma protein profiling by matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Shevchenko, Valeriy E; Arnotskaya, Natalia E; Zaridze, David G

    2010-01-01

    There are no satisfactory plasma biomarkers which are available for the early detection and monitoring of lung cancer, one of the most frequent cancers worldwide. The aim of this study is to explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) to plasma proteomic patterns to distinguish lung cancer patients from healthy individuals. The EDTA plasma samples have been pre-fractionated using magnetic bead kits functionalized with weak cation exchange coatings. We compiled MS protein profiles for 90 patients with squamous cell carcinomas (SCC) and compared them with profiles from 187 healthy controls. The MALDI-ToF spectra were analyzed statistically using ClinProTools bioinformatics software. Depending on the sample used, up to 441 peaks/spectrum could be detected in a mass range of 1000-20,000 Da; 33 of these proteins had statistically differential expression levels between SCC and control plasma (P 90%) in external validation test. These results suggest that plasma MALDI-ToF MS protein profiling can distinguish patients with SCC and also from healthy individuals with relatively high sensitivity and specificity and that MALDI- ToF MS is a potential tool for the screening of lung cancer.

  6. Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

    Directory of Open Access Journals (Sweden)

    Erhui Xiong

    Full Text Available The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1 is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize and dicot (soybean and pea seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

  7. Protein Comparability Assessments and Potential Applicability of High Throughput Biophysical Methods and Data Visualization Tools to Compare Physical Stability Profiles

    Directory of Open Access Journals (Sweden)

    Mohammad A. Alsenaidy

    2014-03-01

    Full Text Available In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs, radar charts, and comparative signature diagrams (CSDs for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1 mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF. Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress. Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

  8. Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles.

    Science.gov (United States)

    Alsenaidy, Mohammad A; Jain, Nishant K; Kim, Jae H; Middaugh, C Russell; Volkin, David B

    2014-01-01

    In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions (i.e., no stress). Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies.

  9. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as effici......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...... as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we...

  10. Apoptosis induced by low-intensity ultrasound in vitro: Alteration of protein profile and potential molecular mechanism

    Science.gov (United States)

    Feng, Yi; Wan, Mingxi

    2017-03-01

    To analyze the potential mechanism related to the apoptosis induced by low intensity focused ultrasound, comparative proteomic method was introduced in the study. After ultrasound irradiation (3.0 W/cm2, 1 minute, 6 hours incubation post-irradiation), the human SMMC-7721 hepatocarcinoma cells were stained by trypan blue to detect the morphologic changes, and then the percentage of early apoptosis were tested by the flow cytometry with double staining of FITC-labelled Annexin V/Propidium iodide. Two-dimensional SDS polyacrylamide gel electrophoresis was used to get the protein profile and some proteins differently expressed after ultrasound irradiation were identified by MALDI-TOF mass spectrometry. It's proved early apoptosis of cells were induced by low intentisy focused ultrasound. After ultrasound irradiation, the expressing characteristics of several proteins changed, in which protein p53 and heat shock proteins are associated with apoptosis initiation. It is suggested that the low-intensity ultrasound-induced apoptotic cancer therapy has the potential application via understanding its relevant molecular signaling and key proteins. Moreover, the comparative proteomic method is proved to be useful to supply information about the protein expression to analyze the metabolic processes related to bio-effects of biomedical ultrasound.

  11. Surface analysis of gold nanoparticles functionalized with thiol-modified glucose SAMs for biosensor applications.

    Directory of Open Access Journals (Sweden)

    Valentina eSpampinato

    2016-02-01

    Full Text Available In this work, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS, Principal Component Analysis (PCA and X-ray Photoelectron Spectroscopy (XPS have been used to characterize the surface chemistry of gold substrates before and after functionalization with thiol-modified glucose self-assembled monolayers and subsequent biochemical specific recognition of maltose binding protein (MBP.The results indicate that the surface functionalization is achieved both on flat and nanoparticles gold substrates thus showing the potential of the developed system as biodetection platform. Moreover, the method presented here has been found to be a sound and valid approach to characterize the surface chemistry of nanoparticles functionalized with large molecules.Both techniques were proved to be very useful tools for monitoring all the functionalization steps, including the investigation of the biological behaviour of the glucose-modified particles in presence of the maltose binding protein.

  12. Alterations in membrane protein-profile during cold treatment of alfalfa

    International Nuclear Information System (INIS)

    Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S.

    1988-01-01

    Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [ 35 S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo

  13. Seldi-tof MS Profiling of Plasma Proteins in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Shao-Pai Wu

    2006-03-01

    Conclusion: This study clearly demonstrates that the combined technology of SELDI-TOF MS and artificial intelligence is effective in distinguishing protein expression between normal and ovarian cancer plasma. The identified protein peaks may be candidate proteins for early detection of ovarian cancer or evaluation of therapeutic response.

  14. Eucalyptus tolerance mechanisms to lanthanum and cerium: subcellular distribution, antioxidant system and thiol pools.

    Science.gov (United States)

    Shen, Yichang; Zhang, Shirong; Li, Sen; Xu, Xiaoxun; Jia, Yongxia; Gong, Guoshu

    2014-12-01

    Guanglin 9 (Eucalyptus grandis × Eucalyptus urophlla) and Eucalyptus grandis 5 are two eucalyptus species which have been found to grow normally in soils contaminated with lanthanum and cerium, but the tolerance mechanisms are not clear yet. In this study, a pot experiment was conducted to investigate the tolerance mechanisms of the eucalyptus to lanthanum and cerium. Cell walls stored 45.40-63.44% of the metals under lanthanum or cerium stress. Peroxidase and catalase activities enhanced with increasing soil La or Ce concentrations up to 200 mg kg(-1), while there were no obvious changes in glutathione and ascorbate concentrations. Non-protein thiols concentrations increased with increasing treatment levels up to 200 mg kg(-1), and then decreased. Phytochelatins concentrations continued to increase under La or Ce stress. Therefore, the two eucalyptus species are La and Ce tolerant plants, and the tolerance mechanisms include cell wall deposition, antioxidant system response, and thiol compound synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Thiol functionalized polymethacrylic acid-based hydrogel microparticles for oral insulin delivery.

    Science.gov (United States)

    Sajeesh, S; Vauthier, C; Gueutin, C; Ponchel, G; Sharma, Chandra P

    2010-08-01

    In the present study thiol functionalized polymethacrylic acid-polyethylene glycol-chitosan (PCP)-based hydrogel microparticles were utilized to develop an oral insulin delivery system. Thiol modification was achieved by grafting cysteine to the activated surface carboxyl groups of PCP hydrogels (Cys-PCP). Swelling and insulin loading/release experiments were conducted on these particles. The ability of these particles to inhibit protease enzymes was evaluated under in vitro experimental conditions. Insulin transport experiments were performed on Caco-2 cell monolayers and excised intestinal tissue with an Ussing chamber set-up. Finally, the efficacy of insulin-loaded particles in reducing the blood glucose level in streptozotocin-induced diabetic rats was investigated. Thiolated hydrogel microparticles showed less swelling and had a lower insulin encapsulation efficiency as compared with unmodified PCP particles. PCP and Cys-PCP microparticles were able to inhibit protease enzymes under in vitro conditions. Thiolation was an effective strategy to improve insulin absorption across Caco-2 cell monolayers, however, the effect was reduced in the experiments using excised rat intestinal tissue. Nevertheless, functionalized microparticles were more effective in eliciting a pharmacological response in diabetic animal, as compared with unmodified PCP microparticles. From these studies thiolation of hydrogel microparticles seems to be a promising approach to improve oral delivery of proteins/peptides. Copyright 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

    Science.gov (United States)

    Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

    2013-10-01

    Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

  17. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Science.gov (United States)

    Wan, Cen; Lees, Jonathan G; Minneci, Federico; Orengo, Christine A; Jones, David T

    2017-10-01

    Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  18. Analysis of temporal transcription expression profiles reveal links between protein function and developmental stages of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Cen Wan

    2017-10-01

    Full Text Available Accurate gene or protein function prediction is a key challenge in the post-genome era. Most current methods perform well on molecular function prediction, but struggle to provide useful annotations relating to biological process functions due to the limited power of sequence-based features in that functional domain. In this work, we systematically evaluate the predictive power of temporal transcription expression profiles for protein function prediction in Drosophila melanogaster. Our results show significantly better performance on predicting protein function when transcription expression profile-based features are integrated with sequence-derived features, compared with the sequence-derived features alone. We also observe that the combination of expression-based and sequence-based features leads to further improvement of accuracy on predicting all three domains of gene function. Based on the optimal feature combinations, we then propose a novel multi-classifier-based function prediction method for Drosophila melanogaster proteins, FFPred-fly+. Interpreting our machine learning models also allows us to identify some of the underlying links between biological processes and developmental stages of Drosophila melanogaster.

  19. A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78.

    Science.gov (United States)

    Chaves, Daniela Fojo Seixas; de Souza, Emanuel Maltempi; Monteiro, Rose Adele; de Oliveira Pedrosa, Fábio

    2009-11-02

    Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant-bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.

  20. A secretory system for bacterial production of high-profile protein targets

    DEFF Research Database (Denmark)

    Kotzsch, Alexander; Vernet, Erik; Hammarström, Martin

    2011-01-01

    Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To impr......Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli...... membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly...

  1. Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply

    Directory of Open Access Journals (Sweden)

    Laura eCeballos-Laita

    2015-03-01

    Full Text Available The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164 were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5% changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as

  2. Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

    Directory of Open Access Journals (Sweden)

    Mosley R Lee

    2008-09-01

    Full Text Available Abstract Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS patients before and during immunization with glatiramer acetate (GA in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform.

  3. Computational Identification of Protein Pupylation Sites by Using Profile-Based Composition of k-Spaced Amino Acid Pairs.

    Directory of Open Access Journals (Sweden)

    Md Mehedi Hasan

    Full Text Available Prokaryotic proteins are regulated by pupylation, a type of post-translational modification that contributes to cellular function in bacterial organisms. In pupylation process, the prokaryotic ubiquitin-like protein (Pup tagging is functionally analogous to ubiquitination in order to tag target proteins for proteasomal degradation. To date, several experimental methods have been developed to identify pupylated proteins and their pupylation sites, but these experimental methods are generally laborious and costly. Therefore, computational methods that can accurately predict potential pupylation sites based on protein sequence information are highly desirable. In this paper, a novel predictor termed as pbPUP has been developed for accurate prediction of pupylation sites. In particular, a sophisticated sequence encoding scheme [i.e. the profile-based composition of k-spaced amino acid pairs (pbCKSAAP] is used to represent the sequence patterns and evolutionary information of the sequence fragments surrounding pupylation sites. Then, a Support Vector Machine (SVM classifier is trained using the pbCKSAAP encoding scheme. The final pbPUP predictor achieves an AUC value of 0.849 in 10-fold cross-validation tests and outperforms other existing predictors on a comprehensive independent test dataset. The proposed method is anticipated to be a helpful computational resource for the prediction of pupylation sites. The web server and curated datasets in this study are freely available at http://protein.cau.edu.cn/pbPUP/.

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

    Science.gov (United States)

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

    2008-08-25

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

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

    Science.gov (United States)

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

    2016-02-01

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

  6. Protein Profiles for Muscle Development and Intramuscular Fat Accumulation at Different Post-Hatching Ages in Chickens.

    Directory of Open Access Journals (Sweden)

    Jie Liu

    Full Text Available Muscle development and growth influences the efficiency of poultry meat production, and is closely related to deposition of intramuscular fat (IMF, which is crucial in meat quality. To clarify the molecular mechanisms underlying muscle development and IMF deposition in chickens, protein expression profiles were examined in the breast muscle of Beijing-You chickens at ages 1, 56, 98 and 140 days, using isobaric tags for relative and absolute quantification (iTRAQ. Two hundred and four of 494 proteins were expressed differentially. The expression profile at day 1 differed greatly from those at day 56, 98 and 140. KEGG pathway analysis of differential protein expression from pair-wise comparisons (day 1 vs. 56; 56 vs. 98; 98 vs. 140, showed that the fatty acid degradation pathway was more active during the stage from day 1 to 56 than at other periods. This was consistent with the change in IMF content, which was highest at day 1 and declined dramatically thereafter. When muscle growth was most rapid (days 56-98, pathways involved in muscle development were dominant, including hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac muscle contraction, tight junctions and focal adhesion. In contrast with hatchlings, the fatty acid degradation pathway was downregulated from day 98 to 140, which was consistent with the period for IMF deposition following rapid muscle growth. Changes in some key specific proteins, including fast skeletal muscle troponin T isoform, aldehyde dehydrogenase 1A1 and apolipoprotein A1, were verified by Western blotting, and could be potential biomarkers for IMF deposition in chickens. Protein-protein interaction networks showed that ribosome-related functional modules were clustered in all three stages. However, the functional module involved in the metabolic pathway was only clustered in the first stage (day 1 vs. 56. This study improves our understanding of the molecular mechanisms underlying muscle development and IMF

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

    Science.gov (United States)

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

    2015-05-01

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

  8. AFAL: a web service for profiling amino acids surrounding ligands in proteins

    Science.gov (United States)

    Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S.; Quatrini, Raquel

    2014-11-01

    With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.

  9. Membrane protein profiling of Acidovorax avenae subsp. avenae under various growth conditions.

    Science.gov (United States)

    Li, Bin; Wang, Li; Ibrahim, Muhammad; Ge, Mengyu; Wang, Yanli; Mannan, Shazia; Asif, Muhammad; Sun, Guochang

    2015-06-01

    Membrane proteins (MPs) of plant pathogenic bacteria have been reported to be able to regulate many essential cellular processes associated with plant disease. The aim of the current study was to examine and compare the expression of MPs of the rice bacterial pathogen Acidovorax avenae subsp. avenae strain RS-1 under Luria-Bertani (LB) medium, M9 medium, in vivo rice plant conditions and leaf extract (LE) medium mimicking in vivo plant condition. Proteomic analysis identified 95, 72, 75, and 87 MPs under LB, in vivo, M9 and LE conditions, respectively. Among them, six proteins were shared under all tested growth conditions designated as abundant class of proteins. Twenty-six and 21 proteins were expressed uniquely under in vivo versus LB medium and LE versus M9 medium, respectively, with 17 proteins common among these uniquely induced proteins. Moreover, most of the shared proteins are mainly related to energy metabolism, transport of small molecules, protein synthesis and secretion as well as virulence such as NADH, OmpA, secretion proteins. Therefore, the result of this study not only suggests that it may be an alternate method to analyze the in vivo expression of proteins by using LE medium to mimic plant conditions, but also reveals that the two sets of differentially expressed MPs, in particular the common MPs between them, might be important in energy metabolism, stress response and virulence of A. avenae subsp. avenae strain RS-1.

  10. Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

    Science.gov (United States)

    Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

    2013-11-01

    Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

  11. Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides.

    Science.gov (United States)

    Wang, Jiawen; Yao, Jizong; Sun, Nianrong; Deng, Chunhui

    2017-08-25

    As protein N-glycosylation involved in generation and development of various cancers and diseases, it is vital to capture glycopeptides from complex biological samples for biomarker discovery. In this work, by taking advantages of the interaction between titania and thiol groups, thiol-polyethylene glycol functionalized magnetic titania nanomaterials (denoted as Fe 3 O 4 @TiO 2 @PEG) were firstly fabricated as an excellent hydrophilic adsorbent of N-linked glycopeptides. On one hand, the special interaction of titanium-thiol makes the synthetic manipulation simple and provides a new idea for design and synthesis of novel nanomaterials; on the other hand, strong magnetic response could realize rapid separation and the outstanding hydrophilicity of polyethylene glycol makes Fe 3 O 4 @TiO 2 @PEG nanomaterials show superior performance for glycopeptides enrichment with ultralow limit of detection (0.1mol/μL) and high selectivity (1:100). As a result, 24 and 33 glycopeptides enriched from HRP and IgG digests were identified respectively by MALDI-TOF MS, and 300 glycopeptides corresponding to 106 glycoproteins were recognized from merely 2μL human serum, indicating a great potential of Fe 3 O 4 @TiO 2 @PEG nanomaterials for glycoproteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Comprehensive protein profiling by multiplexed capillary zone electrophoresis using cross-linked polyacrylamide coated capillaries.

    Science.gov (United States)

    Liu, Shaorong; Gao, Lin; Pu, Qiaosheng; Lu, Joann J; Wang, Xingjia

    2006-02-01

    We have recently developed a new process to create cross-linked polyacrylamide (CPA) coatings on capillary walls to suppress protein-wall interactions. Here, we demonstrate CPA-coated capillaries for high-efficiency (>2 x 10(6) plates per meter) protein separations by capillary zone electrophoresis (CZE). Because CPA virtually eliminates electroosmotic flow, positive and negative proteins cannot be analyzed in a single run. A "one-sample-two-separation" approach is developed to achieve a comprehensive protein analysis. High throughput is achieved through a multiplexed CZE system.

  13. Thiol-ene/methacrylate systems for mechanical damping

    Science.gov (United States)

    McNair, Olivia; Senyurt, Askim; Wei, Huanyu; Gould, Trent; Piland, Scott; Hoyle, Charles; Savin, Daniel

    2010-03-01

    Ternary thiol-ene-methacrylate (TEMA) networks as materials for mechanical energy damping are unique to the sports world. Using a photoinitiation process, TEMA systems are formed via an initial thiol-ene step-growth mechanism along with traditional radical polymerization of acrylate and ene monomers. Final networks have two-part morphologies: acrylate homopolymer sectors imbedded in a multi-component mesh. Several (TEMA) systems have been synthesized and analyzed via thermal and mechanical probing. Initial studies on these ternary systems have shown excellent properties compared to traditional ethylene vinyl alcohol (EVA) copolymers. For example, PEMA networks exhibit glass transition temperatures 33 K higher than EVA, resulting in improved damping at room temperature. This research will help develop relationships between tan delta, glass transition and their effects on mechanical energy damping for ternary (TEMA) systems.

  14. Cellular thiol levels and aerobic radiosensitization by BSO

    International Nuclear Information System (INIS)

    Varnes, M.E.; Biaglow, J.E.; Roizin-Towle, L.; Hall, E.J.

    1984-01-01

    It has been previously shown that pretreatment of A549 human lung carcinoma cells and V79 cells with BSO results in enhancement of the aerobic radiation response. The authors and others have found that addition of either N-acetylcysteine (NAC) or the radioprotector WR-2721 to BSO-treated cells, just prior to irradiation, results in a return to control levels of aerobic sensitivity. NAC and WR-2721 have no effect on the aerobic response of control cells. Reversal of the BSO effect appears unrelated to intracellular thiol levels, since neither NAC nor WR-2721 replenish NPSH within the time that the reversal of the radiation effect is observed. In addition, NAC and WR-2721 must be present during irradiation in order to reverse the BSO sensitization. The authors are continuing to investigate the phenomenon of BSO-induced aerobic sensitization and its reversal, with particular emphasis on the role of membrane thiols and pyridine nucleotide reducing species in radiation response

  15. The effect of thermal processing on protein quality and free amino acid profile of Terminalia catappa (Indian Almond) seed.

    Science.gov (United States)

    Adu, O B; Ogundeko, T O; Ogunrinola, O O; Saibu, G M; Elemo, B O

    2015-07-01

    The study examined the effect of various processing methods- boiling, drying and roasting- on the in vitro and in vivo protein digestibility and free amino acid profiles of Terminalia catappa seed. Moisture and crude protein of the various samples were determined. In vitro protein digestibility was determined after pepsin digestion. For the in vivo experiment, defatted T. catappa based diet was fed to 3 weeks old Wistar rats for 4 weeks and compared with animals maintained on casein based and nitrogen- free diets. The biological value (BV), net protein utilisation (NPU) and protein efficiency ratio (PER) of the diets were determined. Free amino acid composition was carried out using thin layer chromatography. Moisture was highest in the boiled T. catappa seed (8.30 ± 0.00 %). The raw, roasted and dried seeds had 5.55 ± 0.07, 3.88 ± 0.22 and 3.75 ± 0.07 % respectively. Crude protein was 19.19, 18.89, 17.62 and 16.36 % in the dried, roasted, boiled and raw seeds respectively. Roasted T. catappa seed had the highest in vitro protein digestibility with 37.52 %, while the dried, boiled and raw samples had digestibility values of 27.57, 27.07 and 24.45 % respectively. All nine essential amino acids were present in T. catappa in high concentrations except methionine and tryptophan. Glutamate was present in the highest concentration. Also, free amino acids were higher in the processed seeds compared to the raw seed. Animals fed T. catappa diet compared favourably with the casein group, thus indicating that the protein is of good quality.

  16. Contrasting bonding behavior of thiol molecules on carbon fullerene structures

    International Nuclear Information System (INIS)

    Mixteco-Sanchez, J.C.; Guirado-Lopez, R.A.

    2003-01-01

    We have performed semiempirical as well as ab initio density-functional theory (DFT) calculations at T=0 to analyze the equilibrium configurations and electronic properties of spheroidal C 60 as well as of cylindrical armchair (5,5) and (8,8) fullerenes passivated with SCH 3 and S(CH 2 ) 2 CH 3 thiols. Our structural results reveal that the lowest-energy configurations of the adsorbates strongly depend on their chain length and on the structure of the underlying substrate. In the low-coverage regime, both SCH 3 and S(CH 2 ) 2 CH 3 molecules prefer to organize into a molecular cluster on one side of the C 60 surface, providing thus a less protective organic coating for the carbon structure. However, with increasing the number of adsorbed thiols, a transition to a more uniform distribution is obtained, which actually takes place for six and eight adsorbed molecules when using S(CH 2 ) 2 CH 3 and SCH 3 chains, respectively. In contrast, for the tubelike arrangements at the low-coverage regime, a quasi-one-dimensional zigzag organization of the adsorbates along the tubes is always preferred. The sulfur-fullerene bond is considerably strong and is at the origin of outward and lateral displacements of the carbon atoms, leading to the stabilization of three-membered rings on the surface (spheroidal structures) as well as to sizable nonuniform radial deformations (cylindrical configurations). The electronic spectrum of our thiol-passivated fullerenes shows strong variations in the energy difference between the highest occupied and lowest unoccupied molecular orbitals as a function of the number and distribution of adsorbed thiols, opening thus the possibility to manipulate the transport properties of these compounds by means of selective adsorption mechanisms

  17. Investigation of thiol derivatized gold nanoparticle sensors for gas analysis

    Science.gov (United States)

    Stephens, Jared S.

    Analysis of volatile organic compounds (VOCs) in air and exhaled breath by sensor array is a very useful testing technique. It can provide non-invasive, fast, inexpensive testing for many diseases. Breath analysis has been very successful in identifying cancer and other diseases by using a chemiresistor sensor or array with gold nanoparticles to detect biomarkers. Acetone is a biomarker for diabetes and having a portable testing device could help to monitor diabetic and therapeutic progress. An advantage to this testing method is it is conducted at room temperature instead of 200 degrees Celsius. 3. The objective of this research is to determine the effect of thiol derivatized gold nanoparticles based on sensor(s) detection of VOCs. The VOCs to be tested are acetone, ethanol, and a mixture of acetone and ethanol. Each chip is tested under all three VOCs and three concentration levels (0.1, 1, and 5.0 ppm). VOC samples are used to test the sensors' ability to detect and differentiate VOCs. Sensors (also referred to as a chip) are prepared using several types of thiol derivatized gold nanoparticles. The factors are: thiol compound and molar volume loading of the thiol in synthesis. The average resistance results are used to determine the VOC selectivity of the sensors tested. The results show a trend of increasing resistance as VOC concentration is increased relative to dry air; which is used as baseline for VOCs. Several sensors show a high selectivity to one or more VOCs. Overall the 57 micromoles of 4-methoxy-toluenethiol sensor shows the strongest selectivity for VOCs tested. 3. Gerfen, Kurt. 2012. Detection of Acetone in Air Using Silver Ion Exchanged ZSM-5 and Zinc Oxide Sensing Films. Master of Science thesis, University of Louisville.

  18. Expression Profiling of Human Genetic and Protein Interaction Networks in Type 1 Diabetes

    DEFF Research Database (Denmark)

    Brunak, Søren; Bergholdt, R; Brorsson, C

    2009-01-01

    Proteins contributing to a complex disease are often members of the same functional pathways. Elucidation of such pathways may provide increased knowledge about functional mechanisms underlying disease. By combining genetic interactions in Type 1 Diabetes (T1D) with protein interaction data we have...

  19. Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.

    Science.gov (United States)

    Tan, Chris Soon Heng; Go, Ka Diam; Bisteau, Xavier; Dai, Lingyun; Yong, Chern Han; Prabhu, Nayana; Ozturk, Mert Burak; Lim, Yan Ting; Sreekumar, Lekshmy; Lengqvist, Johan; Tergaonkar, Vinay; Kaldis, Philipp; Sobota, Radoslaw M; Nordlund, Pär

    2018-03-09

    Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  20. Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

    International Nuclear Information System (INIS)

    Liu, Chih-Wei; Bramer, Lisa; Computational Modeling); Webb-Robertson, Bobbie-Jo; Computational Modeling); Waugh, Kathleen; Rewers, Marian J.; Zhang, Qibin; Biochemistry)

    2017-01-01

    We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.

  1. Independent component analysis for the extraction of reliable protein signal profiles from MALDI-TOF mass spectra.

    Science.gov (United States)

    Mantini, Dante; Petrucci, Francesca; Del Boccio, Piero; Pieragostino, Damiana; Di Nicola, Marta; Lugaresi, Alessandra; Federici, Giorgio; Sacchetta, Paolo; Di Ilio, Carmine; Urbani, Andrea

    2008-01-01

    Independent component analysis (ICA) is a signal processing technique that can be utilized to recover independent signals from a set of their linear mixtures. We propose ICA for the analysis of signals obtained from large proteomics investigations such as clinical multi-subject studies based on MALDI-TOF MS profiling. The method is validated on simulated and experimental data for demonstrating its capability of correctly extracting protein profiles from MALDI-TOF mass spectra. The comparison on peak detection with an open-source and two commercial methods shows its superior reliability in reducing the false discovery rate of protein peak masses. Moreover, the integration of ICA and statistical tests for detecting the differences in peak intensities between experimental groups allows to identify protein peaks that could be indicators of a diseased state. This data-driven approach demonstrates to be a promising tool for biomarker-discovery studies based on MALDI-TOF MS technology. The MATLAB implementation of the method described in the article and both simulated and experimental data are freely available at http://www.unich.it/proteomica/bioinf/.

  2. C-reactive protein in patients with acute coronary syndrome: association with coronary markers, lipid profile and markers of coagulation

    International Nuclear Information System (INIS)

    Munir, T.A.; Afzal, M.N.

    2010-01-01

    To determine levels of C-reactive protein (CRP) and its association with coronary markers, lipid profile and markers of coagulation in patients of acute coronary syndrome (ACS). The study was conducted at Shifa college of Medicine and Shifa international hospital for a period of one year (November 2005-December 2006). Patients and Methods: Sixty nine age matched controls and 133 consecutive patients of ACS were included in the study. CRP were measured by immunoturbidometric method, MB fraction of creatine kinase (CK-MB) and Troponin-1 by micro-particle enzyme immunoassay, lipid levels by Colorimetric Enzymatic methods, platelets by celldyn and coagulation markers were measured by CA-50 Sysmax. At admission mean CRP levels, cardiac biomarkers, lipid profile and coagulation markers were significantly increased in patients of ACS versus controls. Within the patients of ACS the mean levels of CRP, CK-MB, Trop I, prothrombin time (PT) and activated partial thromboplastin time (Am) were significantly raised in patients with ST - elevation myocardial infarction (STEMI) and non STEMI (NSTEMI) versus patients of unstable angina (VA). Association between CRP levels and coronary markers, coagulation markers and lipid profile was found to be non significant. The CRP levels were increased in patients with ACS as compared to controls. The CRP levels were insignificantly correlated with coronary markers (CK-MB, Trop I), coagulation markers (platelet count, PT, Am), and lipid profile (cholesterol, triglyceride, HDL and LDL cholesterol) in patients with ACS. (author)

  3. Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection

    Science.gov (United States)

    Kim, Tae Yun; Song, Kye-Yong; Sohn, Woon-Mok; Kang, Shin-Yong

    2001-01-01

    In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection. PMID:11775331

  4. Simple preparation of thiol-ene particles in glycerol and surface functionalization by thiol-ene chemistry (TEC) and surface chain transfer free radical polymerization (SCT-FRP)

    DEFF Research Database (Denmark)

    Hoffmann, Christian; Chiaula, Valeria; Yu, Liyun

    2018-01-01

    functionalization of excess thiol groups via photochemical thiol-ene chemistry (TEC) resulting in a functional monolayer. In addition, surface chain transfer free radical polymerization (SCT-FRP) was used for the first time to introduce a thicker polymer layer on the particle surface. The application potential...

  5. FULL-LENGTH PEPTIDE ASSAY OF ANTIGENIC PROFILE OF ENVELOPE PROTEINS FROM SIBERIAN ISOLATES OF HEPATITIS C VIRUS

    Directory of Open Access Journals (Sweden)

    A. A. Grazhdantseva

    2010-01-01

    Full Text Available Antigenic profiles of envelope glycoproteins of hepatitis C virus presented by three genotypes 1b, 2a/2c and 3a, which are most widespread in the territory of Russia and, in particular, in Novosibirsk, were studied using a panel of overlapping synthetic peptides. It was shown that highly immunogenic peptide epitopes of Е1 and Е2 proteins common for all HCV genotypes, are located in amino acid positions 250-260, 315-325 (Е1 protein, 390-400 (hypervariable region 1, 430-440, and 680-690 (Е2 protein. The greatest inter-genotypic differences were recorded in positions 280-290, 410-430 and 520-540. A novel antigenic determinant was detected in the region of aa 280-290 of the Е1 protein which was typical only for HCV 2a/2c genotype. A broad variation in the boundaries for the most epitopes suggests a high variability of the Е1 and Е2 viral proteins; however, a similar repertoire of antibodies induced by different HCV genotypes indicates to an opportunity of designing a new generation of cross-reactive HCV vaccines based on mapping of the E1 and E2 antigenic regions.

  6. Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

    2013-06-01

    Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

  7. Dynamic thiol/disulphide homeostasis in patients with basal cell carcinoma.

    Science.gov (United States)

    Demirseren, Duriye Deniz; Cicek, Cagla; Alisik, Murat; Demirseren, Mustafa Erol; Aktaş, Akın; Erel, Ozcan

    2017-09-01

    The aim of this study is to measure and compare the dynamic thiol/disulphide homeostasis of patients with basal cell carcinoma and healthy subjects with a newly developed and original method. Thirty four patients attending our outpatient clinic and clinically and histopathologically diagnosed as nodular basal cell carcinoma, and age and gender matched 30 healthy individuals have been involved in the study. Thiol/disulphide homeostasis tests have been measured with a novel automatic spectrophotometric method developed and the results have been compared statistically. Serum native thiol and disulphide levels in the patient and control group show a considerable variance statistically (p = 0.028, 0.039, respectively). Total thiol levels do not reveal a considerable variation (p = 0.094). Disulphide/native thiol ratios and native thiol/total thiol ratios also show a considerable variance statistically (p = 0.012, 0.013, 0.010, respectively). Thiol disulphide homeostasis in patients with basal cell carcinoma alters in the way that disulphide gets lower and thiols get higher. Thiol/disulphide level is likely to have a role in basal cell carcinoma pathogenesis.

  8. Quantifying changes in the cellular thiol-disulfide status during differentiation of B cells into antibody-secreting plasma cells

    DEFF Research Database (Denmark)

    Hansen, Rosa Rebecca Erritzøe; Otsu, Mieko; Braakman, Ineke

    2013-01-01

    by the differentiation, steady-state levels of glutathionylated protein thiols are less than 0.3% of the total protein cysteines, even in fully differentiated cells, and the overall protein redox state is not affected until late in differentiation, when large-scale IgM production is ongoing. A general expansion......Plasma cells produce and secrete massive amounts of disulfide-containing antibodies. To accommodate this load on the secretory machinery, the differentiation of resting B cells into antibody-secreting plasma cells is accompanied by a preferential expansion of the secretory compartments of the cells...... of the ER does not affect global protein redox status until an extensive production of cargo proteins has started....

  9. Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: Prevention by thiol group protecting agents

    International Nuclear Information System (INIS)

    Custodio, Jose B.A.; Cardoso, Carla M.P.; Santos, Maria S.; Almeida, Leonor M.; Vicente, Joaquim A.F.; Fernandes, Maria A.S.

    2009-01-01

    Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca 2+ -induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20 nmol/mg protein) induced Ca 2+ -dependent mitochondrial swelling, depolarization of membrane potential (ΔΨ), Ca 2+ release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40 nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the ΔΨ, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H 2 O 2 generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca 2+ -induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to

  10. Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes.

    Science.gov (United States)

    Barac, Miroljub; Cabrilo, Slavica; Pesic, Mirjana; Stanojevic, Sladjana; Zilic, Sladjana; Macej, Ognjen; Ristic, Nikola

    2010-01-01

    Extractability, extractable protein compositions, technological-functional properties of pea (Pisum sativum) proteins from six genotypes grown in Serbia were investigated. Also, the relationship between these characteristics was presented. Investigated genotypes showed significant differences in storage protein content, composition and extractability. The ratio of vicilin:legumin concentrations, as well as the ratio of vicilin + convicilin: Legumin concentrations were positively correlated with extractability. Our data suggest that the higher level of vicilin and/or a lower level of legumin have a positive influence on protein extractability. The emulsion activity index (EAI) was strongly and positively correlated with the solubility, while no significant correlation was found between emulsion stability (ESI) and solubility, nor between foaming properties and solubility. No association was evident between ESI and EAI. A moderate positive correlation between emulsion stability and foam capacity was observed. Proteins from the investigated genotypes expressed significantly different emulsifying properties and foam capacity at different pH values, whereas low foam stability was detected. It appears that genotype has considerable influence on content, composition and technological-functional properties of pea bean proteins. This fact can be very useful for food scientists in efforts to improve the quality of peas and pea protein products.

  11. Analysis of protein profile and pollen morphology of guaiacum officinale linn

    International Nuclear Information System (INIS)

    Zeb, S.

    2017-01-01

    Asthma and allergic rhinitis is triggered by the pollen of trees, grasses and weeds. Guaiacum officinale L. tree is widely cultivated along with the road side. This species was selected to check its allergenic role.Pollen morphology of Guaiacum officinale was examined by Light microsco4pe (LM) and Scanning electron microscope (SEM). Pollen grains of Guaiacum officinale were prolate shape, having tricolpate aperture, and rugulate tectum. Pollen protein concentration of G. officinale was determined by Bradford's assay and qualitative protein analysis of pollen was done by SDS-PAGE(Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis).Total protein content in the pollen extract was 24.28mg/g of pollen. The SDS-PAGE pollen grains protein analysis showed 07 different protein bands. The molecular weight of separated proteins ranged from 25 to 65kDa. Biochemical analysis of G. officinale pollen grains revealed the presence of low molecular weight proteins therefore it is strongly suggested that this species must be considered as a potent allergy causing species. This research would help for the proper diagnosis and treatment of the bronchial allergy suffering patients. (author)

  12. Protein and antigen profiles of Leptospira interrogans serovar Hardjo Perfil proteico e antigênico da Leptospira interrogans sorovariedade Hardjo

    Directory of Open Access Journals (Sweden)

    Bárbara Nobre Lafetá

    2009-12-01

    Full Text Available The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.Estudou-se o perfil proteico da membrana externa da Leptospira interrogans sorovariedade Hardjo, amostra hardjoprajitno, associado à resposta imune natural de bovinos infectados. Foram utilizados Triton X114 para a extração das proteínas de membrana externa e acetona para precipitá-las. As proteínas extraídas foram analisadas por SDS-PAGE e western blot contra soro de coelhos hiperimunes e de bovinos naturalmente infectados. Em géis corados com nitrato de prata, 14 bandas proteicas foram identificadas, e quatro dessas bandas, com 22, 29, 47 e 63kDa, foram as mais proeminentes. Os western blots com soro hiperimune de coelho detectaram bandas correspondentes a proteínas com pesos moleculares de 35, 27, 24, 21, 17 e 14kDa, e bandas de 32 e 45kDa destacaram-se nos testes com soros de bovinos naturalmente infectados.

  13. Hmb(off/on) as a switchable thiol protecting group for native chemical ligation.

    Science.gov (United States)

    Qi, Yun-Kun; Tang, Shan; Huang, Yi-Chao; Pan, Man; Zheng, Ji-Shen; Liu, Lei

    2016-05-04

    A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.

  14. Inhibition of the Vacuolar-like ATPase from Halobacterium saccharovorum by Thiol Reagents: Evidence for Different Functional Thiols

    Science.gov (United States)

    Hochstein, L. I.; Stanlotter, H.; Emrich, E.; Morrison, David (Technical Monitor)

    1994-01-01

    N-Ethylmaleimide (NEM) inhibited the vacuolar-like ATPase from Halobacterium saccharovorum (K(sub i) approximately 1 mM) by modifying one or more of the thiols located on the largest of the subunit. ATP protected against inhibition and coincidentally prevented NEM binding which suggested that NEM acts at or near the catalytic site. p-Chloromercuriphenylsulfonate (PCMS) also inhibited this ATPase (K(sub i) approximately 90 microM). ATP did not protect against PCMS inhibition. Dithiothreitol (DTT) partially reversed PCMS inhibition and restored approximately half of the initial activity of 90% inhibited enzyme. DTT did not restore activity of the NEM-inhibited enzyme or the PCMS-inhibited enzyme when it was subsequently incubated with NEM. The failure of ATP to protect against PCMS inhibition and the inability of DTT to restore activity of enzyme incubated in the presence of PCMS and NEM suggests these reagents react with different thiols and that the PCMS-sensitive thiol may have a structural role.

  15. Understanding Alzheimer's disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles

    DEFF Research Database (Denmark)

    Lassen, Pernille S.; Thygesen, Camilla; Larsen, Martin R.

    2017-01-01

    elucidated them in neurodegenerative diseases such as Alzheimer's disease. Here, we comprehensively review Alzheimer's pathology in relation to protein phosphorylation and glycosylation on synaptic plasticity from neuroproteomics data. Moreover, we highlight several mass spectrometry-based sample processing...

  16. Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

    International Nuclear Information System (INIS)

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-01

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  17. Growth inhibition and differences in protein profiles in azadirachtin-treated Drosophila melanogaster larvae.

    Science.gov (United States)

    Wang, Hao; Lai, Duo; Yuan, Mei; Xu, Hanhong

    2014-04-01

    Azadirachtin A is a very effective biopesticide widely used in insect pest control. It has strong antifeeding and growth inhibitory activity against most insects, however, its mode of action is still unclear. Proteomic experiments using 2DE indicate significant effects of Azadirachtin A on the amount of proteins related to growth inhibition in Drosophila melanogaster larvae. Twenty-one spots with different intensity in azadirachtin-treated larvae were identified. These proteins are involved in cytoskeletal organization, transcription and translation, hormonal regulation, and energy metabolism. Protein network analysis reveals heat shock protein 23 to be a potential target of azadirachtin. These results provide new insights into understanding the mechanism of growth inhibition in insects in response to azadirachtin. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

    Directory of Open Access Journals (Sweden)

    Yushen Du

    2016-11-01

    Full Text Available Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp, we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

  19. Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

    Science.gov (United States)

    Correani, Virginia; Di Francesco, Laura; Mignogna, Giuseppina; Fabrizi, Cinzia; Leone, Stefano; Giorgi, Alessandra; Passeri, Alessia; Casata, Roberto; Fumagalli, Lorenzo; Maras, Bruno; Schininà, M Eugenia

    2017-09-01

    In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

    Science.gov (United States)

    Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

    2012-01-01

    Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

  1. Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don Endl

    Directory of Open Access Journals (Sweden)

    Chen Yu-Ting

    2010-12-01

    Full Text Available Abstract Background Restoration of rooting competence is important for rejuvenation in Sequoia sempervirens (D. Don Endl and is achieved by repeatedly grafting Sequoia shoots after 16 and 30 years of cultivation in vitro. Results Mass spectrometry-based proteomic analysis revealed three proteins that differentially accumulated in different rejuvenation stages, including oxygen-evolving enhancer protein 2 (OEE2, glycine-rich RNA-binding protein (RNP, and a thaumatin-like protein. OEE2 was found to be phosphorylated and a phosphopeptide (YEDNFDGNSNVSVMVpTPpTDK was identified. Specifically, the protein levels of OEE2 increased as a result of grafting and displayed a higher abundance in plants during the juvenile and rejuvenated stages. Additionally, SsOEE2 displayed the highest expression levels in Sequoia shoots during the juvenile stage and less expression during the adult stage. The expression levels also steadily increased during grafting. Conclusion Our results indicate a positive correlation between the gene and protein expression patterns of SsOEE2 and the rejuvenation process, suggesting that this gene is involved in the rejuvenation of Sequoia sempervirens.

  2. Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles.

    Science.gov (United States)

    Ayyadevara, Srinivas; Mercanti, Federico; Wang, Xianwei; Mackintosh, Samuel G; Tackett, Alan J; Prayaga, Sastry V S; Romeo, Francesco; Shmookler Reis, Robert J; Mehta, Jawahar L

    2016-05-01

    Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each Phypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual. © 2016 American Heart Association, Inc.

  3. Thiol-based redox signaling in the nitrogen-fixing symbiosis

    Directory of Open Access Journals (Sweden)

    Pierre eFrendo

    2013-09-01

    Full Text Available In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, their spatio-temporally distribution is correlated with the corresponding (homoglutathione synthetase activities, and are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume

  4. Fabrication of antibody-loaded microgels using microfluidics and thiol-ene photoclick chemistry.

    Science.gov (United States)

    Gregoritza, Manuel; Abstiens, Kathrin; Graf, Moritz; Goepferich, Achim M

    2018-06-01

    Reducing burst effects, providing controlled release, and safeguarding biologics against degradation are a few of several highly attractive applications for microgels in the field of controlled release. However, the incorporation of proteins into microgels without impairing stability is highly challenging. In this proof of concept study, the combination of microfluidics and thiol-ene photoclick chemistry was evaluated for the fabrication of antibody-loaded microgels with narrow size distribution. Norbornene-modified eight-armed poly(ethylene glycol) with an average molecular mass of 10,000 Da, 20,000 Da, or 40,000 Da were prepared as macromonomers for microgel formation. For functionalization, either hydrolytically cleavable ester or stable amide bonds were used. A microfluidic system was employed to generate precursor solution droplets containing macromonomers, the cross-linker dithiothreitol and the initiator Eosin-Y. Irradiation with visible light was used to trigger thiol-ene reactions which covalently cross-linked the droplets. For all bond-types, molecular masses, and concentrations gelation was very rapid (<20 s) and a plateau for the complex shear modulus was reached after only 5 min. The generated microgels had a rod-like shape and did not show considerable cellular toxicity. Stress conditions during the fabrication process were simulated and it could be shown that fabrication did not impair the activity of the model proteins lysozyme and bevacizumab. It was confirmed that the average hydrogel network mesh size was similar or smaller than the hydrodynamic diameter of bevacizumab which is a crucial factor for restricting diffusion and delaying release. Finally, microgels were loaded with bevacizumab and a sustained release over a period of 30 ± 4 and 47 ± 7 days could be achieved in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper.

    Science.gov (United States)

    Jiang, Weidong; Mao, Ying Qing; Huang, Ruochun; Duan, Chaohui; Xi, Yun; Yang, Kai; Huang, Ruo-Pan

    2014-01-31

    Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Altered protein expression profiles in umbilical veins: insights into vascular dysfunctions of the children born after in vitro fertilization.

    Science.gov (United States)

    Gao, Qian; Pan, Hai-Tao; Lin, Xian-Hua; Zhang, Jun-Yu; Jiang, Ying; Tian, Shen; Chen, Lu-Ting; Liu, Miao-E; Xiong, Yi-Meng; Huang, He-Feng; Sheng, Jian-Zhong

    2014-09-01

    Cardiovascular dysfunction and remodeling have been found in some children conceived by in vitro fertilization (IVF). However, the underlying mechanisms remain unclear. In this study, the retrospective investigation showed that the blood pressure of IVF-conceived Chinese children was higher than that of naturally conceived (NC) children at ages 3-13 yr. We analyzed the expression profile of proteins in the umbilical veins of IVF and NC newborns by proteomic techniques. Using iTRAQ (isobaric tags for relative and absolute quantitation), 47 differentially expressed proteins (DEPs) were identified by feature selection in IVF umbilical veins compared with NC. Ingenuity Pathway Analysis, which is used to explore the signaling pathways of DEPs, revealed that these DEPs played important roles in vascular system development and carbon metabolism, implying that these DEPs might be potential candidates for further exploration of the mechanism(s) of vascular dysfunction in IVF children. We found that the serum estradiol (E₂) level in the cord blood of IVF newborns was significantly higher than that of NC newborns. High concentrations of E₂ induced alteration of lumican and vimentin expression in human umbilical vein endothelial cells, which was consistent with the proteomic results. These findings suggested that abnormal expression of proteins in umbilical veins might be related to the cardiovascular dysfunction and remodeling in IVF offspring. In conclusion, our data for the first time reveal the protein expression profile in blood vessels of IVF offspring and provide information for further mechanism study and evaluation of risks of cardiovascular abnormality in IVF children. © 2014 by the Society for the Study of Reproduction, Inc.

  7. Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

    Directory of Open Access Journals (Sweden)

    Marius Schild

    2016-01-01

    Full Text Available Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n=19 and untrained (SED, n=17 individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

  8. Simulation studies on structural and thermal properties of alkane thiol capped gold nanoparticles.

    Science.gov (United States)

    Devi, J Meena

    2017-06-01

    The structural and thermal properties of the passivated gold nanoparticles were explored employing molecular dynamics simulation for the different surface coverage densities of the self-assembled monolayer (SAM) of alkane thiol. The structural properties of the monolayer protected gold nanoparticles such us overall shape, organization and conformation of the capping alkane thiol chains were found to be influenced by the capping density. The structural order of the thiol capped gold nanoparticles enhances with the increase in the surface coverage density. The specific heat capacity of the alkane thiol capped gold nanoparticles was found to increase linearly with the thiol coverage density. This may be attributed to the enhancement in the lattice vibrational energy. The present simulation results suggest, that the structural and thermal properties of the alkane thiol capped gold nanoparticles may be modified by the suitable selection of the SAM coverage density. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Predicting protein-ATP binding sites from primary sequence through fusing bi-profile sampling of multi-view features

    Directory of Open Access Journals (Sweden)

    Zhang Ya-Nan

    2012-05-01

    Full Text Available Abstract Background Adenosine-5′-triphosphate (ATP is one of multifunctional nucleotides and plays an important role in cell biology as a coenzyme interacting with proteins. Revealing the binding sites between protein and ATP is significantly important to understand the functionality of the proteins and the mechanisms of protein-ATP complex. Results In this paper, we propose a novel framework for predicting the proteins’ functional residues, through which they can bind with ATP molecules. The new prediction protocol is achieved by combination of sequence evolutional information and bi-profile sampling of multi-view sequential features and the sequence derived structural features. The hypothesis for this strategy is single-view feature can only represent partial target’s knowledge and multiple sources of descriptors can be complementary. Conclusions Prediction performances evaluated by both 5-fold and leave-one-out jackknife cross-validation tests on two benchmark datasets consisting of 168 and 227 non-homologous ATP binding proteins respectively demonstrate the efficacy of the proposed protocol. Our experimental results also reveal that the residue structural characteristics of real protein-ATP binding sites are significant different from those normal ones, for example the binding residues do not show high solvent accessibility propensities, and the bindings prefer to occur at the conjoint points between different secondary structure segments. Furthermore, results also show that performance is affected by the imbalanced training datasets by testing multiple ratios between positive and negative samples in the experiments. Increasing the dataset scale is also demonstrated useful for improving the prediction performances.

  10. The levels of nitrite and nitrate, proline and protein profiles in tomato plants infected with pseudomonas syringae

    International Nuclear Information System (INIS)

    Berber, I.; Onlu, H.

    2012-01-01

    In this study, the contents of nitrite-nitrate and free L-proline, and pathogenesis-related (PR) proteins in tomato plants following inoculation with Pseudomonas syringae pv. tomato strain were examined. The results of the nitrite and nitrate indicated that there was a reduction in the levels of nitrate in the infected tomato plants through 1-8 study days, compared with the healthy plants. On the other hands, when the nitrite amounts increased in the first and second days, the nitrite concentrations reduced in infected plants at subsequent time periods, compared with uninfected plants. The accumulation of free proline increased in the infected plants, according to control plants. The whole-cell protein profiles displayed that the levels of the protein bands of molecular masses 204.6 kDa and 69.9 kDa significantly increased in infected and uninfected plants during 2-10 study days. In additionally, in the quantities of the protein bands of molecular weights 90.3 and 79.4 kDa were observed an increase in the infected and healthy plants after the fourth day. However, the protein band of molecular weight 54.3 kDa was visible only in uninfected plants for the fourth and eighth days. Finally, the study suggest that there were the sophisticate relationships among the proline accumulation, the conversion of nitrate to nitrite and the induction of PR protein genes in the regulation of defense mechanisms toward microbial invaders. Our results also indicated that the increases in nitrite and proline contents might be useful indicator for the response toward pathogen attacks. (author)

  11. Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets

    DEFF Research Database (Denmark)

    Stevenson, L.S.; McCullough, K.; Vincent, I.

    2006-01-01

    The purpose of this study was to determine serum profiles of cytokines at a protein level and C-reactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined...

  12. Comparisons of protein profiles of beech bark disease resistant and susceptible American beech (Fagus grandifolia)

    Science.gov (United States)

    2013-01-01

    Background Beech bark disease is an insect-fungus complex that damages and often kills American beech trees and has major ecological and economic impacts on forests of the northeastern United States and southeastern Canadian forests. The disease begins when exotic beech scale insects feed on the bark of trees, and is followed by infection of damaged bark tissues by one of the Neonectria species of fungi. Proteomic analysis was conducted of beech bark proteins from diseased trees and healthy trees in areas heavily infested with beech bark disease. All of the diseased trees had signs of Neonectria infection such as cankers or fruiting bodies. In previous tests reported elsewhere, all of the diseased trees were demonstrated to be susceptible to the scale insect and all of the healthy trees were demonstrated to be resistant to the scale insect. Sixteen trees were sampled from eight geographically isolated stands, the sample consisting of 10 healthy (scale-resistant) and 6 diseased/infested (scale-susceptible) trees. Results Proteins were extracted from each tree and analysed in triplicate by isoelectric focusing followed by denaturing gel electrophoresis. Gels were stained and protein spots identified and intensity quantified, then a statistical model was fit to identify significant differences between trees. A subset of BBD differential proteins were analysed by mass spectrometry and matched to known protein sequences for identification. Identified proteins had homology to stress, insect, and pathogen related proteins in other plant systems. Protein spots significantly different in diseased and healthy trees having no stand or disease-by-stand interaction effects were identified. Conclusions Further study of these proteins should help to understand processes critical to resistance to beech bark disease and to develop biomarkers for use in tree breeding programs and for the selection of resistant trees prior to or in early stages of BBD development in stands. Early

  13. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    Directory of Open Access Journals (Sweden)

    Adriana FIORINI

    2016-01-01

    Full Text Available Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC and sub-minimal inhibitory concentration (sub-MIC of the butanolic extract (BUTE of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1, amino acid metabolism (ILV5, PDC11 and protein synthesis (ASC1 pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides, it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

  14. Coculture of Bifidobacterium longum and Bifidobacterium breve alters their protein expression profiles and enzymatic activities.

    Science.gov (United States)

    Ruiz, Lorena; Sánchez, Borja; de Los Reyes-Gavilán, Clara G; Gueimonde, Miguel; Margolles, Abelardo

    2009-07-31

    Some strains of the genus Bifidobacterium are probiotic bacteria commonly added to functional dairy products. The influence of coculturing Bifidobacterium longum NCIMB8809 and Bifidobacterium breve NCIMB8807 on their physiology was studied. 2DE separation of protein extracts, coupled to MS protein analysis allowed the identification of 16 proteins whose expression drastically changed when cells were grown in compartmentalized coculture, compared to monoculture. These included ribosomal proteins and proteins involved in carbohydrate metabolism, gene regulation, cell envelope biogenesis and transport processes. Significant changes in some glycoside-hydrolysing activities (beta-d-xylopyranosidase, alpha-l-arabinofuranosidase and beta-d-glucopyranosidase) were also detected. Furthermore, qRT-PCR experiments using as targets the B. breve genes clgR (transcriptional regulator) clpP1, clpP2 and clpC (chaperone- and protease-encoding genes positively regulated by clgR) supported the proteomic results, the four genes displaying a higher expression level in coculture. This study provides new insights to understand the communication among Bifidobacterium species.

  15. Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Christensen, René Depont; Madsen, Jonna S

    2008-01-01

    for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom......Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum......-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard...

  16. Serum peptide/protein profiling by mass spectrometry provides diagnostic information independently of CA125 in women with an ovarian tumor

    DEFF Research Database (Denmark)

    Callesen, Anne; Madsen, Jonna S; Iachina, Maria

    2010-01-01

    In the present study, the use of a robust and sensitive mass spectrometry based protein profiling analysis was tested as diagnostic tools for women with an ovarian tumor. The potential additional diagnostic value of serum protein profiles independent of the information provided by CA125 were also...... investigated. Protein profiles of 113 serum samples from women with an ovarian tumor (54 malign and 59 benign) were generated using MALDI-TOF MS. A total of 98 peaks with a significant difference (pwomen with benign tumors/cysts and malignant ovarian tumors were identified. After...... average linkage clustering, a profile of 46 statistical significant mass peaks was identified to distinguish malignant tumors and benign tumors/cysts. In the subgroup of women with normal CA125 values (

  17. Ester-free Thiol-X Resins: New Materials with Enhanced Mechanical Behavior and Solvent Resistance

    OpenAIRE

    Podgórski, Maciej; Becka, Eftalda; Chatani, Shunsuke; Claudino, Mauro; Bowman, Christopher N.

    2015-01-01

    A series of thiol-Michael and radical thiol-ene network polymers were successfully prepared from ester-free as well as ester-containing monomer formulations. Polymerization reaction rates, dynamic mechanical analysis, and solvent resistance experiments were performed and compared between compositions with varied ester loading. The incorporation of ester-free alkyl thiol, vinyl sulfone and allylic monomers significantly improved the mechanical properties when compared with commercial, mercapto...

  18. Multi-chamber and multi-layer thiol-ene microchip for cell culture

    DEFF Research Database (Denmark)

    Tan, H. Y.; Hemmingsen, Mette; Lafleur, Josiane P.

    2014-01-01

    We present a multi-layer and multi-chamber microfluidic chip fabricated using two different thiol-ene mixtures. Sandwiched between the thiol-ene chip layers is a commercially available membrane whose morphology has been altered with coatings of thiol-ene mixtures. Experiments have been conducted ...... with the microchip and shown that the fabricated microchip is suitable for long term cell culture....

  19. Effect of canning on color, protein and phenolic profile of grains from kidney bean, field pea and chickpea.

    Science.gov (United States)

    Parmar, Naincy; Singh, Narpinder; Kaur, Amritpal; Virdi, Amardeep Singh; Thakur, Sheetal

    2016-11-01

    The aim of the present study was to evaluate the effect of canning on color, protein and phenolic profile of grains of kidney bean, field pea and chickpea varieties/accession. Color of grains of different pulses was enhanced after canning. Grains L* (lightness) decreased while a* (redness to yellowness) and b* (greenness to blueness) increased after canning in all the pulses. Protein profiling of grains of different pulses after canning revealed that kidney bean and chickpea, respectively, had the least and the most thermally susceptible polypeptides. Kidney bean and chickpea showed higher Percentage washed drained weight (PWDW) than field pea. Pulse with more grain hardness and PWDW showed higher degree of grain splitting during canning. Grain splitting was also higher in dark colored accessions/varieties as compared to the light colored. Ferulic acid was the most predominant compound present in raw grains of different pulses. Raw kidney bean grains showed higher accumulation of catechin, chlorogenic, protocatechuic acid, p-coumaric acid and ferulic acid than those of chickpea and field pea. Canning caused reduction in all the phenolic compounds except gallic acid and most prominent effect of canning on protocatechuic acid, chlorogenic and ferulic acid was observed. Copyright © 2016. Published by Elsevier Ltd.

  20. Toxicity of Tributyltin in Juvenile Common Carp (Cyprinus Carpio): Physiological Responses, Hepatic Gene Expression, and Stress Protein Profiling.

    Science.gov (United States)

    Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

    2016-02-01

    In this study, the effects of tributyltin (TBT) on biochemical parameters (antioxidant responses and Na(+) -K(+) -ATPase) in different tissues were investigated by using juvenile common carp (Cyprinus Carpio) as well as growth and ion regulation-related genes expression and stress-related proteins profiling in fish liver. Oxidative stress indices and Na(+) -K(+) -ATPase showed tissues-specific responses in fish exposed to different TBT concentrations. All tested genes related to GH/IGF-I axis and ion-regulation were significantly induced in the TBT group with lower concentrations (except for the igfbp3 in 10 μg/L) and were inhibited in 20 μg/L. In addition, the profiling of two proteins Hsp 70 and MT were increasing in a dose-dependent manner under TBT stress. In short, TBT-induced biochemical and molecular responses in different tissues were reflected in the measured parameters in the test. On the basis of TBT residue levels in the natural environment, more long-term experiments at lower concentrations will be necessary in the future. © 2015 Wiley Periodicals, Inc.

  1. Magnetic field action on outdoor and indoor cultures of Spirulina: Evaluation of growth, medium consumption and protein profile.

    Science.gov (United States)

    Deamici, Kricelle Mosquera; Santos, Lucielen Oliveira; Costa, Jorge Alberto Vieira

    2018-02-01

    This study aimed at evaluating whether a magnetic field (MF) affects the growth of Spirulina sp. when applied to it at different exposure times in indoor and outdoor culture systems. The effects of MF on chlorophyll content, medium consumption and protein profile were also investigated. In raceway tanks, a 25 mT MF was applied for 24 h or for 1 h d -1 . MF for 24 h to outdoor assays increased biomass concentration and chlorophyll-a content besides altering the protein profile. Outdoor Spirulina growth was higher (∼3.65 g L -1 ) than the growth found in indoor assays (∼1.80 g L -1 ), while nitrogen and phosphorus consumption was not enhanced by the application of MF. This is the first study that investigated the influence of MF on outdoor microalga assays, and the results showed that MF affected the metabolism of Spirulina cultured in raceways, especially when it was grown outdoors in uncontrolled environmental conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

    Science.gov (United States)

    Hall, Damien; Zhao, Ran; Dehlsen, Ian; Bloomfield, Nathaniel; Williams, Steven R; Arisaka, Fumio; Goto, Yuji; Carver, John A

    2016-04-01

    Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  3. Prioritizing orphan proteins for further study using phylogenomics and gene expression profiles in Streptomyces coelicolor

    Directory of Open Access Journals (Sweden)

    Takano Eriko

    2011-09-01

    Full Text Available Abstract Background Streptomyces coelicolor, a model organism of antibiotic producing bacteria, has one of the largest genomes of the bacterial kingdom, including 7825 predicted protein coding genes. A large number of these genes, nearly 34%, are functionally orphan (hypothetical proteins with unknown function. However, in gene expression time course data, many of these functionally orphan genes show interesting expression patterns. Results In this paper, we analyzed all functionally orphan genes of Streptomyces coelicolor and identified a list of "high priority" orphans by combining gene expression analysis and additional phylogenetic information (i.e. the level of evolutionary conservation of each protein. Conclusions The prioritized orphan genes are promising candidates to be examined experimentally in the lab for further characterization of their function.

  4. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    DEFF Research Database (Denmark)

    Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...

  5. Chemically modified carbon nanotubes as material enhanced laser desorption ionisation (MELDI) material in protein profiling

    International Nuclear Information System (INIS)

    Najam-ul-Haq, M.; Rainer, M.; Schwarzenauer, T.; Huck, C.W.; Bonn, G.K.

    2006-01-01

    Biomarkers play a potential role in the early detection and diagnosis of a disease. Our aim is to derivatize carbon nanotubes for exploration of the differences in human body fluids e.g. serum, through matrix assisted laser desorption ionisation/time of flight mass spectrometry (MALDI/TOF-MS) that can be related to disease and subsequently to be employed in the biomarker discovery process. This application we termed as the material enhanced laser desorption ionisation (MELDI). The versatility of this technology is meant to increase the amount of information from biological samples on the protein level, which will have a major impact to serve the cause of diagnostic markers. Serum peptides and proteins are immobilized on derivatized carbon nanotubes, which function as binding material. Protein-loaded suspension is placed on a stainless steel target or buckypaper on aluminum target for direct analysis with MALDI-MS. The elution method to wash the bound proteins from carbon nanotubes was employed to compare with the direct analysis procedure. Elution is carried out by MALDI matrix solution to get them out of the entangled nanotubes, which are difficult to desorb by laser due to the complex nanotube structures. The advantage of these optimized methods compared to the conventional screening methods is the improved sensitivity, selectivity and the short analysis time without prior albumin and immunoglobulin depletion. The comparison of similarly modified diamond and carbon nanotubes exhibit differences in their nature to bind the proteins out of serum due to the differences in their physical characteristics. Infrared (IR) spectroscopy provided hint for the presence of tertiary amine peak at the crucial chemical step of iminodiacetic acid addition to acid chloride functionality on carbon nanotubes. Atomic absorption spectroscopy (AAS) was utilized to quantitatively measure the copper capacity of these derivatized carbon nanotubes which is a direct measure of capacity of

  6. Serum protein profile at remission can accurately assess therapeutic outcomes and survival for serous ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Jinhua Wang

    Full Text Available BACKGROUND: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. EXPERIMENTAL DESIGN: Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD, remission (RM and recurrence (RC. The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis. RESULTS: Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC have individually good area-under-the-curve (AUC values (AUC = 0.69-0.86 and more than 10 three-marker combinations have excellent AUC values (0.91-0.93 in distinguishing active cancer samples (PD & RC from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC. Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1 measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10(-3. CONCLUSION: We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

  7. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    Science.gov (United States)

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Serum protein profiles predict coronary artery disease in symptomatic patients referred for coronary angiography

    Directory of Open Access Journals (Sweden)

    LaFramboise William A

    2012-12-01

    Full Text Available Abstract Background More than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial. Methods Serum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery. Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1% and Q value (P value for statistical significance adjusted to ≤ 0.01. Results Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100, C-reactive protein (CRP, fibrinogen, vascular cell adhesion molecule 1 (VCAM-1, myeloperoxidase (MPO, resistin, osteopontin, interleukin (IL-1β, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP and decreased apolipoprotein A1 (APO-A1. Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity while maintaining 95% sensitivity for patients requiring

  9. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Science.gov (United States)

    2012-01-01

    Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed

  10. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

    Directory of Open Access Journals (Sweden)

    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  11. Changes in Thiol-Disulfide Homeostasis of the Body to Surgical Trauma in Laparoscopic Cholecystectomy Patients.

    Science.gov (United States)

    Polat, Murat; Ozcan, Onder; Sahan, Leyla; Üstündag-Budak, Yasemin; Alisik, Murat; Yilmaz, Nigar; Erel, Özcan

    2016-12-01

    We aimed to investigate the short-term effect of laparoscopic surgery on serum thiol-disulfide homeostasis levels as a marker of oxidant stress of surgical trauma in elective laparoscopic cholecystectomy patients. Venous blood samples were collected, and levels of native thiols, total thiols, and disulfides were determined with a novel automated assay. Total antioxidant capacity (measured as the ferric-reducing ability of plasma) and serum ischemia modified albumin, expressed as absorbance units assayed by the albumin cobalt binding test, were determined. The major findings of the present study were that native thiol (283 ± 45 versus 241 ± 61 μmol/L), total thiol (313 ± 49 versus 263 ± 67 μmol/L), and disulfide (14.9 ± 4.6 versus 11.0 ± 6.1 μmol/L) levels were decreased significantly during operation and although they increased, they did not return to preoperation levels 24 hours after laparoscopic surgery compared to the levels at baseline. Disulfide/native thiol and disulfide/total thiol levels did not change during laparoscopic surgery. The decrease in plasma level of native and total thiol groups suggests impairment of the antioxidant capacity of plasma; however, the delicate balance between the different redox forms of thiols was maintained during surgery.

  12. Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2014-06-17

    Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

  13. Impact of thiol and amine functionalization on photoluminescence properties of ZnO films

    International Nuclear Information System (INIS)

    Jayalakshmi, G.; Saravanan, K.; Balasubramanian, T.

    2013-01-01

    In the present study, we have investigated surface functionalization of ZnO films with dodecanethiol (Thiol) and trioctylamine (amine) by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), contact angle (CA) and photoluminescence (PL) measurements. The chemical bondings of thiol and amine with ZnO have been confirmed via the formation of Zn–S and Zn–N bonds by XPS measurements. AFM measurements on ZnO films before and after surface functionalization with thiol and amine provide evidence for the successful functionalization of thiol and amine on ZnO surfaces without any island formation. The CA measurements on ZnO films before and after surface functionalization with thiol and amine show the hydrophobic nature. PL measurements of thiol and amine functionalized ZnO show enhancements of UV emission and quenching of visible emission. The enhanced UV emissions in thiol and amine functionalized ZnO films suggest that the surface defects such as oxygen vacancies are passivated by thiol and amine functionalization. -- Highlights: ► Surface functionalization is a new approach to reduce surface dependent non-radiative process. ► Oxygen vacancies are passivated on surface functionalization. ► Thiol and amine functionalized ZnO show enhancements of UV emission

  14. In Profile: Models of Ribosome Biogenesis Defects and Regulation of Protein Synthesis

    NARCIS (Netherlands)

    Essers, P.B.M.

    2013-01-01

    Ribosomes are the mediators of protein synthesis in the cell and therefore crucial to proper cell function. In addition, ribosomes are highly abundant, with ribosomal RNA making up 80% of the RNA in the cell. A large amount of resources go into maintaining this pool of ribosomes, so ribosome

  15. Studies on effect of additives on protein profile, microstructure and quality characteristics of pasta.

    Science.gov (United States)

    Purnima, C; Ramasarma, P R; Prabhasankar, P

    2012-02-01

    Wheat storage proteins play a vital role in pasta making quality. In the present study, SDS-PAGE, Gel filtration chromatography and Scanning electron microscopy techniques were employed to understand the changes in the wheat protein fractions and their interactions with additives namely Sodium Steroyl Lactate (SSL), Glycerol Monostearate (GMS) and Hydroxy Propyl Methyl Cellulose (HPMC) during processing of pasta. SDS-PAGE studies indicated changes in High Molecular Weight Glutenin (HMW) fractions during drying stages of pasta preparation and in cooked pasta samples. In uncooked pasta, gel filtration patterns showed four peaks corresponding to different storage proteins whereas in the case of cooked pasta, these peaks were merged into three peaks. Pasta quality characteristics studies indicated that pasta with HPMC was found to have minimum percentage of cooking loss (5.6%), increased cooked weight (82 g), firmness (2.97 N) and high overall quality score (27) than GMS, SSL and control. Microstructure studies confirm the beneficial effect of HPMC. The present study indicated that HPMC is better additive for pasta manufacture followed by GMS. This could be due to interaction of HPMC with starch and protein matrix is different from that of GMS and SSL.

  16. Effect of soy protein on serum lipid profile and some lipid ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... serum collected to determine the baseline level of the test para- meters studied. ... Biuret reaction described by Gornall et al. (1949) ... Change in mean body weights (g) of rats fed soy protein and cholesterol diet. Mean body ...

  17. Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer

    NARCIS (Netherlands)

    A. Umar (Arzu); J.W.M. Martens (John); J.A. Foekens (John); L. Paša-Tolić (Ljiljana); H. Kang; A.M. Timmermans (Mieke); M.P. Look (Maxime); M.E. Meijer van Gelder (Marion); N. Jaitly (Navdeep); M.A. den Bakker (Michael)

    2009-01-01

    textabstractTamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards

  18. Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni

    NARCIS (Netherlands)

    Silveira, da M.G.; Baumgärtner, M.; Rombouts, F.M.; Abee, T.

    2004-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol

  19. Functional Properties and Amino Acid Profile of Spirulina Platensis Protein Isolates

    International Nuclear Information System (INIS)

    Bashir, S.; Sharif, M. K.; Butt, M. S.; Shahid, M.

    2016-01-01

    Protein malnutrition and food insecurity represent serious obstructions to sustainable development, poverty reduction and food quality throughout the world. The present study has been designed to evaluate the Spirulina platensis (SP) as a protein alternative source for the utilization in food products. A protein isolate was prepared from S. platensis powder through extraction with 0.1N NaOH, precipitation at pH 3, neutralization of the dispersed precipitate to pH 6.8-7.0, and subsequent freeze drying. The S. platensis isolate amino acids compositions revealed that the total essential amino acids contribution was comparatively higher in SPI (31.16±1.43 g/100 g) as compared with SP (27.75±1.21 g/100 g). Moreover, oil and water absorption capacities, foaming and emulsifying properties, surface hydrophobicity and nitrogen solubility index were found better functional properties under laboratory conditions except emulsion properties. Conclusively, SP and its isolates might be used in various food products to curtail protein energy malnutrition. (author)

  20. Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles

    NARCIS (Netherlands)

    Mokry, Michal; Hatzis, Pantelis; de Bruijn, Ewart; Koster, Jan; Versteeg, Rogier; Schuijers, Jurian; van de Wetering, Marc; Guryev, Victor; Clevers, Hans; Cuppen, Edwin

    2010-01-01

    Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be

  1. Vliv pivovarského procesu na profil proteinů ječmene

    Czech Academy of Sciences Publication Activity Database

    Benkovská, Dagmar; Flodrová, Dana; Psota, V.; Bobálová, Janette

    2011-01-01

    Roč. 57, 7-8 (2011), 260-265 ISSN 0023-5830 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : barley * proteins * brewing process Subject RIV: CB - Analytical Chemistry, Separation

  2. Analysis of the protein profiles of the antibiotic-resistant Salmonella ...

    African Journals Online (AJOL)

    The emergent Salmonella typhimurium definitive phage type (DT) 104 is of particular global concern due to its frequent isolation and multiple antibiotic resistances. There is thus a need to know the kind of proteins expressed by S. typhimurium DT104 so as to provide a basis for developing an intervention. This study ...

  3. Identification of biomarkers for radiation-induced acute intestinal symptoms (RIAISs) in cervical cancer patients by serum protein profiling

    International Nuclear Information System (INIS)

    Chai Yanlan; Wang Juan; Gao Ying

    2015-01-01

    Radiation-induced acute intestinal symptoms (RIAISs) are the most frequent complication of radiotherapy that causes great pain and limits the treatment efficacy. The aim of this study was to identify serum biomarkers of RIAISs in cervical cancer patients by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Serum samples were collected from 66 cervical cancer patients prior to pelvic radiotherapy. In our study, RIAISs occurred in 11 patients. An additional 11 patients without RIAISs were selected as controls, whose age, stage, histological type and treatment methods were matched to RIAISs patients. The 22 sera were subsequently analyzed by SELDI-TOF MS, and the resulting protein profiles were evaluated to identify biomarkers using appropriate bioinformatics tools. Comparing the protein profiles of serum samples from the RIAIS group and the control group, it was found that 22 protein peaks were significantly different (P < 0.05), and six of these peaks with mass-to-charge (m/z) ratios of 7514.9, 4603.94, 6887.41, 2769.21, 3839.72 and 4215.7 were successfully identified. A decision tree model of biomarkers was constructed based on three biomarkers (m/z 1270.88, 1503.23 and 7514.90), which separated RIAIS-affected patients from the control group with an accuracy of 81%. This study suggests that serum proteomic analysis by SELDI-TOF MS can identify cervical cancer patients that are susceptible to RIAISs prior to pelvic radiotherapy. (author)

  4. Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles.

    Science.gov (United States)

    Mota, Rita; Pereira, Sara B; Meazzini, Marianna; Fernandes, Rui; Santos, Arlete; Evans, Caroline A; De Philippis, Roberto; Wright, Phillip C; Tamagnini, Paula

    2015-04-29

    The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers. Copyright © 2015. Published by Elsevier B.V.

  5. Electrodeposition of gold templated by patterned thiol monolayers

    Energy Technology Data Exchange (ETDEWEB)

    She, Zhe [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom); Di Falco, Andrea [SUPA, School of Physics and Astronomy, University of St. Andrews, KY16 9SS (United Kingdom); Hähner, Georg [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom); Buck, Manfred, E-mail: mb45@st-andrews.ac.uk [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom)

    2016-06-15

    Graphical abstract: - Highlights: • First demonstration of electrodeposition/lift-off of gold using thiol monolayers. • Microelectrode structures with large length to width ratio were generated. • Performance of two different patterning techniques was investigated. • Conditions for achieving good contrast in the electrodeposition were established. - Abstract: The electrochemical deposition of Au onto Au substrates modified by self-assembled monolayers (SAMs) was studied by linear sweep voltammetry (LSV), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Patterned SAMs exhibiting electrochemical contrast were prepared by two different methods. One used microcontact printing (μCP) to generate a binary SAM of ω-(4′-methyl-biphenyl-4-yl)-propane thiol (CH{sub 3}-C{sub 6}H{sub 4}-C{sub 6}H{sub 4}-(CH{sub 2}){sub 3}-SH, MBP3) and octadecane thiol (CH{sub 3}(CH{sub 2}){sub 17}SH, ODT). Templated by the SAM, a gold microelectrode structure was electrodeposited featuring a line 15 μm wide and 3 mm long. After transfer to an epoxy substrate the structure proved to be electrically conductive across the full length. The other patterning method applied electron beam lithography (EBL) where electrochemical contrast was achieved by crosslinking molecules in a single component SAM of MBP3. An electron dose above 250 mC/cm{sup 2} results in a high deposition contrast. The choice of parameters for the deposition/lift-off process is found to be more critical for Au compared to Cu studied previously. The origin of the differences and implications for nanoscale patterning are discussed.

  6. Comparative study of the protein profiles of Sunki mandarin and Rangpur lime plants in response to water deficit.

    Science.gov (United States)

    Oliveira, Tahise M; da Silva, Fernanda R; Bonatto, Diego; Neves, Diana M; Morillon, Raphael; Maserti, Bianca E; Filho, Mauricio A Coelho; Costa, Marcio G C; Pirovani, Carlos P; Gesteira, Abelmon S

    2015-03-03

    Rootstocks play a major role in the tolerance of citrus plants to water deficit by controlling and adjusting the water supply to meet the transpiration demand of the shoots. Alterations in protein abundance in citrus roots are crucial for plant adaptation to water deficit. We performed two-dimensional electrophoresis (2-DE) separation followed by LC/MS/MS to assess the proteome responses of the roots of two citrus rootstocks, Rangpur lime (Citrus limonia Osbeck) and 'Sunki Maravilha' (Citrus sunki) mandarin, which show contrasting tolerances to water deficits at the physiological and molecular levels. Changes in the abundance of 36 and 38 proteins in Rangpur lime and 'Sunki Maravilha' mandarin, respectively, were observed via LC/MS/MS in response to water deficit. Multivariate principal component analysis (PCA) of the data revealed major changes in the protein profile of 'Sunki Maravilha' in response to water deficit. Additionally, proteomics and systems biology analyses allowed for the general elucidation of the major mechanisms associated with the differential responses to water deficit of both varieties. The defense mechanisms of Rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as in the activation of reactive oxygen species (ROS) detoxification and in the levels of proteins involved in water stress defense. In contrast, the adaptation of 'Sunki Maravilha' to stress was aided by the activation of DNA repair and processing proteins. Our study reveals that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in the roots of citrus plants. The results show that acclimatization to water deficit involves specific responses in Rangpur lime and 'Sunki Maravilha' mandarin. This study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks. In addition, this work allows for a better understanding of the

  7. DTFP-Growth: Dynamic Threshold-Based FP-Growth Rule Mining Algorithm Through Integrating Gene Expression, Methylation, and Protein-Protein Interaction Profiles.

    Science.gov (United States)

    Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan; Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan; Mallik, Saurav; Bhadra, Tapas; Mukherji, Ayan

    2018-04-01

    Association rule mining is an important technique for identifying interesting relationships between gene pairs in a biological data set. Earlier methods basically work for a single biological data set, and, in maximum cases, a single minimum support cutoff can be applied globally, i.e., across all genesets/itemsets. To overcome this limitation, in this paper, we propose dynamic threshold-based FP-growth rule mining algorithm that integrates gene expression, methylation and protein-protein interaction profiles based on weighted shortest distance to find the novel associations among different pairs of genes in multi-view data sets. For this purpose, we introduce three new thresholds, namely, Distance-based Variable/Dynamic Supports (DVS), Distance-based Variable Confidences (DVC), and Distance-based Variable Lifts (DVL) for each rule by integrating co-expression, co-methylation, and protein-protein interactions existed in the multi-omics data set. We develop the proposed algorithm utilizing these three novel multiple threshold measures. In the proposed algorithm, the values of , , and are computed for each rule separately, and subsequently it is verified whether the support, confidence, and lift of each evolved rule are greater than or equal to the corresponding individual , , and values, respectively, or not. If all these three conditions for a rule are found to be true, the rule is treated as a resultant rule. One of the major advantages of the proposed method compared with other related state-of-the-art methods is that it considers both the quantitative and interactive significance among all pairwise genes belonging to each rule. Moreover, the proposed method generates fewer rules, takes less running time, and provides greater biological significance for the resultant top-ranking rules compared to previous methods.

  8. Examination on the protein profiles of salivary glands of P. berghei infected anopheles Sp. post gamma irradiation using SDS-PAGE technique for developing malaria vaccine

    International Nuclear Information System (INIS)

    Tetriana, D.; Syaifudin, M.

    2014-01-01

    Sporozoite is a step of malaria parasitic live cycle that is most invasive and appropriate vaccine candidate. Result of experiments showed that malaria vaccine created by attenuating Plasmodium sp sporozoites with gamma rays was proven more effective. Study on the effects of irradiation to the profiles of protein in vaccine development is also important. The aim of this research was to examine the protein profile of salivary glands in sporozoite infected Anopheles sp post gamma irradiation using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Examination covered the infection of Anopheles sp with Plasmodium sp, maintenance of infected mosquitoes for 14-16 days to obtain sporozoites, in vivo - in vitro irradiation of mosquitoes, preparation of salivary glands, electrophoresis on 10% SDS-PAGE, and Commassie blue staining. Results showed a different protein profile of infected and non infected salivary glands of Anopheles sp. There was additional protein band numbers at higher dose of irradiation (200 Gy) from sporozoite protein of P. berghei (MW 62 kDa). However, no difference of the profiles of circumsporozoite protein (CSP) observed among gamma irradiation doses of 150, 175 and 200 Gy. These results provide basic information that would lead to further study on the role of sporozoite proteins in malaria vaccine development. (author)

  9. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

    Directory of Open Access Journals (Sweden)

    D Malfacini

    Full Text Available Nociceptin/orphanin FQ (N/OFQ controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP. Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells. In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

  10. Amphiphilic silicone architectures via anaerobic thiol-ene chemistry.

    Science.gov (United States)

    Keddie, Daniel J; Grande, John B; Gonzaga, Ferdinand; Brook, Michael A; Dargaville, Tim R

    2011-11-18

    Despite broad application, few silicone-based surfactants of known structure or, therefore, surfactancy have been prepared because of an absence of selective routes and instability of silicones to acid and base. Herein the synthesis of a library of explicit silicone-poly(ethylene glycol) (PEG) materials is reported. Pure silicone fragments were generated by the B(C(6)F(5))(3)-catalyzed condensation of alkoxysilanes and vinyl-functionalized hydrosilanes. The resulting pure products were coupled to thiol-terminated PEG materials using photogenerated radicals under anaerobic conditions.

  11. Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses.

    Science.gov (United States)

    Harris, Craig; Shuster, Daniel Z; Roman Gomez, Rosaicela; Sant, Karilyn E; Reed, Matthew S; Pohl, Jan; Hansen, Jason M

    2013-10-01

    Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net

  12. Genome-wide identification of VQ motif-containing proteins and their expression profiles under abiotic stresses in maize

    Directory of Open Access Journals (Sweden)

    Weibin eSong

    2016-01-01

    Full Text Available VQ motif-containing proteins play crucial roles in abiotic stress responses in plants. Recent studies have shown that some VQ proteins physically interact with WRKY transcription factors to activate downstream genes. In the present study, we identified and characterized genes encoding VQ motif-containing proteins using the most recent version of the maize genome sequence. In total, 61VQ genes were identified. In a cluster analysis, these genes clustered into nine groups together with their homologous genes in rice and Arabidopsis. Most of the VQ genes (57 out of 61 numbers identified in maize were found to be single-copy genes. Analyses of RNA-seq data obtained using seedlings under long-term drought treatment showed that the expression levels of most ZmVQ genes (41 out of 61 members changed during the drought stress response. Quantitative real-time PCR analyses showed that most of the ZmVQ genes were responsive to NaCl treatment. Also, approximately half of the ZmVQ genes were co-expressed with ZmWRKY genes. The identification of these VQ genes in the maize genome and knowledge of their expression profiles under drought and osmotic stresses will provide a solid foundation for exploring their specific functions in the abiotic stress responses of maize.

  13. Amino acids fortification of low-protein diet for broilers under tropical climate: ideal essential amino acids profile

    Directory of Open Access Journals (Sweden)

    Elmutaz Atta Awad

    2014-05-01

    Full Text Available A three-week trial was conducted to determine the effect of lowering dietary protein level (DPL with optimal amino acid (AA profile on growth performance, blood metabolites, and relative weights of abdominal fat and internal organs in broiler chickens raised under tropical hot and humid environment. Five isocaloric (3023 metabolisable energy/kg starter (1-21 days experimental diets were formulated in a gradual crude protein (CP decline from 22.2 (control to 16.2% by 1.5% interval. All diets were meeting or exceeding National Research Council recommendations except CP and metabolisable energy. The formulations were also adjusted to contain 1.1 digestible Lys to meet the ideal AA ratios concept. Body weights (BW, weight gains (WG, feed intake and feed conversion ratio of groups with 19.2, 20.7 and 22.2% DPL were not significantly different. However, BW and WG suppressed (P<0.05 with 16.2 and 17.7% DPL. Feeding the 16.2% CP diet significantly reduced serum total protein and uric acid, but increased serum triglyceride (P<0.05. Moreover, relative heart weights increased (P<0.05 but no changes occurred in liver and abdominal fat weights in chicks with 16.2% DPL. In summary, CP of broilers starter (1-21 days diet can be reduced till 19.2% with essential AA fortification and without any adverse effect on growth performance under the hot, humid tropics.

  14. Proteomic profiling of human keratinocytes undergoing UVB-induced alternative differentiation reveals TRIpartite Motif Protein 29 as a survival factor.

    Directory of Open Access Journals (Sweden)

    Véronique Bertrand-Vallery

    Full Text Available BACKGROUND: Repeated exposures to UVB of human keratinocytes lacking functional p16(INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. METHODOLOGY/PRINCIPAL FINDINGS: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29. Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCdelta. CONCLUSIONS/SIGNIFICANCE: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.

  15. Maternal deprivation affects the neuromuscular protein profile of the rat colon in response to an acute stressor later in life.

    Science.gov (United States)

    Lopes, Luísa V; Marvin-Guy, Laure F; Fuerholz, Andreas; Affolter, Michael; Ramadan, Ziad; Kussmann, Martin; Fay, Laurent B; Bergonzelli, Gabriela E

    2008-04-30

    Early life stress as neonatal maternal deprivation (MD) predisposes rats to alter gut functions in response to acute psychological stressors in adulthood, mimicking features of irritable bowel syndrome (IBS). We applied proteomics to investigate whether MD permanently changes the protein profile of the external colonic neuromuscular layer that may condition the molecular response to an acute stressor later in life. Male rat pups were separated 3 h/day from their mothers during the perinatal period and further submitted to water avoidance (WA) stress during adulthood. Proteins were extracted from the myenteric plexus-longitudinal muscle of control (C), WA and MD+WA rat colon, separated on 2D gels, and identified by mass spectrometry. MD amplified the WA-induced protein changes involved in muscle contractile function, suggesting that stress accumulation along life imbalances the muscle tone towards hypercontractility. Our results also propose a stress dependent regulation of gluconeogenesis. Secretogranin II - the secretoneurin precursor - was induced by MD. The presence of secretoneurin in myenteric ganglia may partially explain the stress-mediated modulation of gastrointestinal motility and/or mucosal inflammation previously described in MD rats. In conclusion, our findings suggest that neonatal stress alters the responses to acute stress in adulthood in intestinal smooth muscle and enteric neurons.

  16. Relationship of molecular weight distribution profile of unreduced gluten protein extracts with quality characteristics of bread.

    Science.gov (United States)

    Chaudhary, Nisha; Dangi, Priya; Khatkar, B S

    2016-11-01

    A statistical correlation was established among the molecular weight distribution patterns of unreduced gluten proteins and physicochemical, rheological and bread-making quality characteristics of wheat varieties. Size exclusion chromatography fractionated the gluten proteins apparently into five peaks. Peak I signified glutenins (30-130kDa), peak II as gliadins (20-55kDa), peak III as very low molecular weight monomeric gliadins (10-28kDa), peak IV and V, collectively, as albumins and globulins (bread loaf volume (r=0.848(∗∗)); however, peak II had negative (r=-0.818(∗∗)) impact. Bread firmness increased with increment in peak II (r=0.625(∗∗)), and decreased with accretion in peak I (r=-0.623(∗∗)). Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  18. Fractionation, amino acid profiles, antimicrobial and free radical scavenging activities of Citrullus lanatus seed protein.

    Science.gov (United States)

    Dash, Priyanka; Ghosh, Goutam

    2017-12-01

    In the present study, a modified Osborne fractionation method was followed to isolate albumin (C alb ), globulin (C glo ), prolamin (C pro ) and glutelin (C glu ) successively from seeds of Citrullus lanatus (watermelon). This research work was undertaken to investigate the antimicrobial and antioxidant activities of isolated protein fractions of C. lanatus seed. Amino acid composition and molecular weight distribution were determined to establish their relationship with antimicrobial and antioxidant activity. Among all the fractions, C pro was found to be most effective against A. baumannii followed by C alb and C glo . The results showed that growth of inhibition of these protein fractions differ significantly from each other (p ≤ 0.05). In view of antioxidant potential, C glo exhibited strongest antioxidant capacity while C glu showed weakest antioxidant potential.

  19. Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

    DEFF Research Database (Denmark)

    Ngara, Rudo; Ndimba, Roya; Borch-Jensen, Jonas

    2012-01-01

    Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet...... sorghum variety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination...... with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p...

  20. Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis.

    Directory of Open Access Journals (Sweden)

    Sunny Eloot

    Full Text Available We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage.This study included 10 patients on standard (3 x 4 h/week high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V1, total distribution volume (V tot and intercompartment clearance (K21. This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies.Dialyzer clearance K, V1 and V tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K21 was 2.7-5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3 x 4 h dialysis at 300 mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated.When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.

  1. Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis.

    Science.gov (United States)

    Eloot, Sunny; Schneditz, Daniel; Cornelis, Tom; Van Biesen, Wim; Glorieux, Griet; Dhondt, Annemie; Kooman, Jeroen; Vanholder, Raymond

    2016-01-01

    We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage. This study included 10 patients on standard (3 x 4 h/week) high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V1), total distribution volume (V tot) and intercompartment clearance (K21). This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies. Dialyzer clearance K, V1 and V tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K21 was 2.7-5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3 x 4 h dialysis at 300 mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated. When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.

  2. Estimation of Genetic Parameters for the Protein Profile in Danish Holstein Milk

    DEFF Research Database (Denmark)

    Buitenhuis, Albert Johannes; Poulsen, Nina Aagaard; Larsen, Lotte Bach

    in the univariate and bivariate analysis of the protein traits. Hertitabilities ranged from 0.77 for κ-CN% to 0 for αs 1-CN% (SE: 0.13-0.21). The genetic correlation between β-CN and κ-CN was low (0.01 ± 0.53) whereas the genetic correlation of αs 2-CN with both β-CN and κ-CN was high (0.90). Furthermore α...

  3. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  4. Different Polar Metabolites and Protein Profiles between High- and Low-Quality Japanese Ginjo Sake.

    Directory of Open Access Journals (Sweden)

    Kei Takahashi

    Full Text Available Japanese ginjo sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing ginjo sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality ginjo sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with ginjo sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with ginjo sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI leakage was linked to the overall metabolite intensity in ginjo sake. This research provides insight into the relationship between sake components and organoleptic properties.

  5. Implementation of a protein profiling platform developed as an academic-pharmaceutical industry collaborative effort.

    Science.gov (United States)

    Végvári, Akos; Magnusson, Mattias; Wallman, Lars; Ekström, Simon; Bolmsjö, Gunnar; Nilsson, Johan; Miliotis, Tasso; Ostling, Jörgen; Kjellström, Sven; Ottervald, Jan; Franzén, Bo; Hultberg, Hans; Marko-Varga, György; Laurell, Thomas

    2008-06-01

    As much attention has devoted to the proteome research during the last few years, biomarker discovery has become an increasingly hot area, potentially enabling the development of new assays for diagnosis and prognosis of severe diseases. This is the field of research interest where efforts originating from both academic and industrial groups should jointly work on solutions. In this paper, we would like to demonstrate the fruitful combination of both research domains where the scientific crossroads sprout fresh ideas from the basic research domain and how these are refined and tethered to industrial standards. We will present an approach that is based on novel microfluidic devices, utilizing their benefits in processing small-volume samples. Our biomarker discovery strategy, built around this platform, involves optimized samples processing (based on SPE and sample enrichment) and fast MALDI-MS readout. The identification of novel biomarkers at low-abundance level has been achieved by the utilization of a miniaturized sample handling platform, which offers clean-up and enrichment of proteins in one step. Complete automation has been realized in the form of a unique robotic instrumentation that is able to extract and transfer 96 samples onto standard MALDI target plates with high throughput. The developed platform was operated with a 60 sample turnaround per hour allowing sensitivities in femtomol regions of medium- and low-abundant target proteins from clinical studies on samples of multiple sclerosis and gastroesophageal reflux disease. Several proteins have been identified as new biomarkers from cerebrospinal fluid and esophagus epithelial cells.

  6. Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Ines eLassowskat

    2014-10-01

    Full Text Available Mitogen-activated protein kinases (MAPKs target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phosphoproteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known