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Sample records for extensive protein fractionation

  1. Expanding the bovine milk proteome through extensive fractionation.

    PubMed

    Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

    2013-01-01

    Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the

  2. Whey protein fractionation

    USDA-ARS?s Scientific Manuscript database

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  3. Trends in whey protein fractionation.

    PubMed

    El-Sayed, Mayyada M H; Chase, Howard A

    2011-08-01

    Whey is a by-product of cheese manufacture that is normally treated as a waste. However, it contains a mixture of proteins with important nutritional and biological attributes. To extract these valuable proteins, whey fractionation has been developed using three main techniques; namely chromatographic (e.g., ion-exchange and hydrophobic adsorption), membrane (e.g., traditional pressure-driven and electro-separation)-, or combined methods. Recently, new promising techniques have been introduced such as aqueous two-phase separation (ATPS) and magnetic fishing. This article reviews the use of these techniques together with an evaluation of their performance regarding the yield and purity of two major proteins in whey.

  4. Solving fuzzy fractional differential equations using Zadeh's extension principle.

    PubMed

    Ahmad, M Z; Hasan, M K; Abbasbandy, S

    2013-01-01

    We study a fuzzy fractional differential equation (FFDE) and present its solution using Zadeh's extension principle. The proposed study extends the case of fuzzy differential equations of integer order. We also propose a numerical method to approximate the solution of FFDEs. To solve nonlinear problems, the proposed numerical method is then incorporated into an unconstrained optimisation technique. Several numerical examples are provided.

  5. Solving Fuzzy Fractional Differential Equations Using Zadeh's Extension Principle

    PubMed Central

    Ahmad, M. Z.; Hasan, M. K.; Abbasbandy, S.

    2013-01-01

    We study a fuzzy fractional differential equation (FFDE) and present its solution using Zadeh's extension principle. The proposed study extends the case of fuzzy differential equations of integer order. We also propose a numerical method to approximate the solution of FFDEs. To solve nonlinear problems, the proposed numerical method is then incorporated into an unconstrained optimisation technique. Several numerical examples are provided. PMID:24082853

  6. Extension of many-body theory and approximate density functionals to fractional charges and fractional spins.

    PubMed

    Yang, Weitao; Mori-Sánchez, Paula; Cohen, Aron J

    2013-09-14

    The exact conditions for density functionals and density matrix functionals in terms of fractional charges and fractional spins are known, and their violation in commonly used functionals has been shown to be the root of many major failures in practical applications. However, approximate functionals are designed for physical systems with integer charges and spins, not in terms of the fractional variables. Here we develop a general framework for extending approximate density functionals and many-electron theory to fractional-charge and fractional-spin systems. Our development allows for the fractional extension of any approximate theory that is a functional of G(0), the one-electron Green's function of the non-interacting reference system. The extension to fractional charge and fractional spin systems is based on the ensemble average of the basic variable, G(0). We demonstrate the fractional extension for the following theories: (1) any explicit functional of the one-electron density, such as the local density approximation and generalized gradient approximations; (2) any explicit functional of the one-electron density matrix of the non-interacting reference system, such as the exact exchange functional (or Hartree-Fock theory) and hybrid functionals; (3) many-body perturbation theory; and (4) random-phase approximations. A general rule for such an extension has also been derived through scaling the orbitals and should be useful for functionals where the link to the Green's function is not obvious. The development thus enables the examination of approximate theories against known exact conditions on the fractional variables and the analysis of their failures in chemical and physical applications in terms of violations of exact conditions of the energy functionals. The present work should facilitate the calculation of chemical potentials and fundamental bandgaps with approximate functionals and many-electron theories through the energy derivatives with respect to the

  7. An Extensive Analysis of Preservice Elementary Teachers' Knowledge of Fractions

    ERIC Educational Resources Information Center

    Newton, Kristie Jones

    2008-01-01

    The study of preservice elementary teachers' knowledge of fractions is important because fractions are notoriously difficult to learn and teach. Unfortunately, studies of preservice teachers' fraction knowledge are limited and have focused primarily on division. The present study included all four operations to provide a more comprehensive…

  8. An Extensive Analysis of Preservice Elementary Teachers' Knowledge of Fractions

    ERIC Educational Resources Information Center

    Newton, Kristie Jones

    2008-01-01

    The study of preservice elementary teachers' knowledge of fractions is important because fractions are notoriously difficult to learn and teach. Unfortunately, studies of preservice teachers' fraction knowledge are limited and have focused primarily on division. The present study included all four operations to provide a more comprehensive…

  9. Two endogenous proteins that induce cell wall extension in plants

    NASA Technical Reports Server (NTRS)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  10. Two endogenous proteins that induce cell wall extension in plants

    NASA Technical Reports Server (NTRS)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  11. 21 CFR 862.1630 - Protein (fractionation) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Protein (fractionation) test system. 862.1630... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation) test system is a device intended to measure protein fractions in blood, urine, cerebrospinal fluid, and...

  12. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

  13. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  14. 21 CFR 862.1630 - Protein (fractionation) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Protein (fractionation) test system. 862.1630 Section 862.1630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation)...

  15. 21 CFR 862.1630 - Protein (fractionation) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Protein (fractionation) test system. 862.1630 Section 862.1630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation)...

  16. 21 CFR 862.1630 - Protein (fractionation) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Protein (fractionation) test system. 862.1630 Section 862.1630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation)...

  17. 21 CFR 862.1630 - Protein (fractionation) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Protein (fractionation) test system. 862.1630 Section 862.1630 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Systems § 862.1630 Protein (fractionation) test system. (a) Identification. A protein (fractionation)...

  18. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

  19. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

  20. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall be...

  1. Facilitating protein solubility by use of peptide extensions

    DOEpatents

    Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

    2013-09-17

    Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

  2. Isolation of Oat (Avena sativa L.) Total Proteins and Their Prolamin Fractions for 2D Electrophoresis.

    PubMed

    Nałęcz, Dorota; Dziuba, Marta; Szerszunowicz, Iwona

    2017-01-01

    Appropriate sample preparation is essential to obtaining good results of two-dimensional gel electrophoresis (2-DE). For various reasons (particularly phenolic compounds, proteolytic enzymes, and cell-wall mucilages) the extraction of proteins from plant material, among them oat proteins, is difficult. During isolation all soluble substances that may interfere with the analysis (especially isoelectric focusing) are removed, and proteins of interest are separated from the remains. However, the applied procedure of isolation cannot be too extensive, because additional stages cause loss of the proteins.In this chapter, we describe a simple procedure for the isolation of oat total proteins and their prolamin fractions prior to 2-DE, without necessity of considerable purification. It can be used for oat protein fractionation, measurement of oat protein concentration, and their 2-DE analysis, with particular reference to prolamin fractions. The presented routine includes modified methods of plant seed proteins extraction and sequential Osborne extraction, based on oat protein solubility differences.

  3. Method for voltage-gated protein fractionation

    DOEpatents

    Hatch, Anson [Tracy, CA; Singh, Anup K [Danville, CA

    2012-04-24

    We report unique findings on the voltage dependence of protein exclusion from the pores of nanoporous polymer exclusion membranes. The pores are small enough that proteins are excluded from passage with low applied electric fields, but increasing the field enables proteins to pass through. The requisite field necessary for a change in exclusion is protein-specific with a correlation to protein size. The field-dependence of exclusion is important to consider for preconcentration applications. The ability to selectively gate proteins at exclusion membranes is also a promising means for manipulating and characterizing proteins. We show that field-gated exclusion can be used to selectively remove proteins from a mixture, or to selectively trap protein at one exclusion membrane in a series.

  4. Protein microarrays using liquid phase fractionation of cell lysates.

    PubMed

    Yan, Fang; Sreekumar, Arun; Laxman, Bharathi; Chinnaiyan, Arul M; Lubman, David M; Barder, Timothy J

    2003-07-01

    We describe an approach in which protein microarrays are produced using a two-dimensional (2-D) liquid phase fractionation of cell lysates. The method involves a pI-based fractionation using chromatofocusing in the first dimension followed by nonporous reversed-phase high-performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero-diagnostic markers for prostate cancer. Importantly, this method allows for use of post-translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.

  5. Whey protein fractionation using supercritical carbon dioxide

    USDA-ARS?s Scientific Manuscript database

    Sweet whey, a coproduct of the cheesemaking process, can be concentrated using ultrafiltration and ion-exchange to produce whey protein isolates (WPI). WPI contains approximately 32% alpha-lactalbumin (alpha-LA) and 61% beta-lactoglobulin (beta-LG), plus a small amount of minor whey proteins. Whil...

  6. Developing new fluorescent proteins with stagger extension process

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Lu, Jinling; Luo, Haiming; Luo, Qingming; Zhang, Zhihong

    2009-02-01

    The Stagger Extension Process (StEP), a recombination of DNA technique, has been used as a rapid molecular mutagenesis strategy. In this study, for obtaining the fluorescence proteins with new properties, six fluorescence proteins (EYFP, EGFP, ECFP, mCitrine, mCerulean and Venus) were used as the templates to recombine the mutation library by the Stagger Extension Process (StEP) technique. Through screening this mutation library, we have obtained some useful new FPs which are different fluorescent properties with ancestor. These protein will extend fluorescent proteins application.

  7. Antioxidant activity of cod (Gadus morhua) protein hydrolysates: Fractionation and characterisation of peptide fractions.

    PubMed

    Sabeena Farvin, K H; Andersen, Lisa Lystbæk; Otte, Jeanette; Nielsen, Henrik Hauch; Jessen, Flemming; Jacobsen, Charlotte

    2016-08-01

    This study aimed to characterise peptide fractions (>5kDa, 3-5kDa and <3kDa) with antioxidative activity obtained from a cod protein hydrolysate. The free amino acids in all fractions were dominated by Ala, Gly, Glu and Ser. The total amino acid composition had high proportions of Lys, Ala and Glu. The 3-5kDa and <3kDa fractions were further fractionated by size exclusion chromatography. All sub-fractions showed high Fe(2+) chelating activity. The DPPH radical-scavenging activity of the 3-5kDa fraction was exerted mainly by one sub-fraction dominated by peptides with masses below 600Da. The DPPH radical-scavenging activity of the <3kDa fraction was exerted by sub-fractions with low molecular weight. The highest reducing power was found in a sub-fraction containing peptides rich in Arg, Tyr and Phe. Both free amino acids and low molecular weight peptides thus seemed to contribute to the antioxidative activity of the peptide fractions, and Tyr seemed to play a major role in the antioxidant activity.

  8. Characterization of pea (Pisum sativum) seed protein fractions.

    PubMed

    Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

    2014-01-30

    Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

  9. Fractionation of liver proteins by preparative electrophoresis.

    PubMed

    Fountoulakis, M; Juranville, J-F; Tsangaris, G; Suter, L

    2004-02-01

    Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.

  10. [Protein fraction distribution in milling and screened physical fractions of grain amaranth].

    PubMed

    Búcaro Segura, María Ester; Bressani, Ricardo

    2002-06-01

    The purpose of the study was to establish the protein distribution based on solubility in physical fractions of amaranth flour, in particular between the flour from the germ and that from the perisperm. The protein distribution was obtained applying a series of solvents sequentially utilized in the classical methodology of Osborne & Mendel. The sample of A. cruentus weighing 2000 g was divided into 4 subsamples of 500 g each. One was left as the control while the other 3 were ground individually with a mill. Each flour was screened through 18, 20, 30 and 40 mesh screens, so that 5 fractions were obtained from each of the whole grain flours. Samples of each screened fractions were observed by stereoscopy and analyzed for moisture, fat and protein. This characterization suggested that the fraction above the 30 mesh screen and the flour which passed the 40 mesh screen probably were the perisperm and germ respectively. The 30 mesh sample contained 2.34 fat and 9.05% protein while the 40 mesh contained 16.18% fat and 26.46% protein. The extraction and partitioning of the proteins indicated that the most important fractions in germ and perisperm were the water soluble and glutelins measured by Kjeldahl. The relationship of the water soluble + globulin to glutelins ratio was 2.1 to 1 in the whole grain, 1.9 to 1 in the perisperm and 1.7 to 1 in the germ. The distribution of proteins was very much alike between germ and perisperm. The levels of prolamines were quite low. The protein extraction of the perisperm proteins retained on the 30 mesh screen was low (71.1%) measured by Kjeldahl and 47.4% with the Bradford method to measure protein.

  11. Fractionation and characterization of brewers' spent grain protein hydrolysates.

    PubMed

    Celus, Inge; Brijs, Kristof; Delcour, Jan A

    2009-06-24

    Protein hydrolysates with a low and high degree of hydrolysis were enzymatically produced from brewers' spent grain (BSG), the insoluble residue of barley malt resulting from the manufacture of wort in the production of beer. To that end, BSG protein concentrate (BPC), prepared by alkaline extraction of BSG and subsequent acid precipitation, was enzymatically hydrolyzed with Alcalase during both 1.7 and 120 min. Because these hydrolysates contained many different peptides, fractionation of the hydrolysates with graded ammonium sulfate or ethanol precipitation was performed to obtain fractions homogeneous in terms of molecular weight (MW) and hydrophobicity. The emulsifying and foaming capacities of the resultant fractions were determined. MW distributions and surface hydrophobicities of fractions with protein contents exceeding 75% were investigated to determine relationships between technofunctional and physicochemical properties. It was found that the emulsifying and foaming properties are determined by different physicochemical properties of the proteins or peptides. Neither MW nor hydrophobicity alone determines the emulsifying and foaming properties of protein hydrolysates. BSG protein hydrolysates with good emulsifying properties contained less than 40% of fragments with MW exceeding 14 500. Moreover, these hydrolysates had a high surface hydrophobicity. BSG protein hydrolysates with good foaming properties contained less than 10% of material with MW lower than 1700. Hydrolysates with good foaming properties showed low surface hydrophobicities, except for protein hydrolysates with higher levels of protein fragments with MW exceeding 14 500 than of such fragments with MW in a 1700-14 500 range.

  12. Nutritional quality of sunflower seed protein fraction extracted with isopropanol.

    PubMed

    Sen, M; Bhattacharyya, D K

    2000-01-01

    This study investigated the nutritional effect of sunflower seed protein fraction (SSPF) extracted with isopropanol on growth, plasma and tissue lipid profile, protein content and erythrocyte membrane lipid profile of rats. Dehulled sunflower seeds were extracted with isopropanol at 50 +/- 1 degree C resulting in a protein fraction (71.5%) with low residual chlorogenic acid (0.07%) and fiber (3.3%) contents. Rats fed the sunflower seed protein fraction had a similar body weight gain and food efficiency ratios in comparison to those fed casein. Rats fed SSPF in contrast had a significantly higher growth and food efficiency ratio than the rats fed sunflower meal (SM), extracted with hexane. However, dietary proteins exerted a separate effect on plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, low density lipoprotein to high density lipoprotein cholesterol (LDL-C/HDL-C) ratio and triglyceride content. Sunflower seed protein fraction resulted in a significant decrease in plasma cholesterol (p < 0.05) and LDL-cholesterol (p < 0.02) levels compared to the casein fed rats. Membrane phospholipid profile also showed a marked variation with the type of dietary protein. Rats fed SSPF and SM did not show much variation in plasma lipids, plasma proteins, liver and brain lipids and membrane phospholipid concentrations. Protein content, liver and brain lipid profile of the groups fed SSPF and casein were comparable, suggesting that the nutritional value of SSPF is better than SM and equivalent to that of casein.

  13. Preparation of bean curds from protein fractions of six legumes.

    PubMed

    Cai, R; Klamczynska, B; Baik, B K

    2001-06-01

    Chickpeas, lentils, smooth peas, mung beans, and faba beans were milled into flours and fractionated to protein and starch fractions. Compositions of the seeds, cotyledons, and flours were compared for each legume and the weight and protein recovery of each fraction analyzed. Bean curds were prepared from the protein fractions through heat denaturation of protein milk, followed by coagulation with calcium sulfate or magnesium sulfate. The effect of chickpea protein concentration and coagulant dosage on the texture of bean curds was evaluated using a texture analyzer. Textural analysis indicated that curd prepared at 2.3-3.0% protein concentration and 1.5% CaSO(4) dosage had better yield and better texture than curds prepared under other conditions. Bean curds prepared from chickpeas and faba beans exhibited the second highest springiness and cohesiveness after those from soybeans. Curds of mung beans and smooth peas, on the other hand, had the highest yields and the highest moisture contents. The protein yield of the first and second soluble extracts used for curd preparation accounted for approximately 90% of the total protein of the seeds.

  14. Antiproliferative and immunostimulatory protein fraction from edible mushrooms.

    PubMed

    Maiti, Swatilekha; Bhutia, Sujit K; Mallick, Sanjaya K; Kumar, Alok; Khadgi, Niyati; Maiti, Tapas K

    2008-09-01

    Fruit bodies and mycelia of various higher Basidiomycetes were studied in search of biological effector molecules. In this study, we evaluated the antiproliferative and immunomodulatory properties of a protein fraction designated as Cibacron blue affinity eluted protein (CBAEP) isolated from five different species of edible mushrooms (Termitomyces clypeatus, Pleurotus florida, Calocybe indica, Astraeus hygrometricus, and Volvariella volvacea). This protein fraction (10-100μg/ml) mediated antiproliferative activity on several tumor cell lines through the induction of apoptosis. Also the isolated protein fraction from all five mushrooms had a stimulatory effect on splenocytes, thymocytes and bone marrow cells. Further it enhanced mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide (NO). The highest immunostimulatory activity was determined in the CBAEP from T. clypeatus and the highest antiproliferative activity from C. indica.

  15. Oxidatively Responsive Chain Extension to Topologically Entangle Engineered Protein Hydrogels

    NASA Astrophysics Data System (ADS)

    Olsen, Bradley; Tang, Shengchang; Glassman, Matthew; Li, Shuaili; Socrate, Simona

    2014-03-01

    Hydrogels with increased toughness and extensibility have attracted a great deal of interest as mimics for natural tissues in biomedical applications. Artificial protein polymers provide particularly attractive systems for these applications due to their similarity to the chemistry of the natural extracellular matrix. Here, we show that entanglements can be incorporated into physically associating protein gels using simple oxidative chain extension chemistries, producing hydrogels with multiple time and length scales of relaxation. These oxidative chemistries follow the Jacobson-Stockmayer theory and are fully reversible, enabling responsive formation of entanglements within a material. The entangled protein gels demonstrate extensibility up to engineering strains of greater than 3,000%, a toughness of 65,000 J/m⌃3, and significant reductions in creep compliance and increases in elastic recovery. The rheology of the materials is compared to sticky reptation theory as a function of gel concentration, providing insights into the effect of network structure on different modes of molecular relaxation.

  16. Composition and molecular weight distribution of carob germ protein fractions.

    PubMed

    Smith, Brennan M; Bean, Scott R; Schober, Tilman J; Tilley, Michael; Herald, Thomas J; Aramouni, Fadi

    2010-07-14

    Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.

  17. [Electrophoretic studies of serum protein fractions in horses with laminitis].

    PubMed

    Edinger, H; Miller, I; Stanek, C; Gemeiner, M

    1992-10-01

    The spectrum of serum proteins was evaluated in 46 horses affected with spontaneous laminitis and correlations between the severity of the disease and changes of the protein pattern were analyzed. The investigation was made in two groups; group A consisted of 21 horses of various breeds (warmblood, thoroughbred, standardbred) and group B of 25 ponys. Each group was subdivided according to the severity of the disease, using the OBEL-grade (OG) classification system. Serum proteins were separated by different one- and two-dimensional electrophoretic methods. Sera analysed by cellulose acetate electrophoresis showed a significant difference in the alpha 1-globulin fraction between OG II and OG IV affected horses. An increasing severity of the disease was correlated with a decrease of the alpha 1-globulins. The other protein fractions didn't show a uniform tendency. In group B there was a significant difference in the alpha 1-globulin fractions of OG II and OG III and in the beta 2-globulin fractions of OG I and OG II affected ponys. The acute phase proteins C3c, C4, Hp and fibronectin could be determined in a preliminary study in horse serum using the cross-reactivity of antibodies against the homologous human proteins.

  18. THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS.

    PubMed

    A, Güllü; S, Dede

    2016-01-01

    TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control's (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group's (p<0.05). From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins.

  19. THE SERUM PROTEIN FRACTIONS IN THYMOQUINONE TREATED RATS

    PubMed Central

    A, Güllü; S, Dede

    2016-01-01

    Background: TQ has been used as treatment and preventive agent for many diseases over the years. The goal of this study was to investigate the effects of TQ supplement on fractions of serum proteins. Materials and methods: Fourteen male Wistar-Albino rats (200-250 g weight) were used as material for two groups; (control (C) and thymoquinone (TQ) respectively. Each group contained seven rats. The control group had only corn oil, while the TQ group was dissolved in corn oil. 30 mg/kg/day were given by oral gavage for four weeks. The serum protein fractions were identified using cellulose acetate technique. Results: The total protein level and albumin, α-1, α-2 fractions and A/G ratio have showed no difference between groups (p>0.05). β-globulin fractions of TQ group were higher than control’s (p<0.05). In addition, it was observed that the γ-globulin levels of TQ group were lower than that of the control group’s (p<0.05). Conclusion: From the results, it was observed that the changes of these fractions may have originated from elevation or decline synthesis, or activities of containing proteins. PMID:28480357

  20. Native antigen fractionation protein microarrays for biomarker discovery.

    PubMed

    Caiazzo, Robert J; O'Rourke, Dennis J; Barder, Timothy J; Nelson, Bryce P; Liu, Brian C-S

    2011-01-01

    In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

  1. Interactions of soy protein fractions with high-methoxyl pectin.

    PubMed

    Lam, Monica; Paulsen, Paul; Corredig, Milena

    2008-06-25

    A protein-binding technique was employed to visualize, using scanning electron microscopy, the soy protein as well as the association between HMP and soy protein fractions. Image analysis indicated that at pH 7.5 and 3.5 soy protein isolate showed a bimodal distribution of sizes with an average [ d(0.5)] of about 0.05 microm, but at pH 3.8 the proteins formed larger aggregates than at high pH. Addition of HMP at pH 3.8 changed the surface charge of the particles from +20 to -15 mV. A small addition of HMP caused bridging of the pectin between soy protein aggregates and destabilization. With sufficient HMP, the suspensions showed improved stability to precipitation. The microscopy images are the first direct evidence of the interactions between soy proteins with high-methoxyl pectin (HMP).

  2. Fractionation and recovery of whey proteins by hydrophobic interaction chromatography.

    PubMed

    Santos, Maria João; Teixeira, José A; Rodrigues, Lígia R

    2011-03-01

    A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.

  3. Storage of Fractional Flow Reserve Hemodynamic Waveforms Using Semantic Extension of the DICOM Standard.

    PubMed

    Kakouros, Nikolaos

    2016-06-01

    Visual assessment of coronary stenoses by coronary angiography remains widely used but correlates poorly with ischemia, particularly for moderate lesions. Fractional flow reserve (FFR) is a cardiac catheterization procedure that aims to provide objective measures of coronary lesion hemodynamic significance and involves the acquisition of phasic pressure and electrocardiographic waveforms. The dataset from these procedures currently remains in proprietary systems with restricted data access, inability for data exchange, and often inadequate archiving. Digital Imaging and Communications in Medicine (DICOM) includes a waveform information object definition. We describe the method of encapsulating FFR procedural information into a DICOM waveform file. We define private data elements to capture modality-specific data that is not represented by standard DICOM data elements. We propose the adoption of this semantic extension of the DICOM waveform information object for exchange and archiving of data from studies of pressure-derived indices of coronary stenoses.

  4. Isolation and characterization of camelina protein fractions from camelina meal

    USDA-ARS?s Scientific Manuscript database

    Camelina protein fractions — albumin, globulin, and glutelins — were isolated from camelina meal using three different methods: the traditional Osborne sequence (S2), a modified Osbron sequence plus a degumming step (S1), and the isolation method without a degumming step (S0). The isoelectric points...

  5. Deaths observed in Medicare beneficiaries: average attributable fraction and its longitudinal extension for many diseases.

    PubMed

    Murphy, T E; McAvay, G; Carriero, N J; Gross, C P; Tinetti, M E; Allore, H G; Lin, H

    2012-11-30

    Calculating the longitudinal extension of the average attributable fraction (LE-AAF) for many risk factors (RFs) requires a two-stage computational process using only those combinations of RFs observed in the dataset. We first screen candidates RFs in a Cox Model, and assuming piecewise constant hazards, use pooled logistic regression to model the probability of death as a function of combinations of selected RFs. We average the iterative differencing of the attributable fractions calculated for all overlapping subsets of co-occurring RFs to obtain a LE-AAF for each RF that is additive and symmetrical. We illustrate by partitioning the additive proportions of death from 10 different groupings of acute and chronic diseases, on a national sample of older persons from the US (Medicare Beneficiary Survey) over a 4-year period and compare with results reported by the National Center for Healthcare Statistics. We conclude that careful screening of RFs with analysis restricted to extant combinations greatly reduces computational burden. LE-AAF accounted for a cumulative total of 66% of the deaths in our sample, compared with the 83% accounted for by the National Center for Healthcare Statistics. Copyright © 2012 John Wiley & Sons, Ltd.

  6. Effect of flecainide on the extension and localization of complex fractionated electrogram during atrial fibrillation.

    PubMed

    De Bortoli, Alessandro; Shi, Li-Bin; Wang, Yu-Chuan; Hoff, Per Ivar; Solheim, Eivind; Ohm, Ole-Jørgen; Chen, Jian

    2015-06-01

    Complex fractionated electrogram (CFE) ablation in addition to pulmonary vein isolation is an accepted strategy for the treatment of non-paroxysmal atrial fibrillation (AF). We sought to determine the effect of flecainide on the distribution and extension of CFE areas. Twenty-three non-paroxysmal AF patients were enrolled in this prospective study. A first CFE map was obtained under baseline conditions by sampling 5 s of continuous recording from the distal electrodes of the ablation catheter. Intravenous flecainide (1 mg/kg) was administered over 10 min and followed by 30-min observation time. A second CFE map was obtained with the same modalities. CFE-mean values, CFE areas, and atrial electrogram amplitude were retrieved from the electro-anatomical mapping system (Ensite NavX). After flecainide administration, CFE-mean values increased (111.5 ± 55.3 vs. 132.3 ± 65.0 ms, p < 0.001) with a decrease of CFE area (32.9%) in all patients. Atrial electrogram amplitude decreased significantly (0.30 ± 0.31 vs. 0.25 ± 0.20 mV, p < 0.001). We observed 80.9% preservation of CFE areas. A CFE mean of 78 ms was the best cutoff for predicting stable CFE areas. Flecainide reduces the extension of CFE areas while preserving their spatial localization. A CFE-mean value <80 ms may be crucial to define and locate stable CFE areas.

  7. Protein fractionation byproduct from canola meal for dairy cattle.

    PubMed

    Heendeniya, R G; Christensen, D A; Maenz, D D; McKinnon, J J; Yu, P

    2012-08-01

    Fiber-protein is a byproduct arising from a process for fractionating high-quality protein from canola meal. The objective of this study was to evaluate the fiber-protein fraction by examining the chemical profiles, rumen degradation, and intestinal digestive characteristics and determining the nutritive value of the fiber-protein fraction as dietary components for dairy cattle in comparison with commercial canola meal and soybean meal. Available energy values were estimated based on National Research Council guidelines, whereas total true protein content potentially absorbable in the small intestine (DVE) were predicted using the predicted DVE/degraded protein balance (OEB) model. The results show that fiber-protein was a highly fibrous material [neutral detergent fiber (NDF): 556; acid detergent fiber (ADF): 463; acid detergent lignin: 241 g/kg of dry matter (DM)] compared with canola meal (NDF: 254; ADF: 212; acid detergent lignin: 90 g/kg of DM) due to the presence of a higher level of seed hulls in fiber-protein. Compared with canola meal, fiber-protein contained 90 g/kg of DM less crude protein (CP), 25% of which consisted of undegradable acid detergent-insoluble CP. Most of the ruminally undegradable nutrient components present in canola meal appeared to be concentrated into fiber-protein during the manufacturing process and, as a result, fiber-protein showed a consistently lower effective degradability of DM, organic matter, CP, NDF, and ADF compared with both canola meal and soybean meal. Available energy content in fiber-protein contained two-thirds of that of canola meal. The DVE was one-third that of soybean meal and one-fifth that of canola meal [DVE value: 58 vs. 180 (soybean) and 291 g/kg of DM (canola meal)]. The OEB value of fiber protein was positive and about half of that of soybean and canola meal [OEB value: 74 vs. 162 (soybean) and 137 g/kg of DM (canola meal)]. Fiber-protein can be considered as a secondary source of protein in ruminant feed.

  8. Angiogenic inhibitor protein fractions derived from shark cartilage.

    PubMed

    Bargahi, Afshar; Rabbani-Chadegani, Azra

    2008-02-01

    Development of therapies based on the growth inhibition of new blood vessels is among the most intensively studied approaches to the treatment of cancer and other angiogenesis-related diseases. Shark cartilage has been proven to have inhibitory effects on the endothelial cell angiogenesis, metastasis, cell adhesion and MMP (matrix metalloprotease) activity. In the present study, we have used a chromatography-based procedure for the isolation and partial purification of a shark cartilage protein fraction containing anti-angiogenesis activity. Proteins were extracted in 4 M guanidinium chloride, followed by sequential anion- and cation-exchange column chromatography. Angiogenesis assays were performed using the rat aortic ring and chick CAM (chorioallantoic membrane) assay models. The results show that the final fraction contains two proteins with molecular masses of 14.7 and 16 kDa. The protein fraction is able to block microvessel sprouting in the collagen-embedded rat aortic ring assay in vitro and inhibition of capillary sprouting in the CAM assay in vivo. It is suggested that these are partially purified anti-angiogenesis proteins, which have further biotechnological or biomedical applications.

  9. [Obtaining protein fractions from commercial sesame cakes (Sesamum indicum)].

    PubMed

    Guerra, M J; Jaffe, W G; Sangronis, E

    1984-09-01

    Sesame press cake represents an important potential protein source for human consumption. Some of the limiting factors are its high crude fiber content, oxalic acid content, and its bitter taste. By fractionation of solvent-extracted sesame meal, several preparations were obtained which were analyzed for their nutrient content, protein utilization and digestibility. PER values were low, and supplementation with lysine, skim-milk powder, soymeal or fish meal, improved the PER values considerably. Based on these findings, formulas for use as a protein supplement for children are presented.

  10. Protein composition of seminal plasma in fractionated stallion ejaculates.

    PubMed

    Kareskoski, A M; del Alamo, M M Rivera; Güvenc, K; Reilas, T; Calvete, J J; Rodriguez-Martinez, H; Andersson, M; Katila, T

    2011-02-01

    Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH-2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion's own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse-phase liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry. The area-under-the-curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS-PAGE and densitometry. No significant differences emerged between fractions in the AUC-values of the Horse Seminal Protein-1 (HSP-1) and HSP-2 peaks, or the peak containing HSP-3 and HSP-4 (HSP-3/4). Levels of HSP-1, HSP-2 and HSP-3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60-70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.

  11. Extraction and characterisation of protein fractions from five insect species.

    PubMed

    Yi, Liya; Lakemond, Catriona M M; Sagis, Leonard M C; Eisner-Schadler, Verena; van Huis, Arnold; van Boekel, Martinus A J S

    2013-12-15

    Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus and Blaptica dubia were evaluated for their potential as a future protein source. Crude protein content ranged from 19% to 22% (Dumas analysis). Essential amino acid levels in all insect species were comparable with soybean proteins, but lower than for casein. After aqueous extraction, next to a fat fraction, a supernatant, pellet, and residue were obtained, containing 17-23%, 33-39%, 31-47% of total protein, respectively. At 3% (w/v), supernatant fractions did not form stable foams and gels at pH 3, 5, 7, and 10, except for gelation for A. domesticus at pH 7. At 30% w/v, gels at pH 7 and pH 10 were formed, but not at pH 3 and pH 5. In conclusion, the insect species studied have potential to be used in foods due to: (1) absolute protein levels; (2) protein quality; (3) ability to form gels.

  12. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  13. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  14. Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels

    PubMed Central

    Tang, Shengchang; Glassman, Matthew J.; Li, Shuaili; Socrate, Simona; Olsen, Bradley D.

    2014-01-01

    Engineering artificial protein hydrogels for medical applications requires precise control over their mechanical properties, including stiffness, toughness, extensibility and stability in the physiological environment. Here we demonstrate topological entanglement as an effective strategy to robustly increase the mechanical tunability of a transient hydrogel network based on coiled-coil interactions. Chain extension and entanglement are achieved by coupling the cysteine residues near the N- and C- termini, and the resulting chain distribution is found to agree with the Jacobson-Stockmayer theory. By exploiting the reversible nature of the disulfide bonds, the entanglement effect can be switched on and off by redox stimuli. With the presence of entanglements, hydrogels exhibit a 7.2-fold enhanced creep resistance and a suppressed erosion rate by a factor of 5.8, making the gels more mechanically stable in a physiologically relevant open system. While hardly affecting material stiffness (only resulting in a 1.5-fold increase in the plateau modulus), the entanglements remarkably lead to hydrogels with a toughness of 65,000 J m-3 and extensibility to approximately 3,000% engineering strain, which enables the preparation of tough yet soft tissue simulants. This improvement in mechanical properties resembles that from double-network hydrogels, but is achieved with the use of a single associating network and topological entanglement. Therefore, redox-triggered chain entanglement offers an effective approach for constructing mechanically enhanced and responsive injectable hydrogels. PMID:24910474

  15. Fractional Brownian motion and fractional Gaussian noise in subsurface hydrology: A review, presentation of fundamental properties, and extensions

    NASA Astrophysics Data System (ADS)

    Molz, F. J.; Liu, H. H.; Szulga, J.

    Recent studies have shown that fractional Brownian motion (fBm) and fractional Gaussian noise (fGn) are useful in characterizing subsurface heterogeneities in addition to geophysical time series. Although these studies have led to a fairly good understanding of some aspects of fBm/fGn, a comprehensive introduction to these stochastic, fractal functions is still lacking in the subsurface hydrology literature. In this paper, efforts have been made to define fBm/fGn and present a development of their mathematical properties in a direct yet rigorous manner. Use of the spectral representation theorem allows one to derive spectral representations for fBm/fGn even though these functions do not have classical Fourier transforms. The discrete and truncated forms of these representations have served as a basis for synthetic generation of fBm/fGn. The discrete spectral representations are developed and various implications discussed. In particular, it is shown that a discrete form of the fBm spectral representation is equivalent to the well known Weierstrass-Mandelbrot random fractal function. Although the full implications are beyond the scope of the present paper, it is observed that discrete spectral representations of fBm constitute stationary processes even though fBm is nonstationary. A new and general spectral density function is introduced for construction of complicated, anisotropic, (3-D) fractals, including those characterized by vertical fGn and horizontal fBm. Such fractals are useful for modeling anisotropic subsurface heterogeneities but cannot be generated with existing schemes. Finally, some basic properties of fractional Lévy motion and concepts of universal multifractals, which can be considered as generalizations of fBm/fGn, are reviewed briefly.

  16. Initial rates of degradation of protein fractions from fresh, wilted, and ensiled alfalfa.

    PubMed

    Makoni, N F; Shelford, J A; Fisher, L J

    1994-06-01

    Initial rates of in vitro degradation of alfalfa proteins were studied. Fresh, 24-h wilted, and ensiled forages were homogenized before analysis for total proteins. Some of the homogenates were fractionated by differential solubility in 10 and 40% ammonium sulfate, followed by ultrafiltration of the 40% salt-saturated solution. The protein fractions obtained were chloroplast membrane proteins (fraction 3), soluble proteins from plant cell cytoplasm and the chloroplast (fraction 2), and proteins remaining soluble in the extracted 40% salt-saturated solution (fraction 2B), respectively. Total and fraction 3 silage proteins were degraded faster than the respective fresh and wilted proteins. There were no treatment effects on the rates of degradation of the soluble proteins of fractions 2 and 2B. Protein fractions from fresh and 24-h wilted alfalfa degraded, from greatest to least, in the following order: fractions 2, 2B, and 3. Degradation rates for fractions 2 and 2B of ensiled forages were similar but greater than that of fraction 3. Alfalfa proteins were degraded rapidly in the rumen, and soluble proteins were degraded faster than the chloroplast membrane proteins. Ensiling of alfalfa increased the rate of degradation of chloroplast membrane proteins, but neither wilting nor ensiling affected degradation rates of the soluble protein fractions 2 and 2B.

  17. Proteomic tools to characterize the protein fraction of Equidae milk.

    PubMed

    Miranda, Guy; Mahé, Marie-Françoise; Leroux, Christine; Martin, Patrice

    2004-08-01

    The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and

  18. Improved feed protein fractionation schemes for formulating rations with the cornell net carbohydrate and protein system.

    PubMed

    Lanzas, C; Broderick, G A; Fox, D G

    2008-12-01

    Adequate predictions of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) supplies are necessary to optimize performance while minimizing losses of excess nitrogen (N). The objectives of this study were to evaluate the original Cornell Net Carbohydrate Protein System (CNCPS) protein fractionation scheme and to develop and evaluate alternatives designed to improve its adequacy in predicting RDP and RUP. The CNCPS version 5 fractionates CP into 5 fractions based on solubility in protein precipitant agents, buffers, and detergent solutions: A represents the soluble nonprotein N, B1 is the soluble true protein, B2 represents protein with intermediate rates of degradation, B3 is the CP insoluble in neutral detergent solution but soluble in acid detergent solution, and C is the unavailable N. Model predictions were evaluated with studies that measured N flow data at the omasum. The N fractionation scheme in version 5 of the CNCPS explained 78% of the variation in RDP with a root mean square prediction error (RMSPE) of 275 g/d, and 51% of the RUP variation with RMSPE of 248 g/d. Neutral detergent insoluble CP flows were overpredicted with a mean bias of 128 g/d (40% of the observed mean). The greatest improvements in the accuracy of RDP and RUP predictions were obtained with the following 2 alternative schemes. Alternative 1 used the inhibitory in vitro system to measure the fractional rate of degradation for the insoluble protein fraction in which A = nonprotein N, B1 = true soluble protein, B2 = insoluble protein, C = unavailable protein (RDP: R(2) = 0.84 and RMSPE = 167 g/d; RUP: R(2) = 0.61 and RMSPE = 209 g/d), whereas alternative 2 redefined A and B1 fractions as the non-amino-N and amino-N in the soluble fraction respectively (RDP: R(2) = 0.79 with RMSPE = 195 g/d and RUP: R(2) = 0.54 with RMSPE = 225 g/d). We concluded that implementing alternative 1 or 2 will improve the accuracy of predicting RDP and RUP within the CNCPS framework.

  19. Methods of dry fractionation to produce barley fractions varying in protein and/or beta-glucan contents

    USDA-ARS?s Scientific Manuscript database

    On average, barley contains 64% starch, 11% protein, and 5% glucan. The remaining 20% includes moisture, fiber, ash and other minor components. Among them, protein and beta-glucan are of higher values. Dry fractionation has been the primary and low cost means to enrich protein and other nutrients ...

  20. Determination of phosphorus fractions in animal protein ingredients.

    PubMed

    Hua, Katheline; Liu, Lijuan; Bureau, Dominique P

    2005-03-09

    Phosphorus (P) is present in different chemical compounds in animal feeds, and the solubility and digestibility of these different compounds are known to differ significantly. Animal protein ingredients generally have a high P content and are major contributors to total P of feeds for fish and other domestic animals. Estimation of different P compounds in these ingredients could help to improve the accuracy of estimates of digestible P contents of feeds. Bone P and organic P contents were quantified in 32 animal protein ingredients, including 10 fish meals, 14 meat and bone meals, and 8 poultry byproducts meals, using a fractionation protocol. The total P contents of the ingredients ranged from 2.1 to 8.3% on a dry matter (DM) basis. Organic P contents varied between 0.3 and 1.3% of DM. Highly significant (p < 0.001) linear relationships were observed between total P and ash and between bone P and ash for all ingredients combined: total P (%) = 0.185 x ash (%) (R (2) = 0.88), and bone P (%) = 0.188 x ash (%) - 0.852 (R (2) = 0.94). These results suggest that bone P can be easily and reliably estimated on the basis of ash content in animal protein ingredients.

  1. Immunomodulatory properties of the protein fraction from Phorphyra columbina.

    PubMed

    Cian, Raúl E; López-Posadas, Rocío; Drago, Silvina R; de Medina, Fermín Sánchez; Martínez-Augustin, Olga

    2012-08-22

    The phycobiliproteins from Rhodophyta , R-phycoerythrin (R-PE) and C-phycocyanin (C-PC), have been shown to exert immunomodulatory effects. This study evaluated the effects of a Phorphyra columbina protein fraction (PF) and R-PE and C-PC on rat primary splenocytes, macrophages, and T-lymphocytes in vitro. PF featured various protein species, including R-PE and C-PC. PF showed mitogenic effects on rat splenocytes and was nontoxic to cells except at 1 g L(-1) protein. IL-10 secretion was enhanced by PF in rat splenocytes, macrophages, and especially T-lymphocytes, whereas it was markedly diminished by R-PE and C-PC. The production of pro-inflammatory cytokines by macrophages was inhibited. The effect of PF on IL-10 was evoked by JNK/p38 MAPK and NF-κB-dependent pathways in macrophages and T-lymphocytes. It was concluded that PF has immunomodulatory effects on macrophages and lymphocytes that appear to be predominantly anti-inflammatory via up-regulated IL-10 production and cannot be accounted for by R-PE and C-PC.

  2. Quantitative aspects of protein fractional synthesis rates in ruminants.

    PubMed

    Lescoat, P; Sauvant, D; Danfaer, A

    1997-01-01

    Protein fractional synthesis rate (FSR) is a key-factor in the characterisation of ruminant metabolism. Published data from the literature were collected and statistically analysed to isolate the factors influencing FSR. FSR varied largely depending on the tissue considered, over a range from 1 to 20. FSR, with the plasma as the precursor pool for protein synthesis, was halved compared to that of the intracellular pool. The method for supplying the amino acid also significantly affects FSR since the flooding dose technique gave higher FSR estimates than the constant infusion technique. The choice of the labelled amino acid infused influenced FSR. There is a ranking order depending on the tissue or organ. The protein and energy levels of the diets significantly increased FSR, which raises the question of the body nitrogen requirements. Moreover, FSR values were dependent on the physiological status of the animals. To conclude, FSR values should be determined simultaneously with other biological parameters in order to obtain a realistic quantitative estimate of the nitrogen turnover rates during intermediary metabolism.

  3. Generation, Fractionation, and Characterization of Iron-Chelating Protein Hydrolysate from Palm Kernel Cake Proteins.

    PubMed

    Zarei, Mohammad; Ghanbari, Rahele; Tajabadi, Naser; Abdul-Hamid, Azizah; Bakar, Fatimah Abu; Saari, Nazamid

    2016-02-01

    Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron-chelating activity of each hydrolysate were evaluated using O-phthaldialdehyde-based spectrophotometric method and ferrozine-based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron-chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron-chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain-generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron-chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron-chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain-generated peptides, GGIF and YLLLK showed among the highest iron-chelating activities of 56% and 53%, whereas their IC50 were 1.4 and 0.2 μM, respectively. © 2015 Institute of Food Technologists®

  4. Solubility of soy lipophilic proteins: comparison with other soy protein fractions.

    PubMed

    Sirison, Jiraporn; Matsumiya, Kentaro; Samoto, Masahiko; Hidaka, Hiroshi; Kouno, Mitsutaka; Matsumura, Yasuki

    2017-04-01

    Solubility of soy lipophilic proteins (LP) was studied as compared with that of other soy protein fractions. LP, β-conglycinin, glycinin, and soy protein isolate (N-SPI) were prepared under the condition to avoid heat denaturation. Solubility of LP was lower than that of other soy protein fractions under all the tested conditions varying in pH values and ionic strength. The solubility of LP was increased constantly by elevating temperature until 90 °C, whereas that of β-conglycinin and glycinin dropped at high temperature. Temperature-dependent change in solubility of N-SPI might reflect the balance among that of glycinin, β-conglycinin and LP. Based on the results of SDS-PAGE, determination of phospholipid content and Fourier Transform Infrared spectroscopy, we discussed the solubilization behavior of LP relating to its origin and composition.

  5. Aminoguanidine inhibits protein browning without extensive Amadori carbonyl blocking.

    PubMed

    Requena, J R; Vidal, P; Cabezas-Cerrato, J

    1993-01-01

    It has been proposed that aminoguanidine reacts extensively with Amadori carbonyl groups of glycated proteins thus blocking them and inhibiting the further reactions which lead to browning and fluorescence development. We have glycated bovine serum albumin in the presence of 1, 5, 10 and 25 mM aminoguanidine and measured fluorescence development at 440 nm upon excitation at 370 nm, free (unblocked) Amadori groups as fructosamine with a colorimetric assay and furosine by HPLC, as an index of total Amadori products. Aminoguandine significantly inhibited fluorescence development at all the tested concentrations (31%, 65%, 69% and 82% inhibitions, respectively) (P < 0.001). Blocking of Amadori groups was demonstrated by decreased fructosamine and unchanged furosine yields but only at the higher concentrations and to a very limited extent (13% and 27% blocking, respectively) (P < 0.01). Incubation of Aminoguanidine with albumin produced the appearance of 320 nm absorbing yellow chromophores, quite increased in the presence of glucose. These results suggest that Aminoguanidine is able to block Amadori groups, as previously hypothesized, but question the importance of this mechanism as an explanation of its capacity to inhibit browning. Scavenging of glucose seems to have no impact on glycation as seen by unchanged furosine yields.

  6. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

    PubMed Central

    Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

    2013-01-01

    Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

  7. Partial purification of fatty-acid binding protein by ammonium sulphate fractionation.

    PubMed

    Avanzati, B; Catalá, A

    1983-07-01

    By fractionation of rat liver cytosol with 70% saturation ammonium sulphate, a soluble fraction showing high affinity for oleic acid was obtained. The binding of oleic acid to this fraction was inhibited by flavaspidic acid. The molecular weight of the main protein present in this fraction was 12 000 as determined by SDS-poly-acrylamide-gel electrophoresis. This soluble fraction stimulated the transfer of oleic acid from microsomes to phosphatidylcholine liposomes as demonstrated by a transfer assay in vitro. The behaviour of this fraction is similar to that described for fatty-acid binding protein.

  8. Protein adsorption to hydrophobic Zeolite Y: salt effects and application to protein fractionation.

    PubMed

    Ghose, S; Mattiasson, B

    1993-12-01

    The binding equilibria of proteins with a hydrophobic variety of crystalline Zeolite Y is affected by salt and is a function of the type of salt and its concentration. The behaviour does not always follow the conventional pattern of increased binding at high salt concentrations and varies also for the different proteins involved. The overall process may be looked upon as a salting-in/salting-out mechanism. This material can be used as a surface for the selective adsorption of proteins and has been applied for the fractionation of ox heart homogenate in multi-stage operations. The presence of NaCl influences the protein binding, and this can be seen by monitoring the activity profile of lactate dehydrogenase. The bound protein can be reversed by treating the equilibrium mixture with low-molecular-mass poly(ethylene glycol)s.

  9. ACUTE PHASE PROTEIN AND ELECTROPHORESIS PROTEIN FRACTION VALUES FOR CAPTIVE AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER).

    PubMed

    Delk, Katie W; Wack, Raymund F; Burgdorf-Moisuk, Anne; Kass, Philip H; Cray, Carolyn

    2015-12-01

    Protein electrophoresis has recognized applications in determining the health status of various species. While reference intervals for electrophoresis have been determined for psittacine and raptor species, there are none reported for Phoenicopteriformes species. Reference intervals for haptoglobin and protein fractions obtained by electrophoresis were determined for the American flamingo (Phoenicopterus ruber) based on plasma samples from 39 captive birds. The reference intervals were as follows: haptoglobin, 0.17-0.8 mg/ml; total protein, 3.65-6.38 g/dl; prealbumin, 0.26-1.9 g/dl; albumin, 1.51-3.12 g/dl; α-1 globulin, 0.06-0.38 g/dl; α-2 globulin, 0.17-0.67 g/dl; β globulin, 0.38-1.33 g/dl; γ globulin, 0.26-0.68 g/dl; albumin : globulin ratio, 0.93-2.17. As captive flamingos often suffer from pododermatitis, feet of all flamingos were scored to determine if pododermatitis would be reflected in the acute phase proteins. Spearman rank correlation was performed on each of the protein fractions and pododermatitis scores, and only albumin had a significant correlation. This indicates that albumin, as a negative acute phase protein, may be a marker for this disease process.

  10. Protein bodies of castor bean endosperm: isolation, fractionation, and the characterization of protein components.

    PubMed

    Tully, R E; Beevers, H

    1976-12-01

    Protein bodies in the endosperm of castor bean seeds (Ricinus communis L.) contain phytin globoids and protein crystalloids embedded in an amorphous proteinaceous matrix. The protein bodies are apparently surrounded by a single membrane. The protein bodies were isolated by grinding and centrifuging in glycerol. Such isolated protein bodies were almost identical (after cytological fixation) to those observed in situ, except that the globoids were lost. However, membrane-like structures appear to have surrounded the globoids. Histochemical analysis of the isolated protein bodies showed that carbohydrates (glycoproteins) are localized only in the matrix region.Addition of water to protein bodies in glycerol caused dissolution of the matrix, and release of the globoids and crystalloids. When the crystalloids were centrifuged on sucrose density gradients, they were recovered at an equilibrium density of 1.29 to 1.30 g/ml. The crystalloids were only slightly soluble in most aqueous buffers but were very soluble in sodium dodecyl sulfate, urea, or NaOH solutions.Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and chromatography on ion exchange celluloses show that the protein bodies are composed of one major and several minor anodic proteins. The major protein, along with a few of the minor proteins, is localized in the crystalloids.The major protein (molecular weight 65,000) was converted by mercaptoethanol into subunits with molecular weights of 32,000 and 15,800. It is proposed that the protein is made up of two of the smaller subunits and one of the larger, linked by disulfide bridges. None of the crystalloid proteins appear to be glycosylated.The water-soluble matrix fraction is composed mainly of two proteins, with molecular weights of 12,500 and 10,300 on the gels. Neither is a glycoprotein, and neither can be reduced with mercaptoethanol to give subunits. The soluble fraction also contains other lesser components among which are

  11. Protein-protein interactions prediction based on iterative clique extension with gene ontology filtering.

    PubMed

    Yang, Lei; Tang, Xianglong

    2014-01-01

    Cliques (maximal complete subnets) in protein-protein interaction (PPI) network are an important resource used to analyze protein complexes and functional modules. Clique-based methods of predicting PPI complement the data defection from biological experiments. However, clique-based predicting methods only depend on the topology of network. The false-positive and false-negative interactions in a network usually interfere with prediction. Therefore, we propose a method combining clique-based method of prediction and gene ontology (GO) annotations to overcome the shortcoming and improve the accuracy of predictions. According to different GO correcting rules, we generate two predicted interaction sets which guarantee the quality and quantity of predicted protein interactions. The proposed method is applied to the PPI network from the Database of Interacting Proteins (DIP) and most of the predicted interactions are verified by another biological database, BioGRID. The predicted protein interactions are appended to the original protein network, which leads to clique extension and shows the significance of biological meaning.

  12. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  13. Protein synthesis of muscle fractions from the small intestine in alcohol fed rats.

    PubMed Central

    Preedy, V R; Peters, T J

    1990-01-01

    The effects of chronic ethanol feeding on the amounts and synthesis rates of cytoplasmic, contractile, and stromal protein fractions were investigated in the small intestine of eight pairs of immature and seven pairs of mature rats. Treated rats were fed ethanol as 36% of total energy in a nutritionally adequate liquid diet. Paired controls were fed isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose. After six weeks the total cytoplasmic and contractile protein content in immature rats was reduced by 18% and 31%, respectively (p less than or equal to 0.007). The decline in the stromal protein content (26%) was not statistically significant (p = 0.130). In mature rats the protein contents were also reduced in the cytoplasmic (25%, p = 0.035) and contractile (27%, p = 0.005) protein fractions, though the stromal protein fraction was unaltered (p = 0.913). In immature rats fractional rates of protein synthesis in cytoplasmic and contractile protein fractions of the small intestine were unaltered by chronic ethanol feeding (p less than or equal to 0.853). In mature rats, the synthesis rates of corresponding fractions declined, by 18% and 31%, respectively, but were also not statistically significant (p less than or equal to 0.369). Absolute rates of protein synthesis in immature rats fell by 6% (p = 0.549) in the cytoplasmic and 31% in the contractile protein fraction (p = 0.045). In mature rats, the corresponding reductions were 38% (p = 0.106) and 48% (p = 0.033), respectively. Virtually no radioactivity could be detected in the stromal fraction, signifying very low synthesis rates. Chronic ethanol feeding reduces the amount of protein in the small intestine of the immature and mature rat with the contractile protein fraction showing the greatest decrease. In the absence of statistically significant reductions in fractional synthesis rates a partial adaptation in turnover rates may have occurred. PMID:2323594

  14. Protein precipitating capacity and antioxidant activity of Turkish Tombul hazelnut phenolic extract and its fractions.

    PubMed

    Pelvan Pelitli, Ebru; Janiak, Michał Adam; Amarowicz, Ryszard; Alasalvar, Cesarettin

    2017-03-01

    Natural (raw) hazelnut was extracted with 80% (v/v) acetone to obtain crude phenolic extract that was then fractionated for elution of low-molecular weight (LMW) and high-molecular weight (HMW) fractions. LMW fraction was further purified (LWM-FP) to remove sugars and organic acids. The crude extract and its fractions were determined by measuring their protein precipitating capacity (PPC) using two different proteins [bovine serum albumin (BSA) and gelatin], molecular weights, total phenolics, condensed tannins, and various antioxidant activities. Significant differences (p<0.05) existed in the contents of total phenolics, condensed tannins, antioxidant activities, and PPC among the crude extract and fractions, albeit to different extends. BSA and gelatin was effectively precipitated by HMW fraction. HMW fraction had the highest total phenolics, condensed tannins, and antioxidant activities, followed by crude extract, LWM-FP, and LMW, respectively. The present study suggests that HMW fraction could be utilised as a source of polyphenols for the food industry.

  15. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  16. Pilot-scale fractionation of whey proteins with supercritical CO2

    USDA-ARS?s Scientific Manuscript database

    A new pilot-scale process is being developed and optimized for the separation of whey proteins into two enriched, highly functional fractions that are free of contaminants. The fractionation of whey protein isolate (WPI), which contains approximately 32% alpha-lactalbumin (alpha-LA) and 61% beta-lac...

  17. Fractionation of whey protein isolate with supercritical carbon dioxide – process modeling and cost estimation

    USDA-ARS?s Scientific Manuscript database

    An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of alpha-lactalbumin (alpha-La) and beta-lactoglobulin (beta-Lg) from a commercial whey protein isolate (WPI) containing 55% ...

  18. Serum protein fractionation using supported molecular matrix electrophoresis.

    PubMed

    Dong, Weijie; Matsuno, Yu-ki; Kameyama, Akihiko

    2013-08-01

    Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states.

  19. Identification of a novel mitochondrial interacting protein of C1QBP using subcellular fractionation coupled with CoIP-MS.

    PubMed

    Chen, Ruibing; Xiao, Mingming; Gao, Huajun; Chen, Yajing; Li, Yongmei; Liu, Yunde; Zhang, Ning

    2016-02-01

    The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.

  20. Protein fractions in forage legumes containing protein-binding polyphenols: Freeze-drying vs. conservation as hay or silage.

    USDA-ARS?s Scientific Manuscript database

    We compared protein fractions in freeze-dried herbage to hay or silage of forage legumes containing about 200 g/kg of crude protein. Protein was partitioned with buffer and detergents into rapidly (A and B1), moderately (B2), and slowly (B3) degraded and undegradable acid-detergent insoluble protein...

  1. Meat protein fractions enhance nonheme iron absorption in humans.

    PubMed

    Hurrell, Richard F; Reddy, Manju B; Juillerat, Marcel; Cook, James D

    2006-11-01

    The nature of the enhancing effect of muscle tissue on nonheme iron absorption in humans is unclear but thought to be related to muscle proteins. We conducted radioiron absorption studies to compare iron absorption from proteins isolated from beef and chicken muscle with that from freeze-dried beef and chicken muscle and from egg albumin. All meals contained an equivalent amount of protein as part of a semisynthetic liquid formula. Freeze-dried beef and chicken muscle increased iron absorption 180% (P < 0.001) and 100% (P < 0.001), respectively, relative to egg albumin. When added to the meal at an equivalent protein level (15 g), the isolated beef protein and the isolated heme-free beef protein with 94 and 98% protein content, respectively, increased iron absorption to the same extent as the native beef muscle. Similarly, when added to the meal at an equivalent protein level (30 g), isolated chicken muscle protein (94% protein) increased iron absorption similarly to native chicken muscle. Iron absorption from the meal containing the isolated heme-free chicken protein, however, was 120% (P < 0.01) greater than from the meal containing freeze-dried chicken muscle, indicating that a nonprotein component of muscle tissue with iron-binding potential may have been removed or concentrated by the protein extraction and separation procedures. Our results support the hypothesis that the enhancing effect of muscle tissue on iron absorption is mainly protein related but indicate that other factors may also play a role.

  2. Amino acid composition of Lagenaria siceraria seed flour and protein fractions.

    PubMed

    Ogunbusola, Moriyike Esther; Fagbemi, Tayo Nathaniel; Osundahunsi, Oluwatooyin Faramade

    2010-12-01

    Defatted seed flours of Lagenaria siceraria (calabash and bottle gourd) were fractionated into their major protein fractions. The amino acid composition of seed flours and their protein fractions were determined and the protein quality was evaluated. Glutamic acid (139-168 mg/g protein) was the most abundant amino acid followed by aspartic acid (89.0-116 mg/g protein) in both the seed flours and their protein fractions. The total essential amino acid ranged from 45.8 to 51.5%. The predicted protein efficiency ratio and the predicted biological value ranged from 2.4 to 2.9 and 8.7 to 44.0, respectively. Lysine and sulphur amino acids were mostly concentrated in the globulin fractions. The first and second limiting amino acids in seed flours and protein fractions were methionine and valine or threonine. The seed flours contained adequate essential amino acids required by growing school children and adults. The seed has potential as protein supplement in cereal based complementary diets or in the replacement of animal proteins in conventional foods.

  3. IDENTIFICATION OF PROTEIN FRACTIONS OF MILK COWS CASEIN COMPLEX.

    PubMed

    Iukalo, A V

    2015-01-01

    To date, dozens of biologically active peptides formed during proteolysis of casein fractions have been discovered. The use of these peptides is closely related to the necessity of their rapid identification. The aim of this work was the development of an electrophoresis system for rapid identification of individual fractions in serial studies and the separation of the milk casein complex. Considering the abnormal nature of the interaction of caseins with the sodium dodecyl sulfate and similar values of their molecular masses, the anode electrophoresis system in a homogeneous polyacrylamide gel was taken as a basis. Caseins, in this system, are separated according to their charge and located on the electrophoregram in accordance with the modern classification. Urea was used as a disaggregating agent in gel. It was shown that the use of Studier type apparatus for electrophoresis with changeable dimensions of electrophoretic chamber significantly reduces (to 45 min) the time for identification of casein fractions. This method may be useful for rapid identification of casein fractions, as well as for rapid analysis of natural milk and milk products.

  4. Uncharged compactlike and fractional Lorentz-violating BPS vortices in the CPT-even sector of the standard model extension

    NASA Astrophysics Data System (ADS)

    Miller, C.; Casana, R.; Ferreira, M. M., Jr.; da Hora, E.

    2012-09-01

    We have investigated and verified the existence of stable uncharged Bogomol’nyi-Prasad-Sommerfeld (BPS) vortices in the framework of an Abelian Maxwell-Higgs model supplemented with CPT-even and Lorentz-violating (LV) terms belonging to the gauge and Higgs sectors of the standard model extension. The analysis is performed in two situations: first, by considering the Lorentz violation only in the gauge sector, and then in both gauge and Higgs sectors. In the first case, it is observed that the model supports vortices somehow equivalent to the ones appearing in a dielectric medium. The Lorentz violation controls the radial extension (core of the solution) and the magnetic field amplitude of the Abrikosov-Nielsen-Olesen vortices, yielding compactlike defects in an alternative and simpler way than that of k-field models. At the end, we consider the Lorentz-violating terms in the gauge and Higgs sectors. It is shown that the full model also supports compactlike uncharged BPS vortices in a modified vacuum, but this time there are two LV parameters controlling the defect structure. Moreover, an interesting novelty is introduced by the LV-Higgs sector: fractional vortex solutions.

  5. An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

    PubMed Central

    Cao, Zheng; Bowie, James U

    2014-01-01

    Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α-helices compared to β-sheets by ∼0.2 kcal/mol. [2] Charge-stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ∼2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies. PMID:24501090

  6. Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites

    PubMed Central

    Seliverstov, Alexandr V.; Zverkov, Oleg A.; Istomina, Svetlana N.; Pirogov, Sergey A.; Kitsis, Philip S.

    2015-01-01

    In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues. PMID:26114107

  7. Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

    PubMed Central

    Martinez, Maria Guadalupe

    2016-01-01

    Alphaviruses are highly organized enveloped RNA viruses with an internal nucleocapsid surrounded by a membrane containing the E2 and E1 transmembrane proteins. Alphavirus budding takes place at the plasma membrane and requires the interaction of the cytoplasmic domain of E2 with the capsid protein. Here we used WT alphaviruses and Sindbis virus in which E2 was fused to a fluorescent protein to characterize virus exit from host cells. Our results show that alphavirus infection induced striking modifications of the host cell cytoskeleton and resulted in the formation of stable intercellular extensions that emanated exclusively from the infected cell. The intercellular extensions were long (> 10 μM), contained actin and tubulin, and formed flattened contacts with neighboring cells, but did not mediate membrane or cytoplasmic continuity between cells. Receptor down-regulation studies indicated that formation of stable extensions did not require the virus receptor, and that extensions promoted cell-to-cell virus transmission to receptor-depleted cells. Virus mutant experiments demonstrated that formation of extensions required the E2-capsid interaction but not active particle budding, while intercellular transmission of infection required the production of fusion-active virus particles. Protein expression studies showed that even in the absence of virus infection, the viral structural proteins alone induced intercellular extensions, and that these extensions were preferentially targeted to non-expressing cells. Together, our results identify a mechanism for alphavirus cell-to-cell transmission and define the key viral protein interactions that it requires. PMID:27977778

  8. Utilization of supercritical carbon dioxide to produce milk protein fractions

    USDA-ARS?s Scientific Manuscript database

    The nutritional, functional and bioactive properties of the individual whey proteins are appreciated by health-conscious consumers, yet few methods have been developed to produce these proteins to satisfy demand. The methods that are available are relatively new technologies that have not been prove...

  9. Supercritical carbon dioxide fractionation of whey protein isolate for new food-grade ingredients

    USDA-ARS?s Scientific Manuscript database

    A new, environmentally benign whey protein fractionation process was developed using supercritical CO2 (SCO2) as an acid aggregating agent to separate a-lactalbumin (a-LA) aggregates from soluble beta-lactoglobulin (beta-LG) protein in concentrated whey protein isolate (WPI) solutions. The process e...

  10. Fractionation of oats into value-added ingredients enriched with protein, beta-Glucan or starch

    USDA-ARS?s Scientific Manuscript database

    Oats contain several valuable nutrients, including protein, beta-glucan (BG, soluble fiber), starch, etc. A key strategy in increasing the value-added utilization of oats is to produce fractions enriched with each key nutrient. Among the reported studies on oat fractionation (dry or wet), most focus...

  11. Fractionation of barley into value-added ingredients enriched with protein, beta-glucan or starch

    USDA-ARS?s Scientific Manuscript database

    Barley contains several valuable nutrients, including protein, beta-glucan (BG) and starch. Each has additional value when concentrated. Among reported studies on barley fractionation (dry or wet), most focused enriching one or two components in term of concentrations in resulting fractions but negl...

  12. Fractionation of oats into products enriched with protein, beta-glucan, starch, or other carbohydrates

    USDA-ARS?s Scientific Manuscript database

    A modified wet method was developed to fractionate ground oat groats into 4 fractions enriched with beta-glucan (BG), protein, starch, and other carbohydrates (CHO), respectively. Effects of defatting oats and centrifuge force for separation were also investigated. Results show that, depending on ...

  13. Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity.

    PubMed

    Marangon, Matteo; Vincenzi, Simone; Lucchetta, Marco; Curioni, Andrea

    2010-02-15

    During the storage, bottled white wines can manifest haziness due to the insolubilisation of the grape proteins that may 'survive' in the fermentation process. Although the exact mechanism of this occurrence is not fully understood, proteins and tannins are considered two of the key factors involved in wine hazing, since their aggregation leads to the formation of insoluble particles. To better understand this complex interaction, proteins and tannins from the same unfined Pinot grigio wine were separated. Wine proteins were then fractionated by hydrophobic interaction chromatography (HIC). A significant correlation between hydrophobicity of the wine protein fractions and the haze formed after reacting with wine tannins was found, with the most reactive fractions revealing (by SDS-PAGE and RP-HPLC analyses) the predominant presence of thaumatin-like proteins. Moreover, the effects of both protein heating and disulfide bonds reduction (with dithiotreithol) on haze formation in the presence of tannins were assessed. These treatments generally resulted in an improved reactivity with tannins, and this phenomenon was related to both the surface hydrophobicity and composition of the protein fractions. Therefore, haze formation in wines seems to be related to hydrophobic interactions occurring among proteins and tannins. These interactions should occur on hydrophobic tannin-binding sites, whose exposition on the proteins can depend on both protein heating and reduction.

  14. Antioxidant activities and functional properties of protein and peptide fractions isolated from salted herring brine.

    PubMed

    Taheri, Ali; Sabeena Farvin, K H; Jacobsen, Charlotte; Baron, Caroline P

    2014-01-01

    In the present study proteins isolated from herring brine, which is a by-product of marinated herring production were evaluated for their functional properties and antioxidant activity. Herring brine was collected from the local herring industry and proteins were precipitated by adjusting the pH to 4.5 and the obtained supernatant was further fractionated by using ultrafiltration membranes with molecular weight cut offs of 50, 10 and 1kDa. The obtained >50kDa, 50-10kDa, 10-1kDa fractions and pH precipitated fraction were studied for their functional properties and antioxidant activity. Functional properties revealed that >50kDa polypeptides showed good emulsion activity index when compared to the other fractions. However all fractions had low emulsion stability index. The pH precipitated fraction showed the highest foaming capacity and stability at pH 10. The 50-10kDa and 10-1kDa peptide fractions showed good radical scavenging activity and reducing power at a concentration of 0.5mg protein/ml. All the fractions demonstrated low iron chelating activity and did not inhibit oxidation in a soybean phosphatidylcholine liposome model system. However all the fractions were to some extent able to delay iron catalyzed lipid oxidation in 5% fish oil in water emulsions and the 10-50kDa fraction was the best. These results show the potential of proteins and peptide fractions recovered from waste water from the herring industry as source of natural antioxidants for use in food products. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Teaching Experimental Design Using an Exercise in Protein Fractionation

    NASA Astrophysics Data System (ADS)

    Loke, J. P.; Hancock, D.; Johnston, J. M.; Dimauro, J.; Denyer, G. S.

    2001-11-01

    This experiment, suitable for introductory biochemistry courses, presents the techniques of protein purification as a problem-solving exercise. Students must identify and purify three proteins from an unknown mixture using the techniques of gel filtration, ion exchange chromatography, UV and visible spectrophotometry, and gel electrophoresis. To aid construction of a strategy, they are given some information about each of the possible proteins: source, function, molecular weight, pI, and UV and visible spectra. From this they must design their own purification protocols and carry out the experimental work. To develop students' computer skills, the experimental results and the logic used in the identification are presented as a short computer-generated report.

  16. A fast method for the determination of fractional contributions to solvation in proteins

    PubMed Central

    Talavera, David; Morreale, Antonio; Meyer, Tim; Hospital, Adam; Ferrer-Costa, Carles; Gelpi, Josep Lluis; de la Cruz, Xavier; Soliva, Robert; Luque, F. Javier; Orozco, Modesto

    2006-01-01

    A fast method for the calculation of residue contributions to protein solvation is presented. The approach uses the exposed polar and apolar surface of protein residues and has been parametrized from the fractional contributions to solvation determined from linear response theory coupled to molecular dynamics simulations. Application of the method to a large subset of proteins taken from the Protein Data Bank allowed us to compute the expected fractional solvation of residues. This information is used to discuss when a residue or a group of residues presents an uncommon solvation profile. PMID:17001031

  17. Comprehensive proteomic analysis of the human milk proteome: contribution of protein fractionation.

    PubMed

    Mangé, A; Bellet, V; Tuaillon, E; Van de Perre, P; Solassol, J

    2008-12-15

    In-depth analysis of the milk proteome by mass spectrometry is challenged by the presence of few high-abundance proteins that interfere with the detection of lower-abundance proteins. Here, we evaluated the proteomic analysis of milk samples following a strong anion exchange fractionation procedure using denaturating conditions ensuring the disruption of protein-protein interactions. Crude whey or skim milk and their different resulting fractions were analyzed by protein chip array mass spectrometry. Using protein chip array mass spectrometry, several high-abundance proteins were localized in distinct fractions increasing the total number of unique peptides and proteins detected. This total number increased by about 20-30% by combining different chromatographic surface arrays used for capture. Reproducible results were obtained in human skim milk and whey; however this approach was not successful with milk fat globule membrane and required refinement. Hence, milk profiling by anion exchange fractionation combined to protein chip array mass spectrometry represents a promising tool to detect unknown low-abundance milk proteins that may ultimately prove useful as biomarkers of diseases transmitted by breastfeeding.

  18. Composition and Molecular Weight Distribution of Carob Germ Proteins Fractions

    USDA-ARS?s Scientific Manuscript database

    Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and electrophoretic analysis. Using a mo...

  19. Impact of carrier fluid composition on recovery of nanoparticles and proteins in flow field flow fractionation.

    PubMed

    Schachermeyer, Samantha; Ashby, Jonathan; Kwon, Minjung; Zhong, Wenwan

    2012-11-16

    Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199, 102, and 45 nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0-0.1M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85 nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0-0.15M) was investigated. Our results suggested that (1) electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; (2) larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and (3) buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using HSA and IgG. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation

    PubMed Central

    Das, Lalatendu Keshary; Kundu, S. S.; Kumar, Dinesh; Datt, Chander

    2015-01-01

    Aim: To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO) and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS). Materials and Methods: Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier) were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash), fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose), primary CHO fractions (CHO, non-structural CHO, structural CHO and starch) and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein). The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - non-degrading or unavailable) and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable) fractions of test feeds. Results: Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Conclusion: Based on our findings, it was

  1. Fractionation of carbohydrate and protein content of some forage feeds of ruminants for nutritive evaluation.

    PubMed

    Das, Lalatendu Keshary; Kundu, S S; Kumar, Dinesh; Datt, Chander

    2015-02-01

    To evaluate some forage feeds of ruminants in terms of their carbohydrate (CHO) and protein fractions using Cornell Net Carbohydrate and Protein System (CNCPS). Eleven ruminant feeds (six green fodders - maize, oat, sorghum, bajra, cowpea, berseem and five range herbages - para grass, guinea grass, hedge lucerne, setaria grass and hybrid napier) were selected for this study. Each feed was chemically analyzed for proximate principles (dry matter, crude protein [CP], ether extract, organic matter and ash), fiber fractions (neutral detergent fiber, acid detergent fiber, acid detergent lignin, cellulose and hemicellulose), primary CHO fractions (CHO, non-structural CHO, structural CHO and starch) and primary protein fractions (neutral detergent insoluble CP, acid detergent insoluble CP, non-protein nitrogen and soluble protein). The results were fitted to the equations of CNCPS to arrive at various CHO (CA - fast degrading, CB1 - intermediate degrading, CB2 - slow degrading and CC - non-degrading or unavailable) and protein (PA - instantaneously degrading, PB1 - fast degrading, PB2 - intermediate degrading, PB3 - slow degrading and PC - non-degrading or unavailable) fractions of test feeds. Among green fodders, cowpea and berseem had higher CA content while except hedge lucerne all range herbages had lower CA values. CB1 content of all feeds was low but similar. All feeds except cowpea, berseem, and hedge lucerne contained higher CB2 values. Oat among green fodders and hybrid napier among range herbages had lower CC fraction. Feeds such as bajra, cowpea, berseem and the setaria grass contained lower PA fraction. All green fodders had higher PB1 content except maize and cowpea while all range herbages had lower PB1 values except hedge lucerne. Para grass and hybrid napier contained exceptionally low PB2 fraction among all feeds. Low PC contents were reported in oat and berseem fodders. Based on our findings, it was concluded that feeds with similar CP and CHO content

  2. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  3. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  4. System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

    PubMed Central

    Veyel, Daniel; Kierszniowska, Sylwia; Kosmacz, Monika; Sokolowska, Ewelina Maria; Michaelis, Aenne; Luzarowski, Marcin; Szlachetko, Jagoda; Willmitzer, Lothar; Skirycz, Aleksandra

    2017-01-01

    Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems. PMID:28205532

  5. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut (Juglans regia L.) proteins and protein fractionations.

    PubMed

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-27

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8-6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  6. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

    PubMed Central

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

  7. The effect of whey acidic protein fractions on bone loss in the ovariectomised rat.

    PubMed

    Kruger, Marlena C; Plimmer, Gabrielle G; Schollum, Linda M; Haggarty, Neill; Ram, Satyendra; Palmano, Kate

    2005-08-01

    Bovine milk has been shown to contain bioactive components with bone-protective properties. Earlier studies on bovine milk whey protein showed that it suppressed bone resorption in the female ovariectomised rat. A new osteotropic component was subsequently identified in the whey basic protein fraction, but bone bioactivity may also be associated with other whey fractions. In the present study, we investigated whether acidic protein fractions isolated from bovine milk whey could prevent bone loss in mature ovariectomised female rats. Six-month-old female rats were ovariectomised (OVX) or left intact (sham). The OVX rats were randomised into four groups. One group remained the control (OVX), whereas three groups were fed various whey acidic protein fractions from milk whey as 3 g/kg diet for 4 months. Outcomes were bone mineral density, bone biomechanics and markers of bone turnover. Bone mineral density of the femurs indicated that one of the whey AF over time caused a recovery of bone lost from OVX. Plasma C-telopeptide of type I collagen decreased significantly in all groups except OVX control over time, indicating an anti-resorptive effect of whey acidic protein. Biomechanical data showed that the AF may affect bone architecture as elasticity was increased by one of the whey AF. The femurs of AF-supplemented rats all showed an increase in organic matter. This is the first report of an acidic whey protein fraction isolated from milk whey that may support the recovery of bone loss in vivo.

  8. pH fractionation and identification of proteins: comparing column chromatofocusing versus liquid isoelectric focusing techniques.

    PubMed

    Gunther, Nereus W; Paul, Moushumi; Nuñez, Alberto; Liu, Yanhong

    2012-06-01

    In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Chromatofocusing fractionation and two-dimensional difference gel electrophoresis for low abundance serum proteins.

    PubMed

    Qin, Shuzhen; Ferdinand, Angeline S; Richie, Jerome P; O'Leary, Michael P; Mok, Samuel C; Liu, Brian C-S

    2005-08-01

    The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.

  10. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    SciTech Connect

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  11. Antioxidant activities of bambara groundnut (Vigna subterranea) protein hydrolysates and their membrane ultrafiltration fractions.

    PubMed

    Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Ijabadeniyi, Oluwatosin A; Aluko, Rotimi E; Amonsou, Eric O

    2016-05-18

    In this study, the bambara protein isolate (BPI) was digested with three proteases (alcalase, trypsin and pepsin), to produce bambara protein hydrolysates (BPHs). These hydrolysates were passed through ultrafiltration membranes to obtain peptide fractions of different sizes (<1, 1-3, 3-5 and 5-10 kDa). The hydrolysates and their peptide fractions were investigated for antioxidant activities. The membrane fractions showed that peptides with sizes <3 kDa had significantly (p < 0.05) reduced surface hydrophobicity when compared with peptides >3 kDa. This is in agreement with the result obtained for the ferric reducing power, metal chelating and hydroxyl radical scavenging activities where higher molecular weight peptides exhibited better activity (p < 0.05) when compared to low molecular weight peptide fractions. However, for all the hydrolysates, the low molecular weight peptides were more effective diphenyl-1-picrylhydrazyl (DPPH) radical scavengers but not superoxide radicals when compared to the bigger peptides. In comparison with glutathione (GSH), BPHs and their membrane fractions had better (p < 0.05) reducing power and ability to chelate metal ions except for the pepsin hydrolysate and its membrane fractions that did not show any metal chelating activity. However, the 5-10 kDa pepsin hydrolysate peptide fractions had greater (88%) hydroxyl scavenging activity than GSH, alcalase and trypsin hydrolysates (82%). These findings show the potential use of BPHs and their peptide fraction as antioxidants in reducing food spoilage or management of oxidative stress-related metabolic disorders.

  12. Dry fractionation methods to produce barley meals varying in protein, beta-glucan, and starch contents.

    PubMed

    Liu, KeShun; Barrows, Frederic T; Obert, Don

    2009-08-01

    Barley contains several valuable nutrients including beta-glucan (BG), protein, and starch. Each has additional value when concentrated. Dehulled and hulless barleys were sequentially pearled for 1 to 6 cycles, each with 8% removal. The 6 pearled kernels and the initial kernel were subjected to impact or abrasive milling, followed by sieving with a series of U.S. standard sieves. Results of pearling fines show that protein was most concentrated in the outer area, and decreased all the way toward the core area (near 100% surface removal). Starch showed an opposite trend. BG followed the starch trend, but reached a peak at about 60% surface removal. Upon milling and sieving of kernel samples, genotype and particle size had significant effects on nutrient contents in sieved fractions. The pearling cycle had significant effects on protein and starch contents but little effect on BG content, while the milling method had significant effects on protein and BG contents but little on starch content. Abrasive milling produced sieved fractions with much higher variation in protein content than impact milling, but the opposite effect was observed for shifting BG content. Mass frequency influenced more on recovery rates of nutrients than their concentrations in individual fractions. When the recovery rate was also taken into consideration, pearling alone was found to be the most effective way to enrich protein in barley kernels. However, a combination of pearling with the method of milling and sieving was needed for maximally shifting BG and starch contents. Although dry fractionation is the method of choice for separating barley into fractions with varying levels of protein, BG, and/or starch, selection of a specific single or combined method is needed for achieving maximum shifts of a particular nutrient. Such information is significant to those who use dry fractionation methods to enrich protein, BG, and/or starch.

  13. [Extraction and characterization of soluble protein fractions from Phaseolus lunatus L seeds].

    PubMed

    Gallegos Tintoré, Santiago; Pacheco Aguirre, Jessé; Betancur Ancona, David; Chel Guerrero, Luis

    2004-03-01

    Legume proteins as a potential source of valuable nutrients, are the object of several studies in order to obtain the best use. A basic knowledge becomes more important for those proteins from species not wholly utilized, before using them as food ingredients. The objective of this work was to determine several structural and nutritional characteristics of the protein fractions from Phaseolus lunatus, separated in different solvents. The relative amount of extraction for the albumins (ALB), globulins (GLB), prolamines (PRL), and glutelins (GLT) was 62.3, 34.8, 1.4 and 1.5%, respectively. The SDS-PAGE electrophoretic profile of both ALB and GLB, showed seven common bands in intervals from 10 to 95 kDa, and 14 to 99 kDa, respectively; the amino acids profile showed that PRL was the rich fraction in sulfurated amino acids (11.5 g/100 g protein); the content of lysine in the fraction of ALB was smaller than expected but the requirement of the FAO in the fractions of GLB and GLT was covered. In general, the fraction of GLB had the best balance of amino acids and digestibility (80%); however, it had a relationship of calculated protein efficiency ratio (C-PER) of 0.11, smaller than the ratio in ALB (0.97). The calorimetric analysis showed denatured temperatures around 90 degrees C for the ALB, GLB, and GLU fractions. The PRL fraction probably did not present a thermal transition because the proteins were denaturalized by the extraction conditions.

  14. HIV-1 gag proteins in virions and in infected cell fractions.

    PubMed

    Sharova, N K; Grigor'ev, V B; Bukrinskaya, A G

    1991-01-01

    The relation of the initial products of the HIV-1 gag gene to the final products was determined in virus samples and cell fractions of infected H9 and Jurkat-tat cell cultures. The proteins were identified by immunoblotting with pooled sera from AIDS patients or monoclonal antibodies. The proportion in the virions of gag precursor proteins and the products of their proteolytic cleavage varied according to the maturity of the virus particles as determined by electron microscopy. The distribution of viral gag proteins in the cell fractions was determined 2, 4, and 24 h after infection. Treatment of cells with cycloheximide to block de novo protein synthesis did not significantly affect the results. Gag proteins containing the N terminus of the precursor p55 (including p55, the intermediate precursors p41(45) and p39, and mature protein p17) were found in the cell nuclei up to 24 h after infection. The major core protein p24 was located in the cytoplasmic fraction. These data strongly suggest that gag precursors from the p55 N terminus and the matrix protein p17 enter the infected cell separately from the major core protein p24, or become separated from it in the cytoplasm.

  15. Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

    PubMed Central

    Borner, Georg H. H.; Hein, Marco Y.; Hirst, Jennifer; Edgar, James R.; Mann, Matthias; Robinson, Margaret S.

    2014-01-01

    We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps. PMID:25165137

  16. 2-DE Separation and Identification of Oat (Avena sativa L.) Proteins and Their Prolamin Fractions.

    PubMed

    Nałęcz, Dorota; Szerszunowicz, Iwona; Dziuba, Marta; Minkiewicz, Piotr

    2017-01-01

    At present two-dimensional polyacrylamide gel electrophoresis (2-DE) is the most widely used proteomic tool, which enables simultaneous separation of even thousands of proteins with a high degree of resolution. The quality of 2-DE separation depends on the type of biological material used as a protein source. The presence of interfering compounds (e.g., phenols, as it is the fact in plant material including oat seeds) impedes 2-DE run. With the use of this technique it is possible to analyze the complex protein mixtures, characteristic protein fractions, as well as individual proteins.The purpose of this chapter is to describe the 2-DE technique (the separate stages of the first and the second dimension) for determining the oat protein composition (oat seed proteome), separation and preliminary identification of oat prolamin fractions. Electrophoretically separated proteins are identified on the basis of pI markers (identifying the location of both ends of an IPG strip) and on 2D SDS-PAGE standards. The gel images of oat proteins are analyzed with the help of ImageMaster 2D Platinum 6.0 program (Amersham Bioscience, part of GE Healthcare, Uppsala, Sweden). It allows finding unique spot identifiers for the occurrence of oat prolamin fractions in oat total proteins. The characteristic spots of similar shape and intensity (anchoring spots) and characteristic groups of spots can be searched for the purpose of identification.

  17. Extensible byssus of Pinctada fucata: Ca2+-stabilized nanocavities and a thrombospondin-1 protein

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Li, Shiguo; Huang, Jingliang; Liu, Yangjia; Jia, Ganchu; Xie, Liping; Zhang, Rongqing

    2015-10-01

    The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca2+ by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the β-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater.

  18. Extensible byssus of Pinctada fucata: Ca2+-stabilized nanocavities and a thrombospondin-1 protein

    PubMed Central

    Liu, Chuang; Li, Shiguo; Huang, Jingliang; Liu, Yangjia; Jia, Ganchu; Xie, Liping; Zhang, Rongqing

    2015-01-01

    The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca2+ by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the β-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater. PMID:26446436

  19. Extensible byssus of Pinctada fucata: Ca(2+)-stabilized nanocavities and a thrombospondin-1 protein.

    PubMed

    Liu, Chuang; Li, Shiguo; Huang, Jingliang; Liu, Yangjia; Jia, Ganchu; Xie, Liping; Zhang, Rongqing

    2015-10-08

    The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca(2+) by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the β-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater.

  20. Fractionation of Whey Protein Isolate with Supercritical Carbon Dioxide—Process Modeling and Cost Estimation

    PubMed Central

    Yver, Alexandra L.; Bonnaillie, Laetitia M.; Yee, Winnie; McAloon, Andrew; Tomasula, Peggy M.

    2012-01-01

    An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) from a commercial whey protein isolate (WPI) containing 20% α-LA and 55% β-LG, through selective precipitation of α-LA. Pilot-scale experiments were performed around the optimal parameter range (T = 60 to 65 °C, P = 8 to 31 MPa, C = 5 to 15% (w/w) WPI) to quantify the recovery rates of the individual proteins and the compositions of both fractions as a function of processing conditions. Mass balances were calculated in a process flow-sheet to design a large-scale, semi-continuous process model using SuperproDesigner® software. Total startup and production costs were estimated as a function of processing parameters, product yield and purity. Temperature, T, pressure, P, and concentration, C, showed conflicting effects on equipment costs and the individual precipitation rates of the two proteins, affecting the quantity, quality, and production cost of the fractions considerably. The highest α-LA purity, 61%, with 80% α-LA recovery in the solid fraction, was obtained at T = 60 °C, C = 5% WPI, P = 8.3 MPa, with a production cost of $8.65 per kilogram of WPI treated. The most profitable conditions resulted in 57%-pure α-LA, with 71% α-LA recovery in the solid fraction and 89% β-LG recovery in the soluble fraction, and production cost of $5.43 per kilogram of WPI treated at T = 62 °C, C = 10% WPI and P = 5.5 MPa. The two fractions are ready-to-use, new food ingredients with a pH of 6.7 and contain no residual acid or chemical contaminants. PMID:22312250

  1. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation.

    PubMed

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development.

  2. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation

    PubMed Central

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development. PMID:26697039

  3. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    PubMed

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p < 0.05) compared with the activity of these proteases from the non-infested one group. The infested drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  4. A novel protein complex identification algorithm based on Connected Affinity Clique Extension (CACE).

    PubMed

    Li, Peng; He, Tingting; Hu, Xiaohua; Zhao, Junmin; Shen, Xianjun; Zhang, Ming; Wang, Yan

    2014-06-01

    A novel algorithm based on Connected Affinity Clique Extension (CACE) for mining overlapping functional modules in protein interaction network is proposed in this paper. In this approach, the value of protein connected affinity which is inferred from protein complexes is interpreted as the reliability and possibility of interaction. The protein interaction network is constructed as a weighted graph, and the weight is dependent on the connected affinity coefficient. The experimental results of our CACE in two test data sets show that the CACE can detect the functional modules much more effectively and accurately when compared with other state-of-art algorithms CPM and IPC-MCE.

  5. Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats

    PubMed Central

    Manjula, Belur N.; Mische, Sheenah M.; Fischetti, Vincent A.

    1983-01-01

    Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of α-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats. PMID:16593365

  6. Intrinsic fluorescence excitation-emission matrix spectral features of cottonseed protein fractions and the effects of denaturants

    USDA-ARS?s Scientific Manuscript database

    To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, an...

  7. Reorganization of low-molecular-weight fraction of plasma proteins in the annual cycle of cyprinidae.

    PubMed

    Andreeva, A M; Lamas, N E; Serebryakova, M V; Ryabtseva, I P; Bolshakov, V V

    2015-02-01

    Reorganization of the low-molecular-weight fraction of cyprinid plasma was analyzed using various electrophoretic techniques (disc electrophoresis, electrophoresis in polyacrylamide concentration gradient, in polyacrylamide with urea, and in SDS-polyacrylamide). The study revealed coordinated changes in the low-molecular-weight protein fractions with seasonal dynamics and related reproductive rhythms of fishes. We used cultured species of the Cyprinidae family with sequenced genomes for the detection of these interrelations in fresh-water and anadromous cyprinid species. The common features of organization of fish low-molecular-weight plasma protein fractions made it possible to make reliable identification of their proteins. MALDI mass-spectrometry analysis revealed the presence of the same proteins (hemopexin, apolipoproteins, and serpins) in the low-molecular-weight plasma fraction in wild species and cultured species with sequenced genomes (carp, zebrafish). It is found that the proteins of the first two classes are organized as complexes made of protein oligomers. Stoichiometry of these complexes changes in concordance with the seasonal and reproductive rhythms.

  8. Proteomics in globe artichoke: protein extraction and sample complexity reduction by PEG fractionation.

    PubMed

    Acquadro, Alberto; Falvo, Sara; Mila, Silvia; Giuliano Albo, Alessandra; Comino, Cinzia; Moglia, Andrea; Lanteri, Sergio

    2009-05-01

    Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.

  9. Fractionation of the Gulf Toadfish Intestinal Precipitate Organic Matrix Reveals Potential Functions of Individual Proteins.

    PubMed

    Schauer, Kevin L; Grosell, Martin

    2017-03-15

    The regulatory mechanisms behind the production of CaCO3 in the marine teleost intestine are poorly studied despite being essential for osmoregulation and responsible for a conservatively estimated 3-15% of annual oceanic CaCO3 production. It has recently been reported that the intestinally derived precipitates produced by fish as a byproduct of their osmoregulatory strategy form in conjunction with a proteinaceous matrix containing nearly 150 unique proteins. The individual functions of these proteins have not been the subject of investigation until now. Here, organic matrix was extracted from precipitates produced by Gulf toadfish (Opsanus beta) and the matrix proteins were fractionated by their charge using strong anion exchange chromatography. The precipitation regulatory abilities of the individual fractions were then analyzed using a recently developed in vitro calcification assay, and the protein constituents of each fraction were determined by mass spectrometry. The different fractions were found to have differing effects on both the rate of carbonate mineral production, as well as the morphology of the crystals that form. Using data collected from the calcification assay as well as the mass spectrometry experiments, individual calcification promotional indices were calculated for each protein, giving the first insight into the functions each of these matrix proteins may play in regulating precipitation.

  10. Almond protein hydrolysate fraction modulates the expression of proinflammatory cytokines and enzymes in activated macrophages.

    PubMed

    Udenigwe, Chibuike C; Je, Jae-Young; Cho, Young-Sook; Yada, Rickey Y

    2013-04-30

    Simulated gastrointestinal treatment of almond proteins with pepsin and pancreatic proteases resulting in 16.6% degree of hydrolysis or 1.33 milliequivalent leucine per g protein yielded a hydrolysate that modulated excessive nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages. After fractionation, a resulting fraction of molecular size > 5 kDa retained the nitric oxide modulatory effect observed initially in the crude hydrolysate. The high molecular size fraction was found to modulate levels of proinflammatory cytokines, interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)-α in the activated cells. Immunoblotting analysis indicated that the hydrolysate fraction decreased the expression levels of inflammatory enzyme indicators, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in the activated cells. RT-PCR analysis showed that treatment of the activated cells with the hydrolysate fraction resulted in the inhibition of relative gene expressions of proinflammatory IL-6, IL-1β, TNF-α, iNOS and COX-2. These results indicate a potential application of almond protein hydrolysates against inflammatory conditions, and will contribute to delineating the possible contributions of proteins to health benefits attributed to almond consumption.

  11. Study of selenium distribution in the protein fractions of the Brazil nut, Bertholletia excelsa.

    PubMed

    Chunhieng, Thavarith; Pétritis, Konstantinos; Elfakir, Claire; Brochier, José; Goli, Thierry; Montet, Didier

    2004-06-30

    The high selenium content of the Brazil nut, Bertholletia excelsa, makes this seed a healthy food qualified as an antiradical protector. The studied nut contained 126 ppm of selenium. Selenium was found to be distributed in the nut protein fractions. The water-extracted fraction, which represented 17.7% of the cake protein, was the richest in selenium with 153 ppm. Analysis by HPLC-MS showed that selenium was linked by a covalent bond to two amino acids to form selenomethionine and selenocystine. The selenomethionine represented a little less than 1% of the total amount of methionine.

  12. Automated frit inlet/frit outlet flow field-flow fractionation for protein characterization with emphasis on polymeric wheat proteins.

    PubMed

    Stevenson, S G; Ueno, T; Preston, K R

    1999-01-01

    A flow field-flow fractionation (FFF) unit fitted with a 254-nm spacer, frit inlet (FI), and frit outlet (FO) was automated for protein analysis by addition of a system controller, autosampler, and computer software to control pumps, sample loading, and data capture. Standard molecular size marker proteins and polymeric wheat storage protein extracts were used to assess the performance of the automated unit. Optimum resolution for these proteins was obtained with a sample inlet flow of 0.2 mL/min, a frit inlet flow (recirculating) of 1.4 mL/min, and a cross-flow (recirculating) of 5 mL/min using 0.05 M acetic acid containing 0.002% FL-70 as a carrier. Use of the FIFO FFF eliminates the requirement for stop-flow relaxation and pressure balancing, results in better reproducibility, and generates a 7-10-fold increase in sensitivity at the detector by concentrating fractions eluting from the channel. These improvements resulted in superior resolution of polymeric wheat protein fractions compared to those obtained previously using a standard channel with manual load and stop-flow relaxation, allowing accurate integration of peak or size range areas. Automation of this system allows unattended sample fractionation and hence markedly increases potential for sample throughput.

  13. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice.

    PubMed

    Marini, Juan C; Didelija, Inka Cajo

    2015-01-01

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [14C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.

  14. Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR Assignments

    PubMed Central

    Wenrich, Broc R.; Sonstrom, Reilly E.; Gupta, Riju A.; Rovnyak, David

    2015-01-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or 13C′, etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%w/w u-13C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200 mg for a 2 g/L culture) u-13C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced 13C incorporation that gives almost the same NMR signal levels as an exact 20% 13C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that 13C incorporation levels no greater than 20%w/w yield 13C and 13C-13C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-13C glucose to expression media at induction, there is poor preservation of 13Cα-13Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible. PMID:26256059

  15. Fractionation of sheep cheese whey by a scalable method to sequentially isolate bioactive proteins.

    PubMed

    Pilbrow, Jodi; Bekhit, Alaa El-din A; Carne, Alan

    2016-07-15

    This study reports a procedure for the simultaneous purification of glyco(caseino)macropeptide, immunoglobulin, lactoperoxidase, lactoferrin, α-lactalbumin and β-lactoglobulin from sheep cheese sweet whey, an under-utilized by-product of cheese manufacture generated by an emerging sheep dairy industry in New Zealand. These proteins have recognized value in the nutrition, biomedical and health-promoting supplements industries. A sequential fractionation procedure using economical anion and cation exchange chromatography on HiTrap resins was evaluated. The whey protein fractionation is performed under mild conditions, requires only the adjustment of pH between ion exchange chromatography steps, does not require buffer exchange and uses minimal amounts of chemicals. The purity of the whey protein fractions generated were analyzed by reversed phase-high performance liquid chromatography and the identity of the proteins was confirmed by mass spectrometry. This scalable procedure demonstrates that several proteins of recognized value can be fractionated in reasonable yield and purity from sheep cheese whey in one streamlined process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Cellular and humoral responses in coeliac disease. 1. Wheat protein fractions.

    PubMed

    Penttila, I A; Devery, J M; Gibson, C E; LaBrooy, J T; Skerritt, J H

    1991-12-31

    The humoral and cellular immune response of coeliac individuals to various wheat protein fractions was studied using serum antibody ELISA assays and the indirect leucocyte migration inhibition factor (LMIF) assays. Greater migration inhibition factor activity was seen in coeliacs on a gluten-free-diet having low serum antibody titres, and using purified T-cells instead of total peripheral blood mononucleocytes. Gliadin was the most active fraction in both assays. Raised antibodies to low-molecular weight and high-molecular weight glutenin polypeptides was observed, though these proteins had little migration inhibition factor activity. No cellular or humoral response was seen to albumins or globulins. Proteins associated with the granules of well-washed wheat starch are distinct from gluten proteins and had little T-cell activity, correlating with clinical observations that properly prepared wheat starch is devoid of coeliac toxicity. The greater specificity of the humoral response for individual wheat protein fractions in this study, compared with the earlier reports, likely results from cross-contamination in the earlier work of each fraction with gliadin.

  17. Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions.

    PubMed

    Creusot, Nathalie; Gruppen, Harry

    2008-11-12

    In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.

  18. Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds

    PubMed Central

    Lima, Ana; Oliveira, Jennifer; Saúde, Filipe; Mota, Joana; Ferreira, Ricardo Boavida

    2017-01-01

    The search for anticancer MMP-9 inhibitors (MMPIs) in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P) and non-protein fractions (NP) isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers. PMID:28264435

  19. Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds.

    PubMed

    Lima, Ana; Oliveira, Jennifer; Saúde, Filipe; Mota, Joana; Ferreira, Ricardo Boavida

    2017-02-27

    The search for anticancer MMP-9 inhibitors (MMPIs) in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P) and non-protein fractions (NP) isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers.

  20. The evolution of fraction i protein during the origin of a new species of Nicotiana.

    PubMed

    Kung, S D; Sakano, K; Gray, J C; Wildman, S G

    1975-12-31

    The polypeptide composition of Fraction I protein from Nicotiana digluta, a synthetic species which arose by chromosome doubling following the interspecific hybridization of N. glutinosa and N. tabacum, has been examined by isoelectric focusing. The composition of the protein from N. digluta, which was identical to the protein from the infertile F1 hybrid N. glutinosa x N. tabacum, showed 3 polypeptides in the large subunit and 4 polypeptides in the small subunit. The large subunit polypeptides were identical to those from N. glutinosa, the maternal parent in the original hybridization, whereas the small subunit polypeptides were a composite of the small subunit polypeptides from both N. glutinosa and N. tabacum. This analysis demonstrates how the polypeptide composition of Fraction I protein evolves during the origin of new species of Nicotiana.

  1. Simultaneous purification and fractionation of nucleic acids and proteins from complex samples using bidirectional isotachophoresis.

    PubMed

    Qu, Yatian; Marshall, Lewis A; Santiago, Juan G

    2014-08-05

    We report on our efforts to create an on-chip system to simultaneously purify and fractionate nucleic acids and proteins from complex samples using isotachophoresis (ITP). We have developed this technique to simultaneously extract extracellular DNA and proteins from human blood serum samples and deliver these to two separate output reservoirs on a chip. The purified DNA is compatible with quantitative polymerase chain reaction (qPCR), and proteins can be extracted so as to exclude albumin, the most abundant protein in serum. We describe significant remaining challenges in making this bidirectional method a robust and efficient technique. These challenges include managing channel surface adsorption of proteins, identifying the cause of observed reductions in low molecular weight proteins, and dealing with nonspecific binding of proteins and DNA.

  2. Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins.

    PubMed

    Malins, Lara R; Payne, Richard J

    2014-10-01

    Native chemical ligation continues to play a pivotal role in the synthesis of increasingly complex peptide and protein targets twenty years after its initial report. This opinion article will highlight a number of recent, powerful extensions of the technology that have expanded the scope of the reaction, accelerated ligation rates, enabled chemoselective post-ligation modifications, and streamlined the ligation of multiple peptide fragments. These advances have facilitated the synthesis of a number of impressive protein targets to date and hold great promise for the continued application of native chemical ligation for the detailed study of protein structure and function. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. A fractionation procedure for identifying novel proteins induced by chill stress in Arabidopsis thaliana.

    PubMed

    Sun, Liwen; Wang, Shichen; Xi, Jinghui; Yang, Shuo; Liu, Xiangguo; Chai, Xin; Xin, Hongbao; An, Baiyi; Hao, Dongyun

    2009-08-01

    Extraction of plant proteins using typical extraction buffers leaves insoluble debris that cannot be investigated by conventional 2-DE technologies. In this paper, we present a scalable, off-line procedure for extraction of Arabidopsis thaliana homogenates that can be used in combination with both in-gel digestion and mass spectrometry. Based on sequential NaCl gradients and strong detergent fractionation, this new strategy allowed detection of 11 novel proteins from Arabidopsis thaliana that were altered in response to chilling stress.

  4. Hollow-Fiber Flow Field-Flow Fractionation for Mass Spectrometry: From Proteins to Whole Bacteria

    NASA Astrophysics Data System (ADS)

    Reschiglian, Pierluigi; Zattoni, Andrea; Rambaldi, Diana Cristina; Roda, Aldo; Hee Moon, Myeong

    Mass spectrometry (MS) provides analyte identification over a wide molar-mass range. However, particularly in the case of complex matrices, this ability is often enhanced by the use of pre-MS separation steps. A separation, prototype technique for the "gentle" fractionation of large/ultralarge analytes, from proteins to whole cells, is here described to reduce complexity and maintain native characteristics of the sample before MS analysis. It is based on flow field-flow fractionation, and it employs a micro-volume fractionation channel made of a ca. 20 cm hollow-fiber membrane of sub-millimeter section. The key advantages of this technique lie in the low volume and low-cost of the channel, which makes it suitable to a disposable usage. Fractionation performance and instrumental simplicity make it an interesting methodology for in-batch or on-line pre-MS treatment of such samples.

  5. Amino acid composition of some Amaranthus sp. grain proteins and of its fractions.

    PubMed

    Correa, A D; Jokl, L; Carlsson, R

    1986-09-01

    This study was carried out to determine the protein content of several Amaranthus sp. grains. Findings revealed this has a high lysine (5.3 to 6.3 of the protein) and sulphur amino acids content (3.4-4.0%), while leucine could well be limiting when those seeds are used as a sole protein source in food. Using the correction for in vitro protein digestibility, the chemical score varied from 50 to 67. The calculated protein efficiency ratios and biological values ranged from 1.39 to 1.80 and 53 to 68, respectively. Considering that amaranth grain is a good supplement to cereal grain, the protein of A. hypochondriacus HH5 (yellow seeds) and A. anclancalius (black seeds) was fractionated into albumin, globulin, prolamin and glutelin. The average proportions between those soluble proteins were 65:17:11:7, respectively. Albumin had the highest lysine content (7.3-8.2%), and globulin the highest methionine (4.1-5.3%) and phenylalanine (6.0-6.1%) content. Prolamin had the highest threonine (4.6-5.4%) and leucine (6.8-6.9%) content, while glutelin had a very low methionine content (0.6-1.0%). Based on the above-mentioned findings, the authors conclude the variation in the amino acid composition of the protein fractions can be used for genetic protein improvement.

  6. Toward the characterization of fractional stochastic processes underlying methyl dynamics in proteins.

    PubMed

    Calligari, Paolo; Abergel, Daniel

    2012-11-01

    In this article, we investigate the multiple-scale structure of methyl side chain dynamics in proteins. We show that the orientational correlation functions of CH(3) methyl groups are well described by a fractional Brownian dynamics model. Typical angular correlation functions involved in NMR relaxation were computed from MD simulations performed on two different proteins. These correlation functions were shown to be very well fitted by a fractional Ornstein-Uhlenbeck process in the presence of effective local potentials at the C-H and C-C methyl bonds. In addition, our analysis highlights the presence of the asymptotic power law decay of the waiting time probability density of the stochastic process involved, thereby illustrating the connection between approaches based on fractional diffusion equations and the continuous time random walk.

  7. Nutritional and functional properties of Vicia faba protein isolates and related fractions.

    PubMed

    Vioque, Javier; Alaiz, Manuel; Girón-Calle, Julio

    2012-05-01

    The goal of this research was the characterisation of Vicia faba (broadbean) protein isolates and related fractions in order to determine whether this grain legume could be used for production of high quality protein products and other fractions rich in functional components. Alkaline extraction of the defatted seed flour, followed by precipitation at the isoelectric pH, yielded a 92% protein isolate with a high oil absorption capacity. The contents of the favism-inducing glycosides, vicine and convicine, in the isolate were reduced by more than 99% as compared to the original flour, although the amino acid composition was similar to that of the flour. Some of the by-products of protein isolate production may also be of interest from a nutritional and functional point of view. Thus, the oil resulting from hexane extraction of the flour is rich in unsaturated fatty acids, and polyphenols (resulting from extraction of the defatted flour with acetone) showed a high ABTS radical-scavenging activity. In addition, the solid residue (resulting from protein solubilisation) was high in fibre and showed good water absorption. These results show good nutritional and functional properties in V. faba protein isolates and related fractions, which may favour the revalorisation of this traditional bean crop.

  8. Extension of the selection of protein chromatography and the rate model to affinity chromatography.

    PubMed

    Sandoval, G; Shene, C; Andrews, B A; Asenjo, J A

    2010-01-01

    The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

  9. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    SciTech Connect

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  10. Immunoprecipitation and Mass Spectrometry Defines an Extensive RBM45 Protein-Protein Interaction Network

    PubMed Central

    Li, Yang; Collins, Mahlon; An, Jiyan; Geiser, Rachel; Tegeler, Tony; Tsantilas, Kristine; Garcia, Krystine; Pirrotte, Patrick; Bowser, Robert

    2016-01-01

    The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized the protein-protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/FTLD. Using immunoprecipitation coupled to mass spectrometry (IP-MS), we identified 132 proteins that specifically interact with RBM45 within HEK293 cells. Select PPIs were validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. Analysis of the biological processes and pathways associated with RBM45-interacting proteins indicates enrichment for nuclear RNA processing/splicing via association with hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact with RBM45, consistent with prior observations of these proteins within intracellular inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, suggest novel functions for this protein, and provide new insights into the contributions of RBM45 to neurodegeneration in ALS/FTLD. PMID:26979993

  11. Denervation alters protein-lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.).

    PubMed

    Barriviera, M L; Louro, S R; Wajnberg, E; Hasson-Voloch, A

    2001-06-15

    Protein-lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.). Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na(+),K(+)-ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na(+),K(+)-ATPase activity, bands a and b. Band b is almost absent in microsomes from the denervated organ, and band a', with the same density as band a has lower Na(+),K(+)-ATPase activity. Band a' presents a larger ratio of protein-interacting lipids than band a. Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein-lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.

  12. Reference intervals for total protein concentration, serum protein fractions, and albumin/globulin ratios in clinically healthy dairy cows.

    PubMed

    Alberghina, Daniela; Giannetto, Claudia; Vazzana, Irene; Ferrantelli, Vincenzo; Piccione, Giuseppe

    2011-01-01

    The aim of the current study was to evaluate total serum protein concentration measured by the biuret reaction as well as albumin and globulin protein fractions determined by agarose gel electrophoresis. These data were used to establish reference intervals in dairy cows of different ages. Blood was collected from 111 clinically healthy Modicana dairy cows by means of jugular venipuncture. Reference intervals (mean ± standard deviation) were determined for total protein (67.54 ± 11.53 g/l), albumin (31.86 ± 4.60 g/l), α(1)-globulin (5.77 ± 2.20 g/l), α(2)-globulin (5.84 ± 1.90 g/l), β-globulin (7.46 ± 1.94 g/l), and γ-globulin (16.73 ± 4.54 g/l) concentrations as well as for albumin/globulin (A/G) ratio (0.88 ± 0.43). Values from 2-, 3-, 4-, 5-, and 6-year-old cows were compared statistically. One-way analysis of variance showed age-related differences for α-globulin and β-globulin fractions only. The results of the current study provide reference intervals for total protein concentration as well as albumin and globulin protein fractions in 2- to 6-year-old dairy cows.

  13. Characterizing Synaptic Vesicle Proteins Using Synaptosomal Fractions and Cultured Hippocampal Neurons

    PubMed Central

    DiGiovanni, Jerome; Sun, Tao; Sheng, Zu-Hang

    2012-01-01

    Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. PMID:22470148

  14. On the detection of functionally coherent groups of protein domains with an extension to protein annotation

    PubMed Central

    McLaughlin, William A; Chen, Ken; Hou, Tingjun; Wang, Wei

    2007-01-01

    Background Protein domains coordinate to perform multifaceted cellular functions, and domain combinations serve as the functional building blocks of the cell. The available methods to identify functional domain combinations are limited in their scope, e.g. to the identification of combinations falling within individual proteins or within specific regions in a translated genome. Further effort is needed to identify groups of domains that span across two or more proteins and are linked by a cooperative function. Such functional domain combinations can be useful for protein annotation. Results Using a new computational method, we have identified 114 groups of domains, referred to as domain assembly units (DASSEM units), in the proteome of budding yeast Saccharomyces cerevisiae. The units participate in many important cellular processes such as transcription regulation, translation initiation, and mRNA splicing. Within the units the domains were found to function in a cooperative manner; and each domain contributed to a different aspect of the unit's overall function. The member domains of DASSEM units were found to be significantly enriched among proteins contained in transcription modules, defined as genes sharing similar expression profiles and presumably similar functions. The observation further confirmed the functional coherence of DASSEM units. The functional linkages of units were found in both functionally characterized and uncharacterized proteins, which enabled the assessment of protein function based on domain composition. Conclusion A new computational method was developed to identify groups of domains that are linked by a common function in the proteome of Saccharomyces cerevisiae. These groups can either lie within individual proteins or span across different proteins. We propose that the functional linkages among the domains within the DASSEM units can be used as a non-homology based tool to annotate uncharacterized proteins. PMID:17937820

  15. [Influence of a moist-heat treatment on protein fractions of wheat].

    PubMed

    Korableva, N V; Kasymova, T D

    2011-01-01

    Influence of different temperature modes of a moist-heat treatment on the protein fractions of wheat, grown in Uzbekistan, has been studied within a temperature range from 40 to 80 degrees C. Using inversed phase and exclusion chromatography, we have revealed that moist-heat treatment reduces the extract content and causes some changes in the ratio between high- and low-molecular components. If the treatment temperature exceeded 60 degrees C, then, in all cases, except the glutenin fraction, the content of high-molecular components decreased, whereas the content of low-molecular components increased. The glutenin fraction was more subjected to heat influence and demonstrated a higher ability to aggregation, occurring mainly due to the component whose molecular weight was 13.42 kDa. Reduction of the number of free sulfhydryl groups in wheat gluten and its fractions in the case of a temperature increase indicates the oxidation of these groups with formation of new intermolecular disulphide bonds, which, in turn, results in the aggregation of proteins and strengthening of gluten. The obtained results agree with data of our earlier studies of gluten microstructure and fractioning during a moist-heat treatment.

  16. INDEX: Incremental depth extension approach for protein-protein interaction networks alignment.

    PubMed

    Mir, Abolfazl; Naghibzadeh, Mahmoud; Saadati, Nayyereh

    2017-08-30

    High-throughput methods have provided us with a large amount of data pertaining to protein-protein interaction networks. The alignment of these networks enables us to better understand biological systems. Given the fact that the alignment of networks is computationally intractable, it is important to introduce a more efficient and accurate algorithm which finds as large as possible similar areas among networks. This paper proposes a new algorithm named INDEX for the global alignment of protein-protein interaction networks. INDEX has multiple phases. First, it computes topological and biological scores of proteins and creates the initial alignment based on the proposed matching score strategy. Using networks topologies and aligned proteins, it then selects a set of high scoring proteins in each phase and extends new alignments around them until final alignment is obtained. Proposing a new alignment strategy, detailed consideration of matching scores, and growth of the alignment core has led INDEX to obtain a larger common connected subgraph with a much greater number of edges compared with previous methods. Regarding other measures such as edge correctness, symmetric substructure score, and runtime, the proposed algorithm performed considerably better than existing popular methods. Our results show that INDEX can be a promising method for identifying functionally conserved interactions. The INDEX executable file is available at https://github.com/a-mir/index/. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Nutrient digestibility and evaluation of protein and carbohydrate fractionation of citrus by-products.

    PubMed

    Lashkari, S; Taghizadeh, A

    2013-08-01

    The protein and carbohydrate fractionation and nutrient digestibility of citrus by-products were determined. Ruminal, intestinal and total tract CP disappearance values were measured by a modified three-step (MTSP) method and in vitro CP disappearance method (IVCP). Test feeds were orange pulp (OP), lime pulp (LP), lemon pulp (LEP), grapefruit pulp (GP), sweet lemon pulp (SLP), bitter lemon pulp (BLP), bergamot orange pulp (BP) and tangerine pulp (TP). The rumen undegradable protein (RUP) fractions of the feedstuffs were obtained by ruminal incubation in three cannulated wethers and incubation in protease solution (protease type xiv, Streptomyces griseus). The data were analysed using completely randomized design. There were significant differences between the tested feeds in protein fractions and acid detergent insoluble nitrogen (ADIN; C fraction) was highest in GP (14.56%) (p<0.001). For carbohydrate fraction, the highest C fraction was also observed in GP (2.67%) and in relation to the other citrus pulps (p<0.001). Ruminal CP disappearance was highest in OP (71.89%) (p<0.001). The level of post-ruminal CP disappearance, measured by MTSP, was highest for BP (34.94%) (p<0.001). The highest in vitro dry matter digestibility (IVDMD) was found for TP (80.44%) followed by that estimated for BP (78.38%) (p<0.001). The estimated metabolizable energy (MJ/kg DM) varied from 9.77 for LP to 12.91 for BP. Tangerine pulp had the highest true rumen digestibility (TRD) (p<0.001). According to the results, it could be concluded that citrus by-products have high nutritive value and also, the in vitro techniques can be easily used to determine of the nutritive value of citrus by-products. © 2012 Blackwell Verlag GmbH.

  18. Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

    PubMed

    Mokhtarian, Kobra; Akhlaghi, Lame; Meamar, Ahmad Reza; Razmjou, Elham; Manouchehri Naeini, Kourosh; Gholami, Samaneh; Najafi Samei, Masoomeh; Falak, Reza

    2016-08-01

    In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

  19. Proteomic Analysis of Escherichia coli Protein Fractions Resistant to Solubilization by Ionic Detergents.

    PubMed

    Antonets, K S; Volkov, K V; Maltseva, A L; Arshakian, L M; Galkin, A P; Nizhnikov, A A

    2016-01-01

    Amyloids are protein fibrils adopting structure of cross-beta spine exhibiting either pathogenic or functionally significant properties. In prokaryotes, there are several groups of functional amyloids; however, all of them were identified by specialized approaches that do not reveal all cellular amyloids. Here, using our previously developed PSIA (Proteomic Screening and Identification of Amyloids) approach, we have conducted a proteomic screening for candidates for novel amyloid-forming proteins in Escherichia coli as one of the most important model organisms and biotechnological objects. As a result, we identified 61 proteins in fractions resistant to treatment with ionic detergents. We found that a fraction of proteins bearing potentially amyloidogenic regions predicted by bioinformatics algorithms was 3-5-fold more abundant among the identified proteins compared to those observed in the entire E. coli proteome. Almost all identified proteins contained potentially amyloidogenic regions, and four of them (BcsC, MukB, YfbK, and YghJ) have asparagine- and glutamine-rich regions underlying a crucial feature of many known amyloids. In this study, we demonstrate for the first time that at the proteome level there is a correlation between experimentally demonstrated detergent-resistance of proteins and potentially amyloidogenic regions predicted by bioinformatics approaches. The data obtained enable further comprehensive characterization of entirety of amyloids (or amyloidome) in bacterial cells.

  20. Hydrophobic Fractionation Enhances Novel Protein Detection by Mass Spectrometry in Triple Negative Breast Cancer

    PubMed Central

    Lu, Ming; Whitelegge, Julian P.; Whelan, Stephen A.; He, Jianbo; Saxton, Romaine E.; Faull, Kym F.; Chang, Helena R.

    2010-01-01

    It is widely believed that discovery of specific, sensitive and reliable tumor biomarkers can improve the treatment of cancer. The goal of this study was to develop a novel fractionation protocol targeting hydrophobic proteins as possible cancer cell membrane biomarkers. Hydrophobic proteins of breast cancer tissues and cell lines were enriched by polymeric reverse phase columns. The retained proteins were eluted and digested for peptide identification by nano-liquid chromatography with tandem mass spectrometry using a hybrid linear ion-trap Orbitrap. Hundreds of proteins were identified from each of these three specimens: tumors, normal breast tissue, and breast cancer cell lines. Many of the identified proteins defined key cellular functions. Protein profiles of cancer and normal tissues from the same patient were systematically examined and compared. Stem cell markers were overexpressed in triple negative breast cancer (TNBC) compared with non-TNBC samples. Because breast cancer stem cells are known to be resistant to radiation and chemotherapy, and can be the source of metastasis frequently seen in patients with TNBC, our study may provide evidence of molecules promoting the aggressiveness of TNBC. The initial results obtained using a combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of these proteins appear promising in the discovery of potential cancer biomarkers. When sufficiently refined, this approach may prove useful for early detection and better treatment of breast cancer. PMID:20596302

  1. Improved Feed Protein Fractional Schemes for Formulating Rations With the Cornell Net Carbohydrate and Protein System

    USDA-ARS?s Scientific Manuscript database

    Adequate predictions of rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) supplies are necessary to optimize performance while minimizing losses of excess nitrogen (N). The objectives of this study were to evaluate the original Cornell Net Carbohydrate Protein System (CNCPS) protei...

  2. Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells.

    PubMed

    Tisdale, E J; Tartakoff, A M

    1988-06-15

    Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.

  3. Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension.

    PubMed Central

    McQueen-Mason, S; Cosgrove, D J

    1994-01-01

    Plant cell enlargement is controlled by the ability of the constraining cell wall to expand. This ability has been postulated to be under the control of polysaccharide hydrolases or transferases that weaken or rearrange the loadbearing polymeric networks in the wall. We recently identified a family of wall proteins, called expansins, that catalyze the extension of isolated plant cell walls. Here we report that these proteins mechanically weaken pure cellulose paper in extension assays and stress relaxation assays, without detectable cellulase activity (exo- or endo- type). Because paper derives its mechanical strength from hydrogen bonding between cellulose microfibrils, we conclude that expansins can disrupt hydrogen bonding between cellulose fibers. This conclusion is further supported by experiments in which expansin-mediated wall extension (i) was increased by 2 M urea (which should weaken hydrogen bonding between wall polymers) and (ii) was decreased by replacement of water with deuterated water, which has a stronger hydrogen bond. The temperature sensitivity of expansin-mediated wall extension suggests that units of 3 or 4 hydrogen bonds are broken by the action of expansins. In the growing cell wall, expansin action is likely to catalyze slippage between cellulose microfibrils and the polysaccharide matrix, and thereby catalyze wall stress relaxation, followed by wall surface expansion and plant cell enlargement. Images PMID:11607483

  4. Lipid Transfer Proteins Enhance Cell Wall Extension in TobaccoW⃞

    PubMed Central

    Nieuwland, Jeroen; Feron, Richard; Huisman, Bastiaan A.H.; Fasolino, Annalisa; Hilbers, Cornelis W.; Derksen, Jan; Mariani, Celestina

    2005-01-01

    Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall–loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall–loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension. PMID:15937228

  5. ACTION ON FIBROBLASTS OF THE PROTEIN FRACTION OF EMBRYONIC TISSUE EXTRACT

    PubMed Central

    Baker, Lillian E.; Carrel, Alexis

    1926-01-01

    The above experiments indicate that the growth-stimulating substance found in embryonic tissue extract, which has been responsible for the continuous growth of fibroblasts in vitro for 14 years, is either protein in nature or closely associated with the protein of the extract and adsorbed by it. If any specific hormone responsible for cell division is present, it is united to the protein or carried along with it in its first precipitation. It seems probable that the tissues utilize this protein for the nitrogen which they build into protoplasm. Whether it is first hydrolyzed before adsorption by the tissues has not been ascertained as yet. It has been shown in other experiments reported in the following paper that the amino acids of the tissue juice do not suffice for the growth of fibroblasts and that hydrolyzed tissue juice is toxic in the same way that a too concentrated mixture of amino acids is toxic. The results of the foregoing experiments may be summarized as follows: 1. Fractionation of embryo tissue juice has shown that it is the protein fraction that contains the activating substance. 2. Tissues continue to grow for a long time in the protein of the extract precipitated by CO2 and at a rate approximately equal to that in the original extract diluted to the same nitrogen concentration. 3. The non-protein nitrogen gives slight stimulation to growth. 4. Purification of the protein by repeated precipitation destroys its growth-promoting properties, but whether this is due to a denaturing of the protein,—which occurs very readily,—or to loss of some substance possibly an enzyme attached to it, has not been ascertained. 5. Preparations of purified proteins from embryonic tissue and egg white have shown no marked nutritive or stimulating action. A number of other pure substances have been tried without effect. PMID:19869191

  6. Polymerization degrees, molecular weights and protein-binding affinities of condensed tannin fractions from a Leucaena leucocephala hybrid.

    PubMed

    Saminathan, Mookiah; Tan, Hui Yin; Sieo, Chin Chin; Abdullah, Norhani; Wong, Clemente Michael Vui Ling; Abdulmalek, Emilia; Ho, Yin Wan

    2014-06-12

    Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1-F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight--from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  7. Differences of protein fractions among fresh, frozen and powdered donkey milk.

    PubMed

    Polidori, Paolo; Vincenzetti, Silvia

    2010-01-01

    Recently donkey milk has been the focus of several studies because of its special nutritional properties and composition, which is very close to human milk. When a mother cannot breastfeed, or chooses not to breastfeed, the use of a milk substitute must provide the best option to meet the nutritional and health needs of the infant. Donkey milk has been widely used in the past to replace human milk, because chemical composition and protein content are close to that of human milk, and also because the allergenicity of donkey milk is low. The recent studies of the paediatric scientists have demonstrated that infant formulae, which are based on dairy cows milk, are less adapted than donkey milk. In fact, donkey's milk digestibility is higher than cows' milk and similar to human milk, because of the high whey proteins content and the few casein content. Since donkey milk supply is related to its seasonal availability during the year, in this study were evaluated the effects of a specific technological treatment (spray-dryer) and a particular storage temperature (-20 degrees C) on the protein fractions of donkey milk. The results obtained in fresh, frozen and powdered donkey milk showed different values in total proteins, caseins, whey proteins and lysozyme content. The article presents some promising patents on protein fractions among fresh, frozen and powdered donkey milk.

  8. Fractionation and evaluation of proteins in roots of Echinacea purpurea (L.) Moench.

    PubMed

    Balciunaite, Gabriele; Juodsnukyte, Jovita; Savickas, Arunas; Ragazinskiene, Ona; Siatkute, Luka; Zvirblyte, Gitana; Mistiniene, Edita; Savickiene, Nijole

    2015-12-01

    Echinacea purpurea (L.) Moench, a member of the Asteraceae family, is a plant rich in flavonoids, essential oils, phenolic compounds, saponins, polysaccharides and glycoproteins. The aim of the study was to evaluate the protein content in dried roots of Echinacea purpurea (L.) Moench after homogenization of roots with liquid nitrogen, extraction in 0.01 mol L-1 phosphate-buffered saline (PBS) and purification followed by fractionation of proteins using gel filtration chromatography. Total concentration of proteins was measured using the Bradford method, and evaluation of the molecular mass of proteins was accomplished by applying the SDS-PAGE gel electrophoresis. The Bradford assay revealed that the highest concentration of proteins in fractions collected after gel filtration chomatography was 4.66-6.07 mg mL-1. Glycoproteins, alkamides and polysaccharides in roots of Echinacea purpurea (L.) Moench are chemical compounds that are responsible for their immunomodulatory properties. However, information about the difference of protein contents in fresh and dried roots of E. purpurea is insufficient.

  9. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  10. Effect of summer season on milk protein fractions in Holstein cows.

    PubMed

    Bernabucci, U; Basiricò, L; Morera, P; Dipasquale, D; Vitali, A; Piccioli Cappelli, F; Calamari, L

    2015-03-01

    Milk characteristics are affected by heat stress, but very little information is available on changes of milk protein fractions and their relationship with cheesemaking properties of milk. The main objective of the study was to evaluate the effect of hot season on milk protein fractions and cheesemaking properties of milk for Grana Padano cheese production. The study was carried out in a dairy farm with a cheese factory for transforming the milk to Grana Padano cheese. The study was carried out from June 2012 to May 2013. Temperature and relative humidity of the inside barn were recorded daily during the study period using 8 electronic data loggers programmed to record every 30 min. Constant managerial conditions were maintained during the experimental periods. During the experimental period, feed and diet characteristics, milk yield, and milk characteristics were recorded in summer (from June 29 to July 27, 2012), winter (from January 25 to March 8, 2013), and spring (from May 17 to May 31, 2013). Milk yield was recorded and individual milk samples were taken from 25 cows selected in each season during the p.m. milking. Content of fat, proteins, caseins (CN), lactose and somatic cell count (SCC), titratable acidity, and milk rennet coagulation properties were determined on fresh samples. Milk protein fraction concentrations were determined by the sodium dodecyl sulfate-PAGE. Data were tested for nonnormality by the Shapiro-Wilk test. In case of nonnormality, parameters were normalized by log or exponential transformation. The data were analyzed with repeated measures ANOVA using a mixed model procedure. For all the main milk components (fat, protein, total solids, and solids-not-fat), the lowest values were observed in the summer and the greatest values were observed in the winter. Casein fractions, with the exception of γ-CN, showed the lowest values in the summer and the greatest values in the winter. The content of IgG and serum albumin was greater in summer

  11. The protein fraction from wheat-based dried distiller's grain with solubles (DDGS): extraction and valorization

    PubMed Central

    Villegas-Torres, M.F.; Ward, J.M.; Lye, G.J.

    2015-01-01

    Nowadays there is worldwide interest in developing a sustainable economy where biobased chemicals are the lead actors. Various potential feedstocks are available including glycerol, rapeseed meal and municipal solid waste (MSW). For biorefinery applications the byproduct streams from distilleries and bioethanol plants, such as wheat-based dried distiller's grain with solubles (DDGS), are particularly attractive, as they do not compete for land use. Wheat DDGS is rich in polymeric sugars, proteins and oils, making it ideal as a current animal feed, but also a future substrate for the synthesis of fine and commodity chemicals. This review focuses on the extraction and valorization of the protein fraction of wheat DDGS as this has received comparatively little attention to date. Since wheat DDGS production is expected to increase greatly in the near future, as a consequence of expansion of the bioethanol industry in the UK, strategies to valorize the component fractions of DDGS are urgently needed. PMID:25644639

  12. Molecular characterization of whey protein hydrolysate fractions with ferrous chelating and enhanced iron solubility capabilities.

    PubMed

    O'Loughlin, Ian B; Kelly, Phil M; Murray, Brian A; FitzGerald, Richard J; Brodkorb, Andre

    2015-03-18

    The ferrous (Fe2+) chelating capabilities of WPI hydrolysate fractions produced via cascade membrane filtration were investigated, specifically 1 kDa permeate (P) and 30 kDa retentate (R) fractions. The 1 kDa-P possessed a Fe2+ chelating capability at 1 g L(-1) equivalent to 84.4 μM EDTA (for 30 kDa-R the value was 8.7 μM EDTA). Fourier transformed infrared (FTIR) spectroscopy was utilized to investigate the structural characteristics of hydrolysates and molecular interactions with Fe2+. Solid-phase extraction was employed to enrich for chelating activity; the most potent chelating fraction was enriched in histidine and lysine. The solubility of ferrous sulfate solutions (10 mM) over a range of pH values was significantly (P<0.05) improved in dispersions of hydrolysate fraction solutions (10 g protein L(-1)). Total iron solubility was improved by 72% in the presence of the 1 kDa-P fraction following simulated gastrointestinal digestion (SGID) compared to control FeSO4·7H2O solutions.

  13. Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

    PubMed Central

    Gibson, David S.; Newell, Keri; Evans, Alexandra N.; Finnegan, Sorcha; Manning, Gwen; Scaife, Caitriona; McAllister, Catherine; Pennington, Stephen R.; Duncan, Mark W.; Moore, Terry L.; Rooney, Madeleine E.

    2012-01-01

    Introduction. Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints. Methods. SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography. Results. A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005). Conclusion. Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients. PMID:22771520

  14. Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

    PubMed Central

    Delfosse, Kathleen; Wozny, Michael R.; Jaipargas, Erica-Ashley; Barton, Kiah A.; Anderson, Cole; Mathur, Jaideep

    2016-01-01

    Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes. PMID:26834765

  15. Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

    PubMed

    Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

    2016-12-01

    The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.

  16. Randomised controlled trial of plasma protein fraction versus dopamine in hypotensive very low birthweight infants.

    PubMed Central

    Gill, A B; Weindling, A M

    1993-01-01

    Around 20% of very low birthweight infants admitted to a neonatal intensive care unit become hypotensive within 24 hours of their admission. Standard treatment is either expansion of the circulating volume by the infusion of plasma protein fraction or by using dopamine to improve cardiac function. The purpose of this study was to investigate by a randomised controlled trial which was the most appropriate treatment. Thirty nine infants were randomised to receive either plasma protein fraction or dopamine as first line treatment if they became hypotensive within 24 hours of admission to the neonatal intensive care unit. Seventeen of 19 (89%) infants responded to dopamine, whereas only 9/20 (45%) responded to plasma protein fraction. The median dose of dopamine needed to increase the blood pressure to at least the 10th centile was 7.5 micrograms/kg/min and was infused for a median duration of 18 hours. These observations suggest that dopamine should be used earlier in the treatment of these infants than has previously been recommended. PMID:8215566

  17. Pumpkin (Cucurbita maxima) seed proteins: sequential extraction processing and fraction characterization.

    PubMed

    Rezig, Leila; Chibani, Farhat; Chouaibi, Moncef; Dalgalarrondo, Michèle; Hessini, Kamel; Guéguen, Jacques; Hamdi, Salem

    2013-08-14

    Seed proteins extracted from Tunisian pumpkin seeds ( Cucurbita maxima ) were investigated for their solubility properties and sequentially extracted according to the Osborne procedure. The solubility of pumpkin proteins from seed flour was greatly influenced by pH changes and ionic strength, with higher values in the alkaline pH regions. It also depends on the seed defatting solvent. Protein solubility was decreased by using chloroform/methanol (CM) for lipid extraction instead of pentane (P). On the basis of differential solubility fractionation and depending on the defatting method, the alkali extract (AE) was the major fraction (42.1 (P), 22.3% (CM)) compared to the salt extract (8.6 (P), 7.5% (CM)). In salt, alkali, and isopropanol extracts, all essential amino acids with the exceptions of threonine and lysine met the minimum requirements for preschool children (FAO/WHO/UNU). The denaturation temperatures were 96.6 and 93.4 °C for salt and alkali extracts, respectively. Pumpkin protein extracts with unique protein profiles and higher denaturation temperatures could impart novel characteristics when used as food ingredients.

  18. Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk.

    PubMed

    Saadaoui, Besma; Bianchi, Leonardo; Henry, Céline; Miranda, Guy; Martin, Patrice; Cebo, Christelle

    2014-05-01

    Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP-HPLC in the first dimension with SDS-PAGE in the second dimension (RP-HPLC/SDS-PAGE). Llama milk proteins, namely caseins (αs1 -, αs2 -, β-, and κ-caseins), α-lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC-ESI-MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS-identified llama milk proteins. Interestingly, α-lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α-lactalbumin protein by LC coupled with MS/MS (LC-MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α-lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk.

  19. Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

    PubMed

    Niu, Mingming; Cho, Ji-Hoon; Kodali, Kiran; Pagala, Vishwajeeth; High, Anthony A; Wang, Hong; Wu, Zhiping; Li, Yuxin; Bi, Wenjian; Zhang, Hui; Wang, Xusheng; Zou, Wei; Peng, Junmin

    2017-02-22

    Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

  20. Theory of force-extension curves for modular proteins and DNA hairpins

    NASA Astrophysics Data System (ADS)

    Bonilla, L. L.; Carpio, A.; Prados, A.

    2015-05-01

    We study a model describing the force-extension curves of modular proteins, nucleic acids, and other biomolecules made out of several single units or modules. At a mesoscopic level of description, the configuration of the system is given by the elongations of each of the units. The system free energy includes a double-well potential for each unit and an elastic nearest-neighbor interaction between them. Minimizing the free energy yields the system equilibrium properties whereas its dynamics is given by (overdamped) Langevin equations for the elongations, in which friction and noise amplitude are related by the fluctuation-dissipation theorem. Our results, both for the equilibrium and the dynamical situations, include analytical and numerical descriptions of the system force-extension curves under force or length control and agree very well with actual experiments in biomolecules. Our conclusions also apply to other physical systems comprising a number of metastable units, such as storage systems or semiconductor superlattices.

  1. The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

    PubMed

    Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

    2014-07-15

    In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. 'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.

    PubMed

    Khanna, Rajesh; Li, Qi; Stanley, Elise F

    2006-12-20

    The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG), has also been implicated in synaptic vesicle (SV) recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE) from presynaptic nerve terminals and have used a novel fractional recovery (FR) assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. The first step in FR analysis was to immunoprecipitate (IP) the complex with an antibody against one protein component (the IP-protein). The immobilized complex was then exposed to a high salt (1150 mM) stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins). A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot) was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized), and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method) at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. We hypothesize that the SAE serves as a recognition site and also as a seed complex for clathrin-mediated synaptic vesicle recovery. The combination of

  3. Comparison of Strong Cation Exchange and SDS/PAGE Fractionation for Analysis of Multi-Protein Complexes

    PubMed Central

    Das, Sudipto; Bosley, Allen D.; Ye, Xiaoying; Chan, King C.; Chu, Isabel; Green, Jeffery E.; Issaq, Haleem J.; Veenstra, Timothy D.; Andresson, Thorkell

    2013-01-01

    Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. While affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes pre-fractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Pre-fractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data shows that pre-fractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels. PMID:20968308

  4. Effect of dietary protein and iron on the fractional turnover rate of rat liver xanthine oxidase

    SciTech Connect

    Cherry, D.M.; Amy, N.K.

    1987-12-01

    Rat liver xanthine oxidase activity is regulated in response to dietary protein and iron. To investigate whether the change in activity was mediated by a change in the rate of protein degradation, we measured the fractional turnover rate using the double-isotope technique with (/sup 3/H)- and (/sup 14/C)leucine and calculated the apparent half-life of xanthine oxidase in rats fed diets containing either 20 or 5% casein with either 35 or 5 mg iron/kg diet. Under control conditions, xanthine oxidase had an apparent half-life of 4.8 d and approximately 65% of the enzyme subunits were active. Rats fed diets with low dietary protein had lower xanthine oxidase activity, but the enzyme had a slower fractional turnover rate, resulting in an apparent half-life of 6.4 d, and only 15-20% of the enzyme was active. The apparent half-life of xanthine oxidase increased to 7.5 d in rats fed diets with low dietary iron, but dietary iron did not affect the specific activity of the enzyme or the percentage of active subunits. These results suggest that the loss of enzyme activity is not due to loss of enzyme protein by increased degradation, but rather to inactivation of the enzyme.

  5. Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.

    PubMed

    Sharma, Ritin; Dill, Brian D; Chourey, Karuna; Shah, Manesh; VerBerkmoes, Nathan C; Hettich, Robert L

    2012-12-07

    The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

  6. PCE-FR: A Novel Method for Identifying Overlapping Protein Complexes in Weighted Protein-Protein Interaction Networks Using Pseudo-Clique Extension Based on Fuzzy Relation.

    PubMed

    Cao, Buwen; Luo, Jiawei; Liang, Cheng; Wang, Shulin; Ding, Pingjian

    2016-10-01

    Identifying overlapping protein complexes in protein-protein interaction (PPI) networks can provide insight into cellular functional organization and thus elucidate underlying cellular mechanisms. Recently, various algorithms for protein complexes detection have been developed for PPI networks. However, majority of algorithms primarily depend on network topological feature and/or gene expression profile, failing to consider the inherent biological meanings between protein pairs. In this paper, we propose a novel method to detect protein complexes using pseudo-clique extension based on fuzzy relation (PCE-FR). Our algorithm operates in three stages: it first forms the nonoverlapping protein substructure based on fuzzy relation and then expands each substructure by adding neighbor proteins to maximize the cohesive score. Finally, highly overlapped candidate protein complexes are merged to form the final protein complex set. Particularly, our algorithm employs the biological significance hidden in protein pairs to construct edge weight for protein interaction networks. The experiment results show that our method can not only outperform classical algorithms such as CFinder, ClusterONE, CMC, RRW, HC-PIN, and ProRank +, but also achieve ideal overall performance in most of the yeast PPI datasets in terms of composite score consisting of precision, accuracy, and separation. We further apply our method to a human PPI network from the HPRD dataset and demonstrate it is very effective in detecting protein complexes compared to other algorithms.

  7. Preparation and antioxidative properties of a rapeseed ( Brassica napus ) protein hydrolysate and three peptide fractions.

    PubMed

    Xue, Zhaohui; Yu, Wancong; Liu, Zhiwei; Wu, Moucheng; Kou, Xiaohong; Wang, Jiehua

    2009-06-24

    This study investigated the possibility of converting the insoluble rapeseed meal protein into functionally active ingredients for food applications. The rapeseed ( Brassica napus ) meal protein isolates were first digested by Alcalase and Flavourzyme, and the resultant rapeseed crude hydrolysate (RSCH) exhibited a dose-dependent reducing antioxidant power and hydroxyl radical scavenging ability. RSCH could also inhibit the malonyldialdehyde (MDA) generation by 50% in blood serum at 150 mg/mL. RSCH was further separated into three fractions (RSP1, RSP2, and RSP3) by Sephadex gel filtration according to their different molecular weights. The amino acid compositions and antioxidant potentials were assessed for RSP1-3 fractions. All three fractions showed inhibiting effects on superoxide anion generation to various extents. They could also inhibit the autohemolysis of rat red blood cells and MDA formation in rat liver tissue homogenate. The results suggested that rapeseed peptide hydrolysate may be useful as a human food addition as a source of bioactive peptides with antioxidant properties.

  8. Nutrient-Dependent Requirement for SOD1 in Lifespan Extension by Protein Restriction in Drosophila melanogaster

    PubMed Central

    Sun, Xiaoping; Komatsu, Toshimitsu; Lim, Jinhwan; Laslo, Mara; Yolitz, Jason; Wang, Cecilia; Poirier, Luc; Alberico, Thomas; Zou, Sige

    2012-01-01

    Summary Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target-of-rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP and low calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high, and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions. PMID:22672579

  9. Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

    PubMed

    Gnegy, M E; Nathanson, J A; Uzunov, P

    1977-03-29

    The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.

  10. Enzymatic hydrolysis of heated whey: iron-binding ability of peptides and antigenic protein fractions.

    PubMed

    Kim, S B; Seo, I S; Khan, M A; Ki, K S; Lee, W S; Lee, H J; Shin, H S; Kim, H S

    2007-09-01

    This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100 degrees C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50 degrees C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (beta-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of beta-lactoglobulin, alpha-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4 x 7 H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of

  11. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    PubMed

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

  12. Fractionation and Structural Characterization of Arabinogalactan-Proteins from the Cell Wall of Rose Cells.

    PubMed Central

    Serpe, M. D.; Nothnagel, E. A.

    1995-01-01

    Arabinogalactan-proteins (AGPs) have been purified from Paul's Scarlet rose (Rosa sp.) cell walls. As estimated by gel permeation chromatography, the apparent molecular masses of the two major cell-wall AGP fractions were 130 and 242 kD. Since the 130-kD AGP had a ratio of arabinose/glucuronic acid that was 12 times higher than that of the 242-kD AGP, the fractions were named cell-wall AGP1 (CW-AGP1) and glucuronogalactan-protein (GGP), respectively. CW-AGP1 and GGP contained predominantly t-arabinofuranosyl residues; 3-linked, 6-linked, and 3,6-branched galactopyranosyl residues; and 4-linked and t-glucuronopyranosyl residues. The 1H-nuclear magnetic resonance spectra of CW-AGP1 and GGP showed that the arabinofuranosyl and galactopyranosyl residues were predominantly in [alpha]- and [beta]-anomeric configuration, respectively, and that GGP contained a few O-acetyl residues. The protein moieties of CW-AGP1 and GGP were both rich in hydroxyproline and alanine but differed in the percentage of various amino acids, including hydroxyproline, alanine, serine, and glycine. Cell-wall AGPs bound to ([beta]-D-glucosyl)3 Yariv phenylglycoside, but the stoichiometry of binding was about 6 times greater in GGP than in other Rosa AGPs. GGP seems to be peculiar to the cell wall, since no similar molecule was found in the culture medium. PMID:12228648

  13. Quantity and functionality of protein fractions in chicken breast fillets affected by white striping.

    PubMed

    Mudalal, S; Babini, E; Cavani, C; Petracci, M

    2014-08-01

    Recently, white striations parallel to muscle fibers direction have been observed on the surface of chicken breast, which could be ascribed to intensive growth selection. The aim of this study was to evaluate the effect of white striping on chemical composition with special emphasis on myofibrillar and sarcoplasmic protein fractions that are relevant to the processing features of chicken breast meat. During this study, a total of 12 pectoralis major muscles from both normal and white striped fillets were used to evaluate chemical composition, protein solubility (sarcoplasmic, myofibrillar, and total protein solubility), protein quantity (sarcoplasmic, myofibrillar, and stromal proteins), water holding capacity, and protein profile by SDS-PAGE analysis. White-striped fillets exhibited a higher percentage of moisture (75.4 vs. 73.8%; P < 0.01), intramuscular fat (2.15 vs. 0.98%; P < 0.01), and collagen (1.36 vs. 1.22%; P < 0.01), and lower content of protein (18.7 vs. 22.8%; P < 0.01) and ash (1.14 vs. 1.34%; P < 0.01), in comparison with normal fillets. There was a great decline in myofibrillar (14.0 vs. 8.7%; P < 0.01) and sarcoplasmic (3.2 vs. 2.6%; P < 0.01) content and solubility as well as an increase in cooking loss (33.7 vs. 27.4%; P < 0.05) due to white striping defects. Moreover, gel electrophoresis showed that the concentration of 3 myofibrillar proteins corresponding to actin (42 kDa); LC1, slow-twitch light chain myosin (27.5 kDa); and LC3, fast-twitch light chain myosin (16 kDa), and almost all sarcoplasmic proteins were lower than normal. In conclusion, the findings of this study revealed that chicken breast meat with white striping defect had different chemical composition (more fat and less protein) and protein quality and quantity (low content of myofibrillar proteins and high content of stromal proteins) with respect to normal meat. Furthermore, white striped fillets had lower protein functionality (higher cooking loss). All the former changes

  14. Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra.

    PubMed

    Liebner, Robert; Mathaes, Roman; Meyer, Martin; Hey, Thomas; Winter, Gerhard; Besheer, Ahmed

    2014-07-01

    Half-life extension (HLE) is becoming an essential component of the industrial development of small-sized therapeutic peptides and proteins. HESylation(®) is a HLE technology based on coupling drug molecules to the biodegradable hydroxyethyl starch (HES). In this study, we report on the synthesis, characterization and pharmacokinetics of HESylated anakinra, where anakinra was conjugated to propionaldehyde-HES using reductive amination, leading to a monoHESylated protein. Characterization using size exclusion chromatography and dynamic light scattering confirmed conjugation and the increase in molecular size, while Fourier transform infrared spectroscopy showed that the secondary structure of the conjugate was not affected by coupling. Meanwhile, microcalorimetry and aggregation studies showed a significant increase in protein stability. Surface plasmon resonance and microscale thermophoresis showed that the conjugate retained its nanomolar affinity, and finally, the pharmacokinetics of the HESylated protein exhibited a 6.5-fold increase in the half-life, and a 45-fold increase in the AUC. These results indicate that HESylation(®) is a promising HLE technology.

  15. Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate.

    PubMed

    Di Bernardini, Roberta; Rai, Dilip K; Bolton, Declan; Kerry, Joseph; O'Neill, Eileen; Mullen, Anne Maria; Harnedy, Pádraigín; Hayes, Maria

    2011-02-01

    Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37°C for 2h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a(w)) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe(2+) chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers.

    PubMed

    Heebøll-Nielsen, Anders; Justesen, Sune F L; Thomas, Owen R T

    2004-09-30

    In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.042 microM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (< or = 0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4-1 M. The anion-exchanger adsorbed beta-lactoglobulin (beta-LG) selectively allowing separation from the remaining protein.

  17. Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

    PubMed Central

    Huang, Da Wei; Fanti, Laura; Pak, Daniel T.S.; Botchan, Michael R.; Pimpinelli, Sergio; Kellum, Rebecca

    1998-01-01

    The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae. PMID:9679132

  18. Coupling detergent lysis/clean-up methodology with intact protein fractionation for enhanced proteome characterization

    SciTech Connect

    Sharma, Ritin; Dill, Brian; Chourey, Karuna; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L

    2012-01-01

    The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four different detergent clean-up methods (Trichloroacetic acid (TCA) precipitation, Chloroform/Methanol/Water (CMW) extraction, commercial detergent removal spin column method (DRS) and filter-aided sample preparation(FASP)) with respect to varying amounts of protein biomass in the samples, and provide efficiency benchmarks with respect to protein, peptide, and spectral identifications for each method. Our results show that for protein limited samples, FASP outperforms the other three clean-up methods, while at high protein amount all the methods are comparable. This information was used in a dual strategy of comparing molecular weight based fractionated and unfractionated lysates from three increasingly complex samples (Escherichia coli, a five microbial isolate mixture, and a natural microbial community groundwater sample), which were all lysed with SDS and cleaned up using FASP. The two approaches complemented each other by enhancing the number of protein identifications by 8%-25% across the three samples and provided broad pathway coverage.

  19. Fractional synthesis rates of DNA and protein in rabbit skin are not correlated.

    PubMed

    Zhang, Xiao-jun; Chinkes, David L; Wu, Zhanpin; Martini, Wenjun Z; Wolfe, Robert R

    2004-09-01

    We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.

  20. Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk.

    PubMed

    Barbiroli, A; Bonomi, F; Benedetti, S; Mannino, S; Monti, L; Cattaneo, T; Iametti, S

    2007-02-01

    The affinity of aflatoxin M1 toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.

  1. Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems.

    PubMed

    Zimmerman, G L; Clark, C R

    1986-03-01

    Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4-6 h at 37 degrees C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot-ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.

  2. Cellular prion protein in mammary gland and milk fractions of domestic ruminants.

    PubMed

    Didier, A; Gebert, R; Dietrich, R; Schweiger, M; Gareis, M; Märtlbauer, E; Amselgruber, W M

    2008-05-09

    The present study shows that PrP(c) is expressed in the mammary gland and milk fractions of domestic ruminants in a species-specific manner. By applying immunohistochemistry, Western blot and ELISA, clear expression differences between bovine, ovine and caprine mammary gland, skimmed milk, acid whey and cream could be demonstrated, the highest relative PrP(c) levels being associated with the cream fraction. In the bovine gland PrP(c) was preferentially detectable at the basolateral surface of mammary gland epithelial cells, whereas in ovine and caprine samples the prion protein was more homogeneously distributed. Moreover, in ovine and caprine bovine mammary gland epithelial cells, apocrine secretory vesicles were strongly stained. Ovine and caprine milk proved to contain PrP(c) in all fractions with an additional truncated form at 12kDa in Western blot. This truncated isoform is the predominate one in caprine acid whey. These results support the hypothesis that the apocrine secretion mode of milk fat globules is a major way of PrP(c) transport into the milk.

  3. Physicochemical characterization of a navy bean (Phaseolus vulgaris) protein fraction produced using a solvent-free method.

    PubMed

    Jafari, Mousa; Rajabzadeh, Amin Reza; Tabtabaei, Solmaz; Marsolais, Frédéric; Legge, Raymond L

    2016-10-01

    A solvent-free electrostatic separation method was employed to separate navy bean flour (NBF) into protein-rich (PR) and starch-rich (SR) fractions. The physicochemical properties of NBF and separated fractions were compared to proteins (navy bean isolate (NBI) and 7S globulin) prepared using a wet process. Gel electrophoresis confirmed that the protein distribution in the isolated fractions was similar to that of NBF. The protein profile of NBI and 7S globulin was found to be devoid of certain proteins that were found in the NBF and PR fraction. Amino acid analysis revealed that the NBI and 7S globulin had a lower content of sulfur-containing amino acids compared to NBF and the electrostatically isolated fractions. CD and fluorescence spectroscopy confirmed that denaturation of the proteins during the acid precipitation is likely. This novel solvent-free electrostatic separation process preserves the native protein structure found in NBF and improves the recovery of some of the smaller MW proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

    PubMed

    Ashby, Jonathan; Schachermeyer, Samantha; Duan, Yaokai; Jimenez, Luis A; Zhong, Wenwan

    2014-09-05

    Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes.

    PubMed

    Cornman, R Scott; Willis, Judith H

    2008-06-01

    Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

  6. Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.

    PubMed Central

    Sottrup-Jensen, L; Folkersen, J; Kristensen, T; Tack, B F

    1984-01-01

    Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier. PMID:6209714

  7. A Compendium of Caenorhabditis elegans RNA Binding Proteins Predicts Extensive Regulation at Multiple Levels

    PubMed Central

    Tamburino, Alex M.; Ryder, Sean P.; Walhout, Albertha J. M.

    2013-01-01

    Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs. PMID:23390605

  8. In vitro antioxidant properties of chicken skin enzymatic protein hydrolysates and membrane fractions.

    PubMed

    Onuh, John O; Girgih, Abraham T; Aluko, Rotimi E; Aliani, Michel

    2014-05-01

    Chicken thigh and breast skin proteins were hydrolysed using alcalase or a combination of pepsin and pancreatin (PP), each at concentrations of 1-4%. The chicken skin protein hydrolysates (CSPHs) were then fractionated by membrane ultrafiltration into different molecular weight peptides (<1, 1-3, 3-5 and 5-10kDa) and analysed for antioxidant properties. Results showed that the CSPHs had a significantly (p<0.05) lower scavenging activity against DPPH radicals when compared to reduced glutathione. The chicken breast skin hydrolysates had significantly higher DPPH scavenging activity than the chicken thigh skin hydrolysates. DPPH scavenging and metal ion chelation increased significantly (p<0.05) from 29-40% to 86-89%, respectively with increasing proteolytic enzyme concentration. In contrast, the antioxidant properties decreased as peptide size increased. We conclude that CSPHs and their peptide fractions may be used as ingredients in the formulation of functional foods and nutraceuticals for the control and management of oxidative stress-related diseases.

  9. The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein.

    PubMed Central

    Roth, G J; Majerus, P W

    1975-01-01

    Aspirin (acetylsalicylic acid) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction. The mechanism of the inhibitory effect is unknown but may involve protein acetylation, since aspirin acetylates a variety of substrates, including platelet protein. We have examined the relationship between protein acetylation and aspirin's physiologic effect on platelets. Suspensions of washed human platelets were incubated at 37 degrees C with (3H)aspirin, and incorporation of radioactivity into protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Exposure to (acetyl-3H)aspirin but not (aromatic ring-3H)aspirin resulted in radioactive labeling of three platelet proteins, suggesting that the drug acetylates these three proteins. The acetylation of two of the proteins (located in the supernatant fraction) was not saturable, implying that these reactions may not be physiologically significant. Acetylation of the third protein, approximate mol wt 85,000 (located in the particulate fraction), saturated at an aspirin concentration of 30 muM and was complete within 20 min. Platelets prepared from aspirin-treated donors did not incorporate any (acetyl-3H)aspirin radioactivity into the particulate protein for 2 days after drug treatment and did not show full pretreatment uptake of radioactivity for 12 days thereafter. The course of increasing incorporation of (acetyl-3H)aspirin radioactivity parralleled that of platelet turnover. Therefore, in addition to its saturability, acetylation of the particulate fraction protein by aspirin was permanent. In two respects, the inhibition of platelet function by aspirin correlates well with the aspirin-mediated acetylation of the particulate fraction protein. Both persist for the life-span of the aspirin-treated platelet, and both occur at a similar saturating aspirin concentration. The evidence suggests that the physiologic effect of aspirin on human platelets is produced

  10. Isolation and characterization of oil palm constitutive promoter derived from ubiquitin extension protein (uep1) gene.

    PubMed

    Masura, Subhi Siti; Parveez, Ghulam Kadir Ahmad; Ismail, Ismanizan

    2010-09-30

    The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm. We have isolated and characterized the 5' region of the oil palm uep1 gene, which contains an 828 bp sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector, which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional analysis of the promoter through transient expression studies. It was found that the 5' region of uep1 functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested, including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf, mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was demonstrated to be capable of driving gusA gene expression in tobacco.

  11. Determining the location of an Arabidopsis chloroplast protein using in vitro import followed by fractionation and alkaline extraction.

    PubMed

    Chu, Chiung-Chih; Li, Hsou-Min

    2011-01-01

    Chloroplasts have one of the most complicated structures among organelles. They have three membrane systems, the outer and inner envelope membranes and the thylakoid membrane, which enclose three aqueous spaces: the intermembrane space between the two envelope membranes, the stroma, and the thylakoid lumen. Each of the chloroplast's sub-organellar compartments houses a distinct set of proteins that perform distinct functions. Determining the sub-organellar location of a protein in the chloroplast is vital for understanding or verifying the function of the protein. Here, we present protocols for determining the sub-organellar location of a chloroplast protein. The protein of interest is synthesized and labeled with [(35)S]methionine by an in vitro translation system, and imported into isolated chloroplasts. The location of the protein is then identified by fractionation of the chloroplasts through differential and sucrose step-gradient centrifugations. The various sub-chloroplast fractions are analyzed by SDS-PAGE and autoradiography, so no specific antibody against the protein of interest is required. For membrane proteins, an alkaline extraction protocol is provided to further determine whether the protein is a peripheral or an integral membrane protein. The fractionation and extraction procedures presented can also be used in conjunction with immunoblotting, if an antibody against the protein of interest is available, enabling analyses of endogenous proteins.

  12. Evaluation of protein fractionation and ruminal and intestinal digestibility of corn milling co-products.

    PubMed

    Kelzer, J M; Kononoff, P J; Tedeschi, L O; Jenkins, T C; Karges, K; Gibson, M L

    2010-06-01

    Novel corn milling co-products developed from technological advancements in ethanol production vary widely in chemical composition and nutrient availability. The objectives of this study were to characterize feed protein fractions and evaluate differences in rumen-undegradable protein (RUP) and its digestible fraction (dRUP), amino acid concentration, and in vitro gas production of 7 corn milling co-products. The crude protein (CP; % of dry matter) of co-products was 12.7 for germ, 26.9 for dried distillers grains plus solubles that had no heat exposure before fermentation (DDGS1), 45.4 for high-protein dried distillers grains (HPDDG), 12.7 for bran, 30.2 for wet distillers grains plus solubles (WDGS), 23.1 for wet corn gluten feed (WCGF), and 26.0 for dried distillers grains plus solubles that had heat exposure before fermentation (DDGS2). Two ruminally and duodenally fistulated Holstein steers weighing 663+/-24 kg were used to determine RUP and dRUP with the in situ and mobile bag techniques. Samples of each feed were ruminally incubated for 16 h, and mobile bags were exposed to simulated abomasal digestion before insertion into the duodenum and subsequent collection in the feces. Protein fractions A, B(1), B(2), B(3), and C were characterized as follows (% CP): germ=30.0, 15.0, 38.1, 13.5, 3.4; DDGS1=17.0, 7.0, 67.0, 4.8, 4.2; HPDDG=7.4, 0.6, 82.4, 8.8, 0.8; bran=33.5, 4.0, 54.3, 6.0, 2.2; WDGS=18.6, 2.4, 53.1, 11.0, 14.9; WCGF=36.6, 15.9, 33.2, 10.1, 4.1; and DDGS2=17.9, 2.1, 41.1, 11.1, 27.9. The proportions of RUP and dRUP were different and are reported as follows (% CP): DDGS2=56.3, 91.9; HPDDG=55.2, 97.7; WDGS=44.7, 93.1; DDGS1=33.2, 92.1; bran=20.7, 65.8; germ=16.5, 66.8; and WCGF=11.5, 51.1. The concentrations of Lys and Met in the RUP were different and are listed as follows (% CP): germ=2.9, 2.0; DDGS1=1.9, 2.0; HPDDG=2.0, 3.2; bran=3.2, 1.5; WDGS=1.9, 2.3; WCGF=3.5, 1.6; and DDGS2=1.9, 2.4. In vitro gas production (mL/48h) was highest for germ (52

  13. Milk from different species: Relationship between protein fractions and inflammatory response in infants affected by generalized epilepsy.

    PubMed

    Albenzio, M; Santillo, A; Ciliberti, M G; Figliola, L; Caroprese, M; Marino, R; Polito, A N

    2016-07-01

    The present study was undertaken to evaluate the effect of protein fractions from bovine, caprine, and ovine milk on production of cytokines and reactive oxygen species (ROS) and reactive nitrogen species (RNS) by cultured peripheral blood mononuclear cells (PMBC) from infants with generalized epilepsy. Bovine, caprine, and ovine bulk milks were pasteurized and analyzed for chemical composition. Then, PBMC were isolated from 10 patients with generalized epilepsy (5 males; mean age 33.6±5.4mo). Production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC (from infants with epilepsy and controls) stimulated by bovine, caprine, and ovine milk and casein and whey protein fractions, and levels of ROS and RNS were measured in the culture supernatant. The ability of PBMC to secrete cytokines in response to milk and protein fraction stimulation may predict the secretion of soluble factor TNF-α in the bloodstream of challenged patients. Bovine, caprine, and ovine bulk milks induced low-level production of IL-10 by cultured PBMC in at least 50% of cases; the same behavior was observed in both casein and whey protein fractions for all species studied. Bovine and ovine milk and their casein fractions induced production of lower levels of IL-1β in 80% of patients, whereas caprine milk and its casein fraction induced the highest levels in 80% of patients. The amount of IL-6 detected after stimulation of PBMC by milk and its fractions for all species was lower than that of other proinflammatory cytokines. In the bovine, total free radicals were higher in bulk milk and lower in the casein fraction, whereas the whey protein fraction showed an intermediate level; in caprine, ROS/RNS levels were not different among milk fractions, whereas ovine had higher levels for bulk milk and casein than the whey protein fraction. Lower levels of ROS/RNS detected in PBMC cultured with caprine milk fraction could be responsible for the lower levels of

  14. A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction.

    PubMed

    Gabig, T G; Eklund, E A; Potter, G B; Dykes, J R

    1990-08-01

    The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.

  15. A structure based plasma protein pre-fractionation using conjoint immobilized metal/chelate affinity (IMA) system.

    PubMed

    Karkra, Karan; Tetala, Kishore K R; Vijayalakshmi, M A

    2017-05-01

    The potential of immobilized metal/chelate affinity (IMA) in a continuous fashion, referred as conjoint approach, to pre-fractionate plasma proteins (in their native state) prior to LC-MS analysis was investigated in this study. Four transition metal-ions (Co (II), Zn (II), Ni (II) and Cu (II)) were individually chelated with IDA (iminodiacetic acid) coated CIM (Convective Interaction Media) disks and placed in a single housing in the following sequential order: IDA-Co (II)→IDA-Zn (II)→IDA-Ni (II)→IDA-Cu (II). The rationale behind this order is to retain proteins based on their specific requirement for surface exposed histidine topography. This structural pre-fractionation hypothesis was successfully proven using four human plasma proteins (fibrinogen, IgG, transferrin, and albumin) with varying histidine topographies. This conjoint IMA pre-fractionation strategy not only fractionated proteins (from plasma) based on their native surface histidine topography, but also identified 157 proteins from human plasma. The advantage of our conjoint IMA is its ability to fractionate proteins in their native state and reduce plasma complexity in a single step by employing single buffer system. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.

    PubMed

    Choi, Megan; Nordmeyer, Robert A; Cornell, Earl; Dong, Ming; Biggin, Mark D; Jin, Jian

    2010-01-01

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.

  17. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    SciTech Connect

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  18. Identification of β-Catenin-Interacting Proteins in Nuclear Fractions of Native Rat Collecting Duct Cells.

    PubMed

    Hwang, Jacqueline R; Chou, Chung-Lin; Medvar, Barbara; Knepper, Mark A; Jung, Hyun Jun

    2017-03-15

    The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that acts as a transcriptional co-regulator in the nucleus. The purpose of this study was to identify β-catenin interacting proteins that may be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells using both experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin binding proteins, including several known β-catenin binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj and several histones). Many of the identified β-catenin binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II pre-initiation complex. To identify sequence-specific TFs that may interact with β-catenin, Bayes' Theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1 and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.

  19. Simvastatin Ameliorates Radiation Enteropathy Development After Localized, Fractionated Irradiation by a Protein C-Independent Mechanism

    SciTech Connect

    Wang Junru; Boerma, Marjan; Fu Qiang; Kulkarni, Ashwini; Fink, Louis M.; Hauer-Jensen, Martin . E-mail: mhjensen@life.uams.edu

    2007-08-01

    Purpose: Microvascular injury plays a key role in normal tissue radiation responses. Statins, in addition to their lipid-lowering effects, have vasculoprotective properties that may counteract some effects of radiation on normal tissues. We examined whether administration of simvastatin ameliorates intestinal radiation injury, and whether the effect depends on protein C activation. Methods and Materials: Rats received localized, fractionated small bowel irradiation. The animals were fed either regular chow or chow containing simvastatin from 2 weeks before irradiation until termination of the experiment. Groups of rats were euthanized at 2 weeks and 26 weeks for assessment of early and delayed radiation injury by quantitative histology, morphometry, and quantitative immunohistochemistry. Dependency on protein C activation was examined in thrombomodulin (TM) mutant mice with deficient ability to activate protein C. Results: Simvastatin administration was associated with lower radiation injury scores (p < 0.0001), improved mucosal preservation (p = 0.0009), and reduced thickening of the intestinal wall and subserosa (p = 0.008 and p = 0.004), neutrophil infiltration (p = 0.04), and accumulation of collagen I (p = 0.0003). The effect of simvastatin was consistently more pronounced for delayed than for early injury. Surprisingly, simvastatin reduced intestinal radiation injury in TM mutant mice, indicating that the enteroprotective effect of simvastatin after localized irradiation is unrelated to protein C activation. Conclusions: Simvastatin ameliorates the intestinal radiation response. The radioprotective effect of simvastatin after localized small bowel irradiation does not appear to be related to protein C activation. Statins should undergo clinical testing as a strategy to minimize side effects of radiation on the intestine and other normal tissues.

  20. Simvastatin ameliorates radiation enteropathy development after localized, fractionated irradiation by a protein C-independent mechanism.

    PubMed

    Wang, Junru; Boerma, Marjan; Fu, Qiang; Kulkarni, Ashwini; Fink, Louis M; Hauer-Jensen, Martin

    2007-08-01

    Microvascular injury plays a key role in normal tissue radiation responses. Statins, in addition to their lipid-lowering effects, have vasculoprotective properties that may counteract some effects of radiation on normal tissues. We examined whether administration of simvastatin ameliorates intestinal radiation injury, and whether the effect depends on protein C activation. Rats received localized, fractionated small bowel irradiation. The animals were fed either regular chow or chow containing simvastatin from 2 weeks before irradiation until termination of the experiment. Groups of rats were euthanized at 2 weeks and 26 weeks for assessment of early and delayed radiation injury by quantitative histology, morphometry, and quantitative immunohistochemistry. Dependency on protein C activation was examined in thrombomodulin (TM) mutant mice with deficient ability to activate protein C. Simvastatin administration was associated with lower radiation injury scores (p < 0.0001), improved mucosal preservation (p = 0.0009), and reduced thickening of the intestinal wall and subserosa (p = 0.008 and p = 0.004), neutrophil infiltration (p = 0.04), and accumulation of collagen I (p = 0.0003). The effect of simvastatin was consistently more pronounced for delayed than for early injury. Surprisingly, simvastatin reduced intestinal radiation injury in TM mutant mice, indicating that the enteroprotective effect of simvastatin after localized irradiation is unrelated to protein C activation. Simvastatin ameliorates the intestinal radiation response. The radioprotective effect of simvastatin after localized small bowel irradiation does not appear to be related to protein C activation. Statins should undergo clinical testing as a strategy to minimize side effects of radiation on the intestine and other normal tissues.

  1. Fractionation and antioxidant properties of rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion.

    PubMed

    Phongthai, Suphat; D'Amico, Stefano; Schoenlechner, Regine; Homthawornchoo, Wantida; Rawdkuen, Saroat

    2018-02-01

    Rice bran was used as a starting material to prepare protein concentrate through enzyme-assisted extraction. The hydrolysis of protein concentrate under in vitro gastrointestinal digestion (pepsin-trypsin system) greatly improved the antioxidant properties. Rice bran protein hydrolysate was further fractionated by membrane ultrafiltration (UF, F1: molecular weight (MW) <3kDa, F2: MW 3-5kDa, and F3: MW 5-10kDa). Peptides with smaller MW possessed higher antioxidant activities (P<0.05). UF showed a great efficacy to selectively separate the metal-chelating peptides. Tyrosine and phenylalanine had positive correlations with their DPPH & ABTS radicals scavenging activities and ferric reducing antioxidant power (r>0.831). A major peptide fragment was detected at m/z 1088 by a MALDI-TOF mass spectrometry. There is high potential that antioxidative peptides from rice bran might also be produced in the gastrointestinal tract of the human body. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Predicting beta-turns in proteins using support vector machines with fractional polynomials

    PubMed Central

    2013-01-01

    Background β-turns are secondary structure type that have essential role in molecular recognition, protein folding, and stability. They are found to be the most common type of non-repetitive structures since 25% of amino acids in protein structures are situated on them. Their prediction is considered to be one of the crucial problems in bioinformatics and molecular biology, which can provide valuable insights and inputs for the fold recognition and drug design. Results We propose an approach that combines support vector machines (SVMs) and logistic regression (LR) in a hybrid prediction method, which we call (H-SVM-LR) to predict β-turns in proteins. Fractional polynomials are used for LR modeling. We utilize position specific scoring matrices (PSSMs) and predicted secondary structure (PSS) as features. Our simulation studies show that H-SVM-LR achieves Qtotal of 82.87%, 82.84%, and 82.32% on the BT426, BT547, and BT823 datasets respectively. These values are the highest among other β-turns prediction methods that are based on PSSMs and secondary structure information. H-SVM-LR also achieves favorable performance in predicting β-turns as measured by the Matthew's correlation coefficient (MCC) on these datasets. Furthermore, H-SVM-LR shows good performance when considering shape strings as additional features. Conclusions In this paper, we present a comprehensive approach for β-turns prediction. Experiments show that our proposed approach achieves better performance compared to other competing prediction methods. PMID:24565438

  3. The effect of pH and gas composition on the bubble fractionation of proteins

    SciTech Connect

    DeSouza, A.H.G.; Tanner, R.D.; Effler, W.T. Jr.

    1991-12-31

    Studies were conducted to establish the effect of the variation of environmental factors on the separation occurring in protein systems, resulting from bubble fractionation in a bioreactor. The measure of separation was selected to be the separation ratio. This is defined to be the ratio of either the top or the middle position concentration in the vessel to the bottom concentration of the vessel. Invertase and Ce-amylase were the two {open_quotes}model{close_quotes} enzymes considered. It was observed that, under certain conditions, i.e., a combination of the nature of the sparging gas and the medium pH, varying degrees of protein separation were achieved. The pH of the system dramatically influenced the separation. It was found that the best separation occurred at a certain pH, assumed to be at or close to the pI of the protein in question. Furthermore, it was observed that systems sparged with CO{sub 2} exhibited greater separation than systems sparged with air. In fact, in the case of invertase, almost threefold separation was observed at the top port when the solution was sparged with CO{sub 2}.

  4. Effects of cow milk versus extensive protein hydrolysate formulas on infant cognitive development

    PubMed Central

    Mennella, Julie A.; Trabulsi, Jillian C.; Papas, Mia A.

    2015-01-01

    Little research has focused on infant developmental effects, other than growth, of formulas that differ substantially in the form of protein. To examine development of infants fed formulas differing in free amino acid content, we randomized 0.5-month-old infants (n=79) to either a control group who fed only cow milk formula (CMF) during the first 8 months (CMF8), or to one of two experimental groups: one experimental group fed extensively protein hydrolyzed formula (EHF) for 1–3 months during first 4.5 months (EHF1-3) of life, and the other fed EHF for 8 months (EHF8). The Mullen Scales of Early Learning were administered monthly from 1.5 to 8.5 months to assess fine (FM) and gross (GM) motor control, receptive (RL) and expressive (EL) language, visual reception (VR), and an early learning composite (ELC). Across the 5.5–8.5 month time period, when compared to CMF8 infants, GM scores in EHF1-3 infants averaged 1.5 points higher (95% CI: 0.1, 3.0) and in EHF8 infants 2.2 points higher (95% CI: 0.3, 4.0). Similarly, VR scores averaged 1.9 points higher (95% CI: 0.1, 3.8) in EHF1-3 infants and 2.2 points higher (95% CI: −0.2, 4.5) in EHF8 infants. EHF8 infants RL scores averaged 1.8 points lower (95% CI: 0.1, 3.6) than CMF8 infants. These data suggest that the form of protein in infant formula may impact cognitive development and that the higher free amino acid content in breast milk may be a contributing factor to the differential cognitive development between breastfed and CMF-fed infants. Clinical Trial Registration: clinicaltrials.gov NCT00994747. PMID:26497857

  5. Serum proteins and their fractions in the Timahdite sheep in Morocco: variations with age and with liver or lung diseases.

    PubMed

    Kessabi, M; Lamnaouer, D

    1981-01-01

    The serum proteins and their fractions were analyzed in the Moroccan "Timahdite" sheep. The values of those parameters in 20 two year-old adult animals were: total proteins 64.2 +/- 1.6 g/l; albumin 458 +/- 31 mumol/l; alpha-glob 11.9 +/- 1.0 g/l; beta-glob 6.2 +/- 1.6 g/l; gamma-glob 14.7 +/- 2.3 g/l. The total proteins increase with age is particularly due to an increase of the globulin fractions. In animals with liver or pulmonary affections the total proteins are increased. The amount of albumin is lower in liver diseases, whereas the globulin fractions are increased in both pathological processes.

  6. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    SciTech Connect

    Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan; Lee, Hyejin; An, Sungkwan; Park, Myung-Jin; Kim, Min-Jung; Lee, Su-Jae

    2010-11-26

    Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and

  7. Effect of Peptide Size on Antioxidant Properties of African Yam Bean Seed (Sphenostylis stenocarpa) Protein Hydrolysate Fractions

    PubMed Central

    Ajibola, Comfort F.; Fashakin, Joseph B.; Fagbemi, Tayo N.; Aluko, Rotimi E.

    2011-01-01

    Enzymatic hydrolysate of African yam bean seed protein isolate was prepared by treatment with alcalase. The hydrolysate was further fractionated into peptide sizes of <1, 1–3, 3–5 and 5–10 kDa using membrane ultrafiltration. The protein hydrolysate (APH) and its membrane ultrafiltration fractions were assayed for in vitro antioxidant activities. The <1 kDa peptides exhibited significantly better (p < 0.05) ferric reducing power, diphenyl-1-picryhydradzyl (DPPH) and hydroxyl radical scavenging activities when compared to peptide fractions of higher molecular weights. The high activity of <1 kDa peptides in these antioxidant assay systems may be related to the high levels of total hydrophobic and aromatic amino acids. In comparison to glutathione (GSH), the APH and its membrane fractions had significantly higher (p < 0.05) ability to chelate metal ions. In contrast, GSH had significantly greater (p < 0.05) ferric reducing power and free radical scavenging activities than APH and its membrane fractions. The APH and its membrane fractions effectively inhibited lipid peroxidation, results that were concentration dependent. The activity of APH and its membrane fractions against linoleic acid oxidation was higher when compared to that of GSH but lower than that of butylated hydroxyl toluene (BHT). The results show potential use of APH and its membrane fractions as antioxidants in the management of oxidative stress-related metabolic disorders and in the prevention of lipid oxidation in food products. PMID:22072912

  8. Effect of peptide size on antioxidant properties of African yam bean seed (Sphenostylis stenocarpa) protein hydrolysate fractions.

    PubMed

    Ajibola, Comfort F; Fashakin, Joseph B; Fagbemi, Tayo N; Aluko, Rotimi E

    2011-01-01

    Enzymatic hydrolysate of African yam bean seed protein isolate was prepared by treatment with alcalase. The hydrolysate was further fractionated into peptide sizes of <1, 1-3, 3-5 and 5-10 kDa using membrane ultrafiltration. The protein hydrolysate (APH) and its membrane ultrafiltration fractions were assayed for in vitro antioxidant activities. The <1 kDa peptides exhibited significantly better (p < 0.05) ferric reducing power, diphenyl-1-picryhydradzyl (DPPH) and hydroxyl radical scavenging activities when compared to peptide fractions of higher molecular weights. The high activity of <1 kDa peptides in these antioxidant assay systems may be related to the high levels of total hydrophobic and aromatic amino acids. In comparison to glutathione (GSH), the APH and its membrane fractions had significantly higher (p < 0.05) ability to chelate metal ions. In contrast, GSH had significantly greater (p < 0.05) ferric reducing power and free radical scavenging activities than APH and its membrane fractions. The APH and its membrane fractions effectively inhibited lipid peroxidation, results that were concentration dependent. The activity of APH and its membrane fractions against linoleic acid oxidation was higher when compared to that of GSH but lower than that of butylated hydroxyl toluene (BHT). The results show potential use of APH and its membrane fractions as antioxidants in the management of oxidative stress-related metabolic disorders and in the prevention of lipid oxidation in food products.

  9. The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway1[W

    PubMed Central

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg

    2008-01-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. PMID:18065559

  10. Immunoreactive fraction 1 leaf protein and dry matter content during wilting and ensiling of ryegrass and alfalfa.

    PubMed

    Mangan, J L; Harrison, F A; Vetter, R L

    1991-07-01

    In four experiments with ryegrass and alfalfa, cut herbage was wilted in the field and silage made in 1- or 200-L silos. Direct-cut (mean DM, 20.3%), low wilt (mean DM, 26.0%), medium wilt (mean DM, 36.2%) and high wilt (mean DM, 47.7%) herbages were used. Fraction 1, the most abundant leaf protein, was measured by crossed immunoelectrophoresis using rabbit anti-Fraction 1 serum. In two ryegrass and one alfalfa experiments in which weather conditions allowed rapid drying to high wilt herbage in 24 h, there was no significant loss of Fraction 1 protein. In the second alfalfa experiment, in which wilting was prolonged to 3 d by adverse weather, there was a 70% loss of Fraction 1. Ensiling proceeded normally in the four experiments, with rapid fall in pH and production of VFA, lactate, and NPN; the extent and rates of production were inversely related to DM content. In alfalfa and ryegrass, pH fell below the isoelectric point of Fraction 1 within 8 d. In each ryegrass experiment, a high proportion (58 to 100%) of Fraction 1 in medium and high wilt silages survived fermentation for 28 and 68 d, with lesser amounts in other silages. With alfalfa, however, almost all Fraction 1 protein was degraded at all DM concentrations during fermentation. Fiber-associated protein increased markedly with increases in DM during wilting, and these differences were present in the mature silage of both ryegrass and alfalfa. Digestibility studies with fistulated sheep showed that appreciable amounts of immunoreactive Fraction 1 protein in ryegrass silages were undegraded in the rumen.

  11. Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

    PubMed Central

    Jafari, Mohieddin; Primo, Vincent; Smejkal, Gary B.; Moskovets, Eugene V.; Kuo, Winston P.; Ivanov, Alexander R.

    2014-01-01

    Fractionation of complex samples at the cellular, subcellular, protein or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e., 1-D SDS PAGE, preparative 1-D SDS PAGE, isoelectric focusing in immobilized pH gradients (IEF-IPG), and 2-D PAGE in their performance as fractionation approaches in nanoLC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1-D SDS PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2-D PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1-D SDS PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput. PMID:22899259

  12. Fluorescence spectroscopy and principal component analysis of soy protein hydrolysate fractions and the potential to assess their antioxidant capacity characteristics.

    PubMed

    Ranamukhaarachchi, Sahan A; Peiris, Ramila H; Moresoli, Christine

    2017-02-15

    The potential of intrinsic fluorescence and principal component analysis (PCA) to characterize the antioxidant capacity of soy protein hydrolysates (SPH) during sequential ultrafiltration (UF) and nanofiltration (NF) was evaluated. SPH was obtained by enzymatic hydrolysis of soy protein isolate. Antioxidant capacity was measured by Oxygen Radical Absorbance Capacity (ORAC) and Folin Ciocalteau Reagent (FCR) assays together with fluorescence excitation-emission matrices (EEM). PCA of the fluorescence EEMs revealed two principal components (PC1-tryptophan, PC2-tyrosine) that captured significant variance in the fluorescence spectra. Regression models between antioxidant capacity and PC1 and PC2 displayed strong linear correlations for NF fractions and a weak linear correlation for UF fractions. Clustering of UF and NF fractions according to ORACFPCA and FCRFPCA was observed. The ability of this method to extract information on contributions by tryptophan and tyrosine amino acid residues to the antioxidant capacity of SPH fractions was demonstrated. Copyright © 2016. Published by Elsevier Ltd.

  13. Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion.

    PubMed

    Liu, Zhao; Fan, Songhua; Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

    2016-01-01

    The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest.

  14. Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

    PubMed Central

    Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

    2016-01-01

    The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. PMID:27832179

  15. Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched alpha-lactalbumin and beta-lactoglobulin food ingredients

    USDA-ARS?s Scientific Manuscript database

    A potentially economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (SCO2) as an acid to produce enriched fractions of alpha-lactalbumin (a-LA) and beta-lactoglobulin (b-LG) from whey protein isolate. To prepare the fractions, so...

  16. Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity pre-fractionation.

    PubMed

    Ma, Guorong; Zhou, Fuqiang; Gao, Lina; Sun, Zhanqiang; Deng, Li; Zhang, Shulin; Li, Rongxiu

    2017-04-09

    The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis (MTB) have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines and diagnostics. The constituents of the MTB CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel-based pre-fractionation is usually performed before MS analysis. In this study, we describe a novel pre-fractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six-column cascade. The LC-MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first-time identifications. Notably, ∼1/3 (20/61) of these novel CFPs are membrane proteins, among which 9 proteins have 2-14 transmembrane domains (TMD). In addition, ∼1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that BiAC pre-fractionation markedly improves protein detection by LC-MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein

    PubMed Central

    Lidón-Moya, María C.; Barrera, Francisco N.; Bueno, Marta; Pérez-Jiménez, Raúl; Sancho, Javier; Mateu, Mauricio G.; Neira, José L.

    2005-01-01

    The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C-terminal domain, CA-C. This self-association process, which is thought to be pH-dependent, seems to constitute a key step in virus assembly. CA-C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA-C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size-exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA-C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration-dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm ~ 325 K) was observed by ANS-binding experiments, fluorescence anisotropy, and near-UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far-UV CD, and NMR revealed a third transition occurring at Tm ~ 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA-C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red-shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule-like conformations, and they resemble the properties of the second species observed in thermal unfolding. PMID:16131662

  18. An extensive thermodynamic characterization of the dimerization domain of the HIV-1 capsid protein.

    PubMed

    Lidón-Moya, María C; Barrera, Francisco N; Bueno, Marta; Pérez-Jiménez, Raúl; Sancho, Javier; Mateu, Mauricio G; Neira, José L

    2005-09-01

    The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C-terminal domain, CA-C. This self-association process, which is thought to be pH-dependent, seems to constitute a key step in virus assembly. CA-C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA-C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size-exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA-C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration-dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm approximately 325 K) was observed by ANS-binding experiments, fluorescence anisotropy, and near-UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far-UV CD, and NMR revealed a third transition occurring at Tm approximately 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA-C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red-shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule-like conformations, and they resemble the properties of the second species observed in thermal unfolding.

  19. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

    PubMed Central

    Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

    1980-01-01

    A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629

  20. Reverse-phase HPLC separation of hemp seed (Cannabis sativa L.) protein hydrolysate produced peptide fractions with enhanced antioxidant capacity.

    PubMed

    Girgih, Abraham T; Udenigwe, Chibuike C; Aluko, Rotimi E

    2013-03-01

    Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60 %) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1 mg/ml when compared to that observed at 0.1 mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

  1. Comparative Composition and Antioxidant Activity of Peptide Fractions Obtained by Ultrafiltration of Egg Yolk Protein Enzymatic Hydrolysates

    PubMed Central

    Chay Pak Ting, Bertrand P.; Mine, Yoshinori; Juneja, Lekh R.; Okubo, Tsutomu; Gauthier, Sylvie F.; Pouliot, Yves

    2011-01-01

    The objective of the study was to compare the antioxidant activity of two distinct hydrolysates and their peptide fractions prepared by ultrafiltration (UF) using membranes with molecular weight cut-off of 5 and 1 kDa. The hydrolysates were a delipidated egg yolk protein concentrate (EYP) intensively hydrolyzed with a combination of two bacterial proteases, and a phosphoproteins (PPP) extract partially hydrolyzed with trypsin. Antioxidant activity, as determined by the oxygen radical absorbance capacity (ORAC) assay, was low for EYP and PPP hydrolysates with values of 613.1 and 489.2 μM TE·g−1 protein, respectively. UF-fractionation of EYP hydrolysate increased slightly the antioxidant activity in permeate fractions (720.5–867.8 μM TE·g−1 protein). However, ORAC values were increased by more than 3-fold in UF-fractions prepared from PPP hydrolysate, which were enriched in peptides with molecular weight lower than 5 kDa. These UF-fractions were characterized by their lower N/P atomic ratio and higher phosphorus content compared to the same UF-fractions obtained from EYP-TH. They also contained high amounts of His, Met, Leu, and Phe, which are recognized as antioxidant amino acids, but also high content in Lys and Arg which both represent target amino acids of trypsin used for the hydrolysis of PPP. PMID:24957729

  2. Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

    PubMed Central

    Tjandrawinata, Raymond R; Trisina, Jessica; Rahayu, Puji; Prasetya, Lorentius Agung; Hanafiah, Aang; Rachmawati, Heni

    2014-01-01

    DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The “enteric coating” formulation showed no leakage in gastric fluid–like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration–versus-time curve, 99mTc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent. PMID:25284988

  3. CD-loop extension in Zika virus envelope protein key for stability and pathogenesis.

    PubMed

    Gallichotte, Emily N; Dinnon, Kenneth H; Lim, Xin-Ni; Ng, Thiam-Seng; Lim, Elisa X Y; Menachery, Vineet D; Lok, Shee-Mei; Baric, Ralph S

    2017-09-08

    With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  4. Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen-related proteins.

    PubMed

    Zhang, Cheng; Glatz, Charles E; Fox, Steven R; Johnson, Lawrence A

    2009-01-01

    Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed-based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen-related proteins; thereby, reducing the amount and type of corn-derived impurities in subsequent protein extraction and purification steps. The two proteins were a full-length human recombinant collagen type I alpha 1(rCIalpha1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44-kDa rCIalpha1 fragment. For each, approximately 60% of the rCIalpha1s in the seed was recovered in the dry-milled germ-rich fractions making up ca. 25% of the total kernel mass. For wet milling, approximately 60% of each was recovered in three fractions accounting for 20-25% of the total kernel mass. The rCIalpha1s in the dry-milled germ-rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIalpha1s were enriched 4-10 times in selected wet-milled fractions. The recovered starch from wet milling was almost free of rCIalpha1. Therefore, it was possible to generate rCIalpha1-enriched fractions by both dry and wet milling along with rCIalpha1-free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on-farm thus minimizing the risk of inadvertent release of viable transgenic seeds.

  5. Occurrence of major whey proteins in the pH 4.6 insoluble protein fraction from UHT-treated milk.

    PubMed

    Pizzano, Rosa; Manzo, Carla; Nicolai, Maria Adalgisa; Addeo, Francesco

    2012-08-15

    A clear picture of the protein rearrangement in milk following UHT-treatment was drawn by a comparative analysis of the pH 4.6 soluble protein fraction (SPF) and the pH 4.6 insoluble protein fraction (IPF) recovered from raw and UHT-treated milk samples. The two protein fractions were analyzed by mono- or bidimensional gel electrophoresis under reducing and nonreducing conditions, and protein bands were identified by specific immunostaining. Results showed that bovine serum albumin, β-lactoglobulin, and, to a lesser extent, α-lactalbumin coprecipitated with caseins in UHT-treated milk samples at pH 4.6. These proteins were almost exclusively involved in high molecular weight aggregates held together by disulfide bonds. Partition of α-lactalbumin and bovine serum albumin in the protein fractions obtained upon acidification of milk at pH 4.6 was evaluated by competitive immunoassays. The ELISA-based results suggested the possibility of using pH 4.6 insoluble α-lactalbumin and bovine serum albumin, in addition to pH 4.6 insoluble β-lactoglobulin, as indicators of the intensity of the heat treatment applied to milk.

  6. Enrichment of distinct microfilament-associated and GTP-binding-proteins in membrane/microvilli fractions from lymphoid cells

    PubMed Central

    Hao, Jian-Jiang; Wang, Guanghui; Pisitkun, Trairak; Patino-Lopez, Genaro; Nagashima, Kunio; Knepper, Mark A.; Shen, Rong-Fong; Shaw, Stephen

    2008-01-01

    Summary Lymphocyte microvilli mediate initial adhesion to endothelium during lymphocyte transition from blood into tissue but their molecular organization is incompletely understood. We modified a shear-based procedure to prepare biochemical fractions enriched for membrane/microvilli (MMV) from both human peripheral blood T-lymphocytes (PBT) and a mouse pre-B lymphocyte line (300.19). Enrichment of proteins in MMV relative to post nuclear lysate was determined by LC/MS/MS analysis and label-free quantitation. Subsequent analysis emphasized the 291 proteins shared by PBT and 300.19 and estimated by MS peak area to be highest abundance. Validity of the label-free quantitation was confirmed by many internal consistencies and by comparison with Western blot analyses. The MMV fraction was enriched primarily for subsets of cytoskeletal proteins, transmembrane proteins and G-proteins, with similar patterns in both lymphoid cell types. The most enriched cytoskeletal proteins were microfilament-related proteins NHERF1, Ezrin/Radixin/Moesin (ERMs), ADF/cofilin and Myosin1G. Other microfilament proteins such as talin, gelsolin, myosin II and profilin were markedly reduced in MMV, as were intermediate filament- and microtubule-related proteins. Heterotrimeric G-proteins and some small G-proteins (especially Ras and Rap1) were enriched in the MMV preparation. Two notable general observations also emerged. There was less overlap between the two cells in their transmembrane proteins than in other classes of proteins, consistent with a special role of plasma membrane proteins in differentiation. Second, unstimulated primary T-lymphocytes have an unusually high concentration of actin and other microfilament related proteins, consistent with the singular role of actin-mediated motility in the immunological surveillance performed by these primary cells. Lymphocyte microvilli initiate adhesion to endothelium during movement from blood into tissue. Using LC/MS/MS and label

  7. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    SciTech Connect

    Zanzoni, Serena; D'Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  8. Induced resistance to Botrytis cinerea in Capsicum annuum by a Fusarium crude elicitor fraction, free of proteins.

    PubMed

    Veloso, J; Díaz, J

    2013-11-01

    Fusarium oxysporum f. sp. lycopersici (FOL) induces resistance in pepper against the airborne pathogen Botrytis cinerea and the soil-borne pathogen Verticillium dahliae. However, its practical use is limited due to its pathogenicity to other crops. In this study we tested several fractions of a heat-sterilised crude FOL-elicitor preparation to protect pepper against B. cinerea and V. dahliae. Only the protein-free insoluble fraction of the preparation reduced B. cinerea infection. However, none of the fractions reduce V. dahliae symptoms. The insoluble protein-free fraction induced expression of defence genes in the plant, namely a chitinase (CACHI2), a peroxidase (CAPO1), a sesquiterpene cyclase (CASC1) and a basic PR1 (CABPR1). Even though the CASC1 gene was not induced directly after treatment with the insoluble fraction in the leaves, it was induced after B. cinerea inoculation, showing a priming effect. The insoluble protein-free FOL-elicitor protected pepper against the airborne pathogen through a mechanism that involves induced responses in the plant, but different to the living FOL. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  9. Complexation of bovine beta-lactoglobulin with 11S protein fractions of soybean (Glycine max) and sesame (Sesamum indicum).

    PubMed

    Anuradha, S N; Prakash, V

    2009-01-01

    Beta-lactoglobulin (beta-Lg) comprises 50% of the whey component of bovine milk. Protein-protein interactions between bovine beta-Lg and 11S protein fractions of soybean and sesame were investigated by turbidity, solubility behaviour and by evaluation of functional properties in the mixed systems. In this work, the aggregation behaviour of soybean and the whey protein (beta-Lg) showed the formation of soluble complexes. Turbidity and solubility studies showed that the proteins interacted at temperatures between 60 and 90+/-5 degrees C. Heating a mixture of beta-Lg and 11S proteins of soybean at higher temperatures formed soluble complexes with beta-Lg. It also reduced the self aggregation behaviour, especially that of 11S protein fraction of soybean. This reduced the precipitation of soybean proteins at higher temperature. The complex formed was resolved by gel filtration using high-performance liquid chromatography. Upon heating beta-Lg at neutral pH, native dimer starts to dissociate into monomers leading to the exposure of previously buried hydrophobic amino acids and the free thiol group. The soluble complex is formed by the exposed thiol groups. But interaction of beta-Lg with sesame 11S protein fractions did not form any soluble complexes. The mechanism of interaction indicates that hydrophobic interactions were preferred over disulfide linkages at the high salt concentrations of the buffer used. During thermal treatment the molecules are unfolded, leading to an exposure of the hydrophobic groups that further enhance the protein-protein interactions that are entropically driven hydrophobic interactions.

  10. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40.

    PubMed

    Morphew, Russell M; Hamilton, Clare M; Wright, Hazel A; Dowling, David J; O'Neill, Sandra M; Brophy, Peter M

    2013-01-31

    Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

  11. Oily fraction of Semecarpus anacardium Linn nuts involves protein kinase C activation for its pro-inflammatory response.

    PubMed

    Tripathi, Yamini B; Pandey, Nidhi; Tripathi, Deepshikha; Tripathi, Pratibha

    2010-12-01

    The oily fraction (non polar fraction-NPF) of S. anacardium (SA) significantly increased the expression of protein kinase C-delta (PKC-delta) in macrophages in concentration dependent manner, which was similar to phorbol myristate acetate (PMA) response. Further, H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine), an inhibitor of PKC significantly inhibited this NPF mediated response in a concentration dependent manner. In the post treatment kinetics, H-7 showed this inhibition only up to 6 min post NPF/PMA addition, but in similar condition, quercetin, a flavone with reported antioxidant property, showed this inhibition only up to 2 min. The results clearly suggest that oily fraction of SA nuts enhances the expression of PKC protein, which may be responsible for its reported pro-inflammatory property.

  12. Emulsifying and Foaming Properties of Different Protein Fractions Obtained from a Novel Lupin Variety AluProt-CGNA(®) (Lupinus luteus).

    PubMed

    Burgos-Díaz, César; Piornos, José A; Wandersleben, Traudy; Ogura, Takahiro; Hernández, Xaviera; Rubilar, Mónica

    2016-07-01

    The use of vegetable proteins as food ingredient is becoming increasingly important due to their high versatility and environmental acceptability. This work describes a chemical characterization and techno-functional properties (emulsifying and foaming properties) of 3 protein fractions obtained from a protein-rich novel lupin variety, AluProt-CGNA(®) . This nongenetically modified variety have a great protein content in dehulled seeds (60.6 g protein/100 g, dry matter), which is higher than soybean and other lupin varieties. A simple procedure was utilized to obtain 3 different fractions by using alkali solubilization and isoelectric precipitation. Fractions 1 and 3 were mainly composed of protein and polysaccharides (NNE), whereas fraction 2 was mainly composed by protein (97%, w/w). Fraction 3 presented interesting and potential foaming properties in comparison to the other fractions evaluated in the study. Besides, its solubility, foaming and emulsifying capacity were practically not affected by pH variations. The 3 fractions also presented good emulsion stability, reaching values above a 95%. SDS-PAGE showed that fractions 1 and 2 contained mainly conglutin α, β, and δ, but in different ratios, whereas fraction 3 contained mainly conglutin γ and albumins. The results of this work will provide better understanding for the utilization of each protein fractions as potential ingredients in food industry.

  13. Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14.

    PubMed

    Ouidir, Tassadit; Jarnier, Frédérique; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2014-10-01

    Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes and signal transduction in bacteria. Bacterial-proteome analysis is required to determine which proteins have been conditionally expressed and whether any post-translational modifications are present. One of the greatest challenges of proteome analysis is the fractionation of these complex protein mixtures to detect low-abundance phosphoproteins. Liquid-phase isoelectric focusing (IEF) is a promising analytical tool in proteomics, but as far as we are aware no work has studied the reproducibility of this approach. In this study, we investigated the phosphoproteome of Pseudomonas aeruginosa strain PA14. We first tested in-solution IEF protein fractionation, and then used this technique to fractionate the proteins in the complex mixture. Next, phosphopeptides were enriched with titanium dioxide and analyzed by high-resolution, high-accuracy liquid chromatography-mass spectrometry. With this approach, we succeeded in characterizing 73 unique phosphorylated peptides belonging to 63 proteins. Interestingly, we observed a higher percentage of modified tyrosine, revealing the importance of this phosphorylated residue in bacteria.

  14. The influence of protein fractions from bovine colostrum digested in vivo and in vitro on human intestinal epithelial cell proliferation.

    PubMed

    Morgan, Alison J; Riley, Lisa G; Sheehy, Paul A; Wynn, Peter C

    2014-02-01

    Colostrum consists of a number of biologically active proteins and peptides that influence physiological function and development of a neonate. The present study investigated the biological activity of peptides released from first day bovine colostrum through in vitro and in vivo enzymatic digestion. This was assessed for proliferative activity using a human intestinal epithelial cell line, T84. Digestion of the protein fraction of bovine colostrum in vitro was conducted with the enzymes pepsin, chymosin and trypsin. Pepsin and chymosin digests yielded protein fractions with proliferative activity similar to that observed with undigested colostrum and the positive control foetal calf serum (FCS). In contrast trypsin digestion significantly (P<0·05) decreased colostral proliferative activity when co-cultured with cells when compared with undigested colostrum. The proliferative activity of undigested colostrum protein and abomasal whey protein digesta significantly increased (P<0·05) epithelial cell proliferation in comparison to a synthetic peptide mix. Bovine colostrum protein digested in vivo was collected from different regions of the gastrointestinal tract (GIT) in newborn calves fed either once (n=3 calves) or three times at 12-h intervals (n=3 calves). Digesta collected from the distal duodenum, jejunum and colon of calves fed once, significantly (P<0·05) stimulated cell proliferation in comparison with comparable samples collected from calves fed multiple times. These peptide enriched fractions are likely to yield candidate peptides with potential application for gastrointestinal repair in mammalian species.

  15. Protein viscosity, mineral fraction and staggered architecture cooperatively enable the fastest stress wave decay in load-bearing biological materials.

    PubMed

    Qwamizadeh, Mahan; Zhang, Zuoqi; Zhou, Kun; Zhang, Yong Wei

    2016-07-01

    One of the key functions of load-bearing biological materials, such as bone, dentin and sea shell, is to protect their inside fragile organs by effectively damping dynamic impact. How those materials achieve this remarkable function remains largely unknown. Using systematic finite element analyses, we study the stress wave propagation and attenuation in cortical bone at the nanoscale as a model material to examine the effects of protein viscosity, mineral fraction and staggered architecture on the elastic wave decay. It is found that the staggered arrangement, protein viscosity and mineral fraction work cooperatively to effectively attenuate the stress wave. For a typical mineral volume fraction and protein viscosity, an optimal staggered nanostructure with specific feature sizes and layouts is able to give rise to the fastest stress wave decay, and the optimal aspect ratio and thickness of mineral platelets are in excellent agreement with experimental measurements. In contrary, as the mineral volume fraction or the protein viscosity goes much higher, the structural arrangement is seen having trivial effect on the stress wave decay, suggesting that the damping properties of the composites go into the structure-insensitive regime from the structure-sensitive regime. These findings not only significantly add to our understanding of the structure-function relationship of load-bearing biological materials, and but also provide useful guidelines for the design of bio-inspired materials with superior resistance to impact loading.

  16. Antiedematogenic and antioxidant properties of high molecular weight protein sub-fraction of Calotropis procera latex in rat.

    PubMed

    Chaudhary, Priyanka; de Araújo Viana, Carolina; Ramos, Marcio V; Kumar, Vijay L

    2015-03-01

    The aim was to evaluate the effect of high molecular weight protein fraction of Calotropis procera latex on edema formation and oxidative stress in carrageenan-induced paw inflammation. A sub-plantar injection of carrageenan was given to induce edema in the hind paw of the rat. The inhibitory effect of high molecular weight protein fraction of C. procera latex was evaluated following intravenous administration (5 and 25 mg/kg body weight) and was compared with that of diclofenac given orally (5 mg/kg). The levels of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) were measured in the inflamed paw tissue at the end of the study. The high molecular weight protein fraction obtained from the latex of C. procera produced a dose-dependent inhibition of edema formation that was accompanied by normalization of levels of oxidative stress markers (GSH and TBARS) and MPO, a marker for neutrophils in the paw tissue. The high molecular weight protein fraction of C. procera latex ameliorates acute inflammation in the paw through its antioxidant effect.

  17. Antiedematogenic and antioxidant properties of high molecular weight protein sub-fraction of Calotropis procera latex in rat

    PubMed Central

    Chaudhary, Priyanka; de Araújo Viana, Carolina; Ramos, Marcio V.; Kumar, Vijay L.

    2015-01-01

    Objectives: The aim was to evaluate the effect of high molecular weight protein fraction of Calotropis procera latex on edema formation and oxidative stress in carrageenan-induced paw inflammation. Methods: A sub-plantar injection of carrageenan was given to induce edema in the hind paw of the rat. The inhibitory effect of high molecular weight protein fraction of C. procera latex was evaluated following intravenous administration (5 and 25 mg/kg body weight) and was compared with that of diclofenac given orally (5 mg/kg). The levels of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) were measured in the inflamed paw tissue at the end of the study. Results: The high molecular weight protein fraction obtained from the latex of C. procera produced a dose-dependent inhibition of edema formation that was accompanied by normalization of levels of oxidative stress markers (GSH and TBARS) and MPO, a marker for neutrophils in the paw tissue. Conclusions: The high molecular weight protein fraction of C. procera latex ameliorates acute inflammation in the paw through its antioxidant effect. PMID:25767367

  18. Extension of the NCAT phantom for the investigation of intra-fraction respiratory motion in IMRT using 4D Monte Carlo

    NASA Astrophysics Data System (ADS)

    McGurk, Ross; Seco, Joao; Riboldi, Marco; Wolfgang, John; Segars, Paul; Paganetti, Harald

    2010-03-01

    The purpose of this work was to create a computational platform for studying motion in intensity modulated radiotherapy (IMRT). Specifically, the non-uniform rational B-spline (NURB) cardiac and torso (NCAT) phantom was modified for use in a four-dimensional Monte Carlo (4D-MC) simulation system to investigate the effect of respiratory-induced intra-fraction organ motion on IMRT dose distributions as a function of diaphragm motion, lesion size and lung density. Treatment plans for four clinical scenarios were designed: diaphragm peak-to-peak amplitude of 1 cm and 3 cm, and two lesion sizes—2 cm and 4 cm diameter placed in the lower lobe of the right lung. Lung density was changed for each phase using a conservation of mass calculation. Further, a new heterogeneous lung model was implemented and tested. Each lesion had an internal target volume (ITV) subsequently expanded by 15 mm isotropically to give the planning target volume (PTV). The PTV was prescribed to receive 72 Gy in 40 fractions. The MLC leaf sequence defined by the planning system for each patient was exported and used as input into the MC system. MC simulations using the dose planning method (DPM) code together with deformable image registration based on the NCAT deformation field were used to find a composite dose distribution for each phantom. These composite distributions were subsequently analyzed using information from the dose volume histograms (DVH). Lesion motion amplitude has the largest effect on the dose distribution. Tumor size was found to have a smaller effect and can be mitigated by ensuring the planning constraints are optimized for the tumor size. The use of a dynamic or heterogeneous lung density model over a respiratory cycle does not appear to be an important factor with a <= 0.6% change in the mean dose received by the ITV, PTV and right lung. The heterogeneous model increases the realism of the NCAT phantom and may provide more accurate simulations in radiation therapy

  19. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

    PubMed

    Okumura, Yuuya; Nakamura, Tsuyoshi S; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong; Tachikawa, Hiroyuki

    2016-01-01

    Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation

  20. Yield of 6,000 proteins by 1D nLC-MS/MS without pre-fractionation.

    PubMed

    Anagnostopoulos, Athanasios K; Stravopodis, Dimitrios J; Tsangaris, George Th

    2017-03-15

    Mass spectrometry (MS) has dominated over other protein analysis methods that aspire to deliver rapid and sensitive protein annotation, due to its ability to acquire high-content biological information from samples of great complexity. Routinely, in-depth analysis of complex biological samples, such as total cell lysates, relies on the high separation power of two-dimensional liquid chromatography-tandem MS (2D LC-MS/MS), often combined with protein pre-fractionation. However, on the basis of recent advances in chromatographic and MS instrumentation, one-dimensional (1D) LC-MS/MS approaches have become the method-of-choice for high-volume/high-throughput protein experiments. Thousands of proteins can be identified in single-run LC-MS/MS experiments. In the present study a 1D LC-MS/MS approach was applied on whole-cell lysates of WM-266-4 human cells leading to identification of more than 5,300 protein groups, 6,000 proteins and 22,00 peptides, in a single run. Using no pre-fractionation steps, method optimization was achieved through experimentation on lysis and protein extraction solutions, as well as nLC gradient parameters.

  1. Multiparametric investigation of competitive and noncompetitive sorption characteristics of SMP fractions (carbohydrate and protein) on activated carbon.

    PubMed

    Dizge, Nadir; Tansel, Berrin

    2011-01-30

    Sorption characteristics of soluble microbial products (SMPs) as carbohydrate and protein on activated carbon were investigated. Batch experiments were conducted to evaluate the sorption kinetics and the equilibrium conditions. The parameters studied included initial SMP concentration (50-200mg/L), activated carbon dosage (0.25-50 g/L), contact time (0.02-4h), particle size of activated carbon used (5-75 μm, 75-850 μm, and 850-1000 μm), and presence of one or both SMP fractions. The equilibrium sorption of carbohydrate and protein were significantly affected by the presence of the second SMP fraction in the solutions. Adsorption isotherms were expressed by the Langmuir and Freundlich models. The adsorption rates under noncompetitive and competitive conditions were analyzed with kinetics-based Lagergren pseudo-first order and pseudo-second order models; and diffusion-based external diffusion and Weber-Morris intraparticle models. Both SMP fractions were removed effectively, however, sorption of protein was significantly better than that of carbohydrate in all cases. The relatively significant effect of particle size on sorption of protein indicates that protein is most likely adsorbed as a single layer on the carbon surface. For the carbohydrate, the increase in particle size did not decrease the sorption significantly indicating that carbohydrate may be adsorbed in multiple layers or may diffuse into the porous matrix more effectively.

  2. Measurement of protein fractional synthesis and breakdown rates in the skin of rabbits using a subflooding dose method.

    PubMed

    Zhang, Xiao-Jun; Chinkes, David L; Herndon, David N; Wolfe, Robert R

    2009-09-01

    The flooding dose method continues to be useful in measuring protein fractional synthetic rate (FSR) in a tissue. However, flooding of free amino acid pools eliminates enrichment difference between plasma and tissue free amino acid pools, which makes it impossible to concomitantly measure protein fractional breakdown rate (FBR). We hypothesized that a subflooding dose of an amino acid reduces the enrichment difference between plasma and tissue free amino acid pool to a minimal measurable level, thus allowing concomitant measurement of protein FSR and FBR. Phenylalanine (40% enriched) at 50 mg/kg was intravenously injected as a bolus in 6 anesthetized rabbits. Arterial blood and chest skin samples were taken before the injection and for 120 minutes after the injection. Fractional breakdown rate of skin protein calculated from 15-60-120-minute sampling times was 11.3%/d +/- 2.0%/d, which was close (P = .66) to the corresponding FSR of 10.0%/d +/- 2.3%/d. The subflooding dose injection did not disturb the FBR approach because modifications on the FBR equation to account for the changes of plasma phenylalanine concentration resulted in the same value (11.5%/d +/- 1.4%/d). The FBR was positively correlated with the FSR (r = 0.80, P < .05). These findings indicate maintenance of protein mass in the skin, which is a metabolic characteristic of the skin. This subflooding dose method provides a methodological choice to concomitantly measure both FSR and FBR in a tissue.

  3. Antinutritional factors and functionality of protein-rich fractions of industrial guar meal as affected by heat processing.

    PubMed

    Nidhina, N; Muthukumar, S P

    2015-04-15

    Proximate composition analysis and antinutritional factor composition of different fractions of industrial guar meal: raw churi (IRC), heated churi (IHC), final churi (IFC) and guar korma (IGK) were studied and compared. Protein content was found to be very high in IGK (52.7%) when compared to the churi fractions (32-33%) and the trypsin inhibitor activities were found to be negligible in all the fractions (0.58-1.8 mg/g). Single fraction (IGK) was selected for further studies, based on the protein content. The antinutritional factors of selected fractions were significantly reduced by different heat treatments. Heat treatments significantly increased the water absorbing capacity of IGK, but reduced the nitrogen solubility, emulsifying and foaming capacity. Highest L(∗) value was observed for boiled IGK, highest a(∗) and b(∗) values for roasted IGK, during colour measurement. FTIR spectral analysis revealed the presence several aromatic groups in IGK and slight modifications in the molecular structure during heat treatments.

  4. Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin-converting enzymes, renin and free radicals.

    PubMed

    Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Malomo, Sunday A; Aluko, Rotimi E; Amonsou, Eric O

    2017-07-01

    An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS(•+) to a three-fold greater extent compared to the isolate. BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  5. [The effect of modified nano-diamonds of detonation synthesis on the protein fractions of human blood].

    PubMed

    Botvich, Iu A; Olkhovskiĭ, I A; Baron, I I; Puzyr', A P; Baron, A V; Bondar', V S

    2013-11-01

    It is established that the modified nano-diamonds of detonation synthesis are able to bind serum proteins of human blood. The relative selectivity is established concerning the effect of modified nano-diamonds of detonation synthesis on beta2- and gamma-globulin fractions of serum. The evidence of concentration dependence of effect of modified nano-diamonds of detonation synthesis from serum proteins is established. The study results make it possible to consider modified nano-diamonds of detonation synthesis as a potential sorbent in technologies of hemodialysis, plasmapheresis, isolation of blood proteins and as a foundation for development of new systems of laboratory diagnostic.

  6. Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry.

    PubMed

    Natera, Siria H A; Ford, Kristina L; Cassin, Andrew M; Patterson, John H; Newbigin, Edward J; Bacic, Antony

    2008-03-01

    To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning beta-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally

  7. Hierarchical description and extensive classification of protein structural changes by Motion Tree.

    PubMed

    Koike, Ryotaro; Ota, Motonori; Kidera, Akinori

    2014-02-06

    The structures of the same protein, determined under different conditions, provide clues toward understanding the role of structural changes in the protein's function. Structural changes are usually identified as rigid-body motions, which are defined using a particular threshold of rigidity, such as domain motions. However, each protein actually undergoes motions with various size and magnitude ranges. In this study, to describe protein structural changes more comprehensively, we propose a method based on hierarchical clustering. This method enables the illustration of a wide range of protein motions in a single tree diagram, named the "Motion Tree". We applied the method to 432 proteins exhibiting large structural changes and classified their Motion Trees in terms of the characteristic indices of the trees. This classification of the Motion Trees revealed clear relationships to their protein functions. Especially, complex structural changes are significantly correlated with multi-step protein functions.

  8. Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins.

    PubMed

    Lange, Andreas; Kistler, Claudia; Jutzi, Tanja B; Bazhin, Alexandr V; Klemke, Claus Detlev; Schadendorf, Dirk; Eichmüller, Stefan B

    2009-06-01

    The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.

  9. Antimutagenic and antioxidant activity of a protein fraction from aerial parts of Urtica dioica.

    PubMed

    Di Sotto, Antonella; Mazzanti, Gabriela; Savickiene, Nijole; Staršelskytė, Rasa; Baksenskaite, Vaida; Di Giacomo, Silvia; Vitalone, Annabella

    2015-06-01

    Urtica dioica L. (Urticaceae), stinging nettle, has been employed as a folklore remedy for a wide spectrum of ailments, including urinary disorders, prostatic hyperplasia, and liver diseases. It has been also used traditionally for cancer treatment. To evaluate the potential chemopreventive properties of a protein fraction from the aerial part of Urtica dioica (namely UDHL30). UDHL30 has been tested for the antimutagenic activity in bacteria (50-800 μg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06-2 mg/mL; 24 and 48 h incubation). Moreover, the antioxidant activity of UDHL30 (0.1-1200 μg/mL; ABTS and superoxide-radical scavenger assays) was evaluated as potential protective mechanisms. UDHL30 was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56, 78, and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 μg/plate). In addition, a remarkable scavenging activity against ABTS radical and superoxide anion (IC50 values of 19.9 ± 1.0 μg/mL and 75.3 ± 0.9 μg/mL, respectively) was produced. UDHL30 possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of UDHL30 can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation. The radical scavenger ability could contribute to 2AA-antimutagenicity. These data encourage further studies in order to better define the potential usefulness of UDHL30 in chemoprevention.

  10. Fractionation of proteins and carbohydrates of extracellular polymeric substances in a membrane bioreactor system.

    PubMed

    Malamis, Simos; Andreadakis, Andreas

    2009-07-01

    The major operational problem associated with membrane bioreactors (MBR) is membrane fouling, for which extracellular polymeric substances (EPS) are primarily responsible. In this work both the soluble and bound EPS (i.e. SMP and EPS) produced in an MBR system operating under sludge retention times (SRT) of 10, 15, 20 and 33 days were fractionized by means of membranes having variable molecular weight cutoffs (300 kDa, 100 kDa, 10 kDa & 1 kDa). The results show that increasing the SRT leads to a reduction of SMP and EPS and that these reductions are more pronounced for the SRTs in the range 10-20 days. This reduction is more significant for carbohydrates than for proteins. The decrease of SMP and EPS with increasing SRT from 10 to 20 days led to a significant decrease of the level of fouling. The further increase of SRT to 33 days did not significantly impact on the level of fouling as the SMP and EPS concentrations did not change much. Under the examined operating conditions, EPS were found to be composed mainly of large macromolecules having a size of 0.45 microm-300 kDa and to a lower extent of very small molecules (<1 kDa) that are not easily decomposed by the biomass activity. The majority of SMP is composed of very small molecules (<1 kDa), while some macromolecules in the range of 0.45 microm-300 kDa are present. Consequently, both EPS and SMP were found to have a bimodal character.

  11. Re-fraction: a machine learning approach for deterministic identification of protein homologues and splice variants in large-scale MS-based proteomics.

    PubMed

    Yang, Pengyi; Humphrey, Sean J; Fazakerley, Daniel J; Prior, Matthew J; Yang, Guang; James, David E; Yang, Jean Yee-Hwa

    2012-05-04

    A key step in the analysis of mass spectrometry (MS)-based proteomics data is the inference of proteins from identified peptide sequences. Here we describe Re-Fraction, a novel machine learning algorithm that enhances deterministic protein identification. Re-Fraction utilizes several protein physical properties to assign proteins to expected protein fractions that comprise large-scale MS-based proteomics data. This information is then used to appropriately assign peptides to specific proteins. This approach is sensitive, highly specific, and computationally efficient. We provide algorithms and source code for the current version of Re-Fraction, which accepts output tables from the MaxQuant environment. Nevertheless, the principles behind Re-Fraction can be applied to other protein identification pipelines where data are generated from samples fractionated at the protein level. We demonstrate the utility of this approach through reanalysis of data from a previously published study and generate lists of proteins deterministically identified by Re-Fraction that were previously only identified as members of a protein group. We find that this approach is particularly useful in resolving protein groups composed of splice variants and homologues, which are frequently expressed in a cell- or tissue-specific manner and may have important biological consequences.

  12. Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

    NASA Astrophysics Data System (ADS)

    Degrève, Léo; Fuzo, Carlos A.; Caliri, Antonio

    2012-12-01

    The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in

  13. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  14. Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

    PubMed

    Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

    2017-09-01

    This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Sonicated Protein Fractions of Mycoplasma hyopneumoniae Induce Inflammatory Responses and Differential Gene Expression in a Murine Alveolar Macrophage Cell Line.

    PubMed

    Damte, Dereje; Lee, Seung-Jin; Birhanu, Biruk Tesfaye; Suh, Joo-Won; Park, Seung-Chun

    2015-12-28

    Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation - only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

  16. Extension of in vivo half-life of biologically active molecules by XTEN protein polymers.

    PubMed

    Podust, Vladimir N; Balan, Sibu; Sim, Bee-Cheng; Coyle, Michael P; Ernst, Ulrich; Peters, Robert T; Schellenberger, Volker

    2016-10-28

    XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

  17. Physical, Chemical and Biochemical Modifications of Protein-Based Films and Coatings: An Extensive Review

    PubMed Central

    Zink, Joël; Wyrobnik, Tom; Prinz, Tobias; Schmid, Markus

    2016-01-01

    Protein-based films and coatings are an interesting alternative to traditional petroleum-based materials. However, their mechanical and barrier properties need to be enhanced in order to match those of the latter. Physical, chemical, and biochemical methods can be used for this purpose. The aim of this article is to provide an overview of the effects of various treatments on whey, soy, and wheat gluten protein-based films and coatings. These three protein sources have been chosen since they are among the most abundantly used and are well described in the literature. Similar behavior might be expected for other protein sources. Most of the modifications are still not fully understood at a fundamental level, but all the methods discussed change the properties of the proteins and resulting products. Mastering these modifications is an important step towards the industrial implementation of protein-based films. PMID:27563881

  18. Evidence for novel tomato seed allergens: IgE-reactive legumin and vicilin proteins identified by multidimensional protein fractionation-mass spectrometry and in silico epitope modeling.

    PubMed

    Bässler, Olivia Y; Weiss, Julia; Wienkoop, Stefanie; Lehmann, Karola; Scheler, Christian; Dölle, Sabine; Schwarz, Dietmar; Franken, Philipp; George, Eckhard; Worm, Margitta; Weckwerth, Wolfram

    2009-03-01

    Tomato fruit and seed allergens were detected by IgE-immunoblotting using sera from 18 adult tomato-sensitized patients selected based on a positive history skin prick test (SPT) and specific Immunglobulin (Ig) E-levels. Isolated tomato seed total protein showed high SPT activity comparable or even higher than tomato fruit protein. For the molecular characterization of tomato seed allergens, a multidimensional protein fractionation strategy and LC-MS/MS was used. Two legumin- and vicilin-proteins were purified and showed strong IgE-reactivity in immunoblots. Individual patient sera exhibited varying IgE-sensitivity against the purified proteins. In silico structural modeling indicates high homology between epitopes of known walnut allergens and the detected IgE-crossreactive tomato proteins.

  19. Determination of the unmetabolised (18)F-FDG fraction by using an extension of simplified kinetic analysis method: clinical evaluation in paragangliomas.

    PubMed

    Barbolosi, Dominique; Hapdey, Sebastien; Battini, Stephanie; Faivre, Christian; Mancini, Julien; Pacak, Karel; Farman-Ara, Bardia; Taïeb, David

    2016-01-01

    Tumours with high (18)F-FDG uptake values on static late PET images do not always exhibit high proliferation indices. These discrepancies might be related to high proportion of unmetabolised (18)F-FDG components in the tissues. We propose a method that enables to calculate different (18)F-FDG kinetic parameters based on a new mathematical approach that integrates a measurement error model. Six patients with diagnosed non-metastatic paragangliomas (PGLs) and six control patients with different types of lesions were investigated in this pilot study using (18)F-FDG PET/CT. In all cases, a whole-body acquisition was followed by four static acquisitions centred over the target lesions, associated with venous blood samplings. We used an extension of the Hunter's method to calculate the net influx rate constant (K H). The exact net influx rate constant and vascular volume fraction (K i and V, respectively) were subsequently obtained by the method of least squares. Next, we calculated the mean percentages of metabolised (PM) and unmetabolised (PUM) (18)F-FDG components, and the times required to reach 80 % of the amount of metabolised (18)F-FDG (T80%). A test-retest evaluation indicated that the repeatability of our approach was accurate; the coefficients of variation were below 2 % regardless of the kinetic parameters considered. We observed that the PGLs were characterised by high dispersions of the maximum standardised uptake value SUVmax (9.7 ± 11, coefficient of variation CV = 114 %), K i (0.0137 ± 0.0119, CV = 87 %), and V (0.292 ± 0.306, CV = 105 %) values. The PGLs were associated with higher PUM (p = 0.02) and T80% (p = 0.02) values and lower k 3 (p = 0.02) values compared to the malignant lesions despite the similar SUVmax values (p = 0.55). The estimations of these new kinetic parameters are more accurate than SUVmax or K i for in vivo metabolic assessment of PGLs at the molecular level.

  20. Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway.

    PubMed

    Formstone, Caroline J; Mason, Ivor

    2005-06-15

    The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.

  1. Effect of the Ethyl Acetate Fraction of Eugenia uniflora on Proteins Global Expression during Morphogenesis in Candida albicans

    PubMed Central

    Silva-Rocha, Walicyranison P.; de Azevedo, Matheus F.; Ferreira, Magda R. A.; da Silva, Julhiany de Fátima; Svidzinski, Terezinha I. E.; Milan, Eveline P.; Soares, Luiz A. L.; Rocha, Keyla B. F.; Uchôa, Adriana F.; Mendes-Giannini, Maria J. S.; Fusco Almeida, Ana M.; Chaves, Guilherme M.

    2017-01-01

    Candida albicans is able to switch from yeast to hyphal growth and this is an essential step for tissue invasion and establishment of infection. Due to the limited drug arsenal used to treat fungal infections and the constant emergence of resistant strains, it is important to search for new therapeutic candidates. Therefore, this study aimed to investigate by proteomic analysis the role of a natural product (Eugenia uniflora) in impairing hypha formation in C. albicans. We also tested the potential action of E. uniflora to prevent and treat oral candidiasis induced in a murine model of oral infection and the ability of polymorphonuclear neutrophils to phagocytize C. albicans cells treated with the ethyl acetate fraction of the extract. We found that this fraction greatly reduced hypha formation after morphogenesis induction in the presence of serum. Besides, several proteins were differentially expressed in cells treated with the fraction. Surprisingly, the ethyl acetate fraction significantly reduced phagocytosis in C. albicans (Mean 120.36 ± 36.71 yeasts/100 PMNs vs. 44.68 ± 19.84 yeasts/100 PMNs). Oral candidiasis was attenuated when C. albicans cells were either pre-incubated in the presence of E. uniflora or when the fraction was applied to the surface of the oral cavity after infection. These results were consistent with the reduction in CFU counts (2.36 vs. 1.85 Log10 CFU/ml) and attenuation of tissue damage observed with histopathological analysis of animals belonging to treated group. We also observed shorter true hyphae by direct examination and histopathological analysis, when cells were treated with the referred natural product. The E. uniflora ethyl acetate fraction was non-toxic to human cells. E. uniflora may act on essential proteins mainly related to cellular structure, reducing the capacity of filamentation and attenuating infection in a murine model, without causing any toxic effect on human cells, suggesting that it may be a future therapeutic

  2. Cataract-specific posttranslational modifications and changes in the composition of urea-soluble protein fraction from the rat lens

    PubMed Central

    Yanshole, Lyudmila V.; Cherepanov, Ivan V.; Snytnikova, Olga A.; Yanshole, Vadim V.; Sagdeev, Renad Z.

    2013-01-01

    Purpose To determine age-related changes in the composition of the urea-soluble (US) protein fraction from lenses of senescence-accelerated OXYS (cataract model) and Wistar (control) rats and to establish posttranslational modifications (PTMs) occurring under enhanced oxidative stress in OXYS lenses. Methods The identity and the relative abundance of crystallins in the US fractions were determined using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MS). The identities and the positions of PTMs were established using MS/MS measurements. Results Two-dimensional gel electrophoresis maps of US protein fractions were obtained for lenses of 3-, 12-, and 62-week-old Wistar and OXYS rats, and the relative abundance of different isoforms of α-, β-, and γ-crystallins was determined. β-Crystallins were the major contributor of the US fraction in 3-week-old lenses (above 50%), γ-crystallins in 12-week-old lenses (50–60%), and in 62-week-old lenses, the contributions from all three crystallin families leveled out. The major interstrain difference was the elevated level of α-crystallins in the US fraction from 12-week-old OXYS lenses. Spots with increased relative abundance in OXYS maps were attributed to the cataract-specific spots of interest. The crystallins from these spots were subjected to MS/MS analysis, and the positions of acetylation, oxidation, deamidation, and phosphorylation were established. Conclusions The increased relative abundance of α-crystallins in the US fraction from 12-week-old OXYS lenses points to the fast insolubilization of α-crystallins under oxidative stress. Most of the PTMs attributed to the cataract-specific modifications also correspond to α-crystallins. These PTMs include oxidation of methionine residues, deamidation of asparagine and glutamine residues, and phosphorylation of serine and threonine residues. PMID:24227915

  3. Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis.

    PubMed

    Grzendowski, Michael; Riemenschneider, Markus J; Hawranke, Eva; Stefanski, Anja; Meyer, Helmut E; Reifenberger, Guido; Stühler, Kai

    2009-11-01

    Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high-molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high-salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2-D DIGE, MS and Western blotting.

  4. A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

    PubMed Central

    Rao, Marie Luise; Rao, Govind S.

    1982-01-01

    1. Binding of l-tri-[125I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25°C. 2. The l-tri-[125I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100°C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at −38°C for several months. 3. It displayed saturability, high affinity (apparent Kd 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3′-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[125I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304±0.11μg/mg of cytosol protein (mean±s.d., n=4) was obtained; the mean concentration in plasma was 0.309±0.07μg/mg of plasma protein (mean±s.d., n=3). 7. The Ka value of 6.3×108m−1 of l-tri-[125I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[125I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin. PMID:6289813

  5. Ions and the protein surface revisited: extensive molecular dynamics simulations and analysis of protein structures in alkali-chloride solutions.

    PubMed

    Friedman, Ran

    2011-07-28

    Proteins interact with ions in various ways. The surface of proteins has an innate capability to bind ions, and it is also influenced by the screening of the electrostatic potential owing to the presence of salts in the bulk solution. Alkali metal ions and chlorides interact with the protein surface, but such interactions are relatively weak and often transient. In this paper, computer simulations and analysis of protein structures are used to characterize the interactions between ions and the protein surface. The results show that the ion-binding properties of protein residues are highly variable. For example, alkali metal ions are more often associated with aspartate residues than with glutamates, whereas chlorides are most likely to be located near arginines. When comparing NaCl and KCl solutions, it was found that certain surface residues attract the anion more strongly in NaCl. This study demonstrates that protein-salt interactions should be accounted for in the planning and execution of experiments and simulations involving proteins, particularly if subtle structural details are sought after. © 2011 American Chemical Society

  6. SITEX 2.0: Projections of protein functional sites on eukaryotic genes. Extension with orthologous genes.

    PubMed

    Medvedeva, Irina V; Demenkov, Pavel S; Ivanisenko, Vladimir A

    2017-04-01

    Functional sites define the diversity of protein functions and are the central object of research of the structural and functional organization of proteins. The mechanisms underlying protein functional sites emergence and their variability during evolution are distinguished by duplication, shuffling, insertion and deletion of the exons in genes. The study of the correlation between a site structure and exon structure serves as the basis for the in-depth understanding of sites organization. In this regard, the development of programming resources that allow the realization of the mutual projection of exon structure of genes and primary and tertiary structures of encoded proteins is still the actual problem. Previously, we developed the SitEx system that provides information about protein and gene sequences with mapped exon borders and protein functional sites amino acid positions. The database included information on proteins with known 3D structure. However, data with respect to orthologs was not available. Therefore, we added the projection of sites positions to the exon structures of orthologs in SitEx 2.0. We implemented a search through database using site conservation variability and site discontinuity through exon structure. Inclusion of the information on orthologs allowed to expand the possibilities of SitEx usage for solving problems regarding the analysis of the structural and functional organization of proteins. Database URL: http://www-bionet.sscc.ru/sitex/ .

  7. Potential antitumor activity of a low-molecular-weight protein fraction from Grifola frondosa through enhancement of cytokine production.

    PubMed

    Kodama, Noriko; Mizuno, Shigeto; Nanba, Hiroaki; Saito, Naoaki

    2010-02-01

    Edible mushrooms contain an abundance of immune-enhancing nutritients. Some of these compounds, referred to as biological response modifiers (BRMs), have been used in biological therapies for cancer treatment. We obtained a low-molecular-weight protein fraction (MLP-Fraction) from the fruiting body of the maitake mushroom Grifola frondosa by multiple sequential steps, including ethanol precipitation, DEAE-exchange chromatography, and gel filtration. The effect of the MLP-Fraction on the immune system was determined using normal mice. This resulted in a simultaneous increase in splenocyte proliferation and production of cytokines such as interleukin (IL)-1alpha, tumor necrosis factor-alpha, IL-10, IL-12, and interferon (IFN)-gamma. The expression levels of IFN-gamma and IL-12 in antigen-presenting cells (APCs) and the activation of natural killer (NK) cells, macrophages, and dendritic cells were observed. These results suggest a mechanism in which NK cells are activated through cytokines produced by APCs. We also confirmed the possibility that the MLP-Fraction acts as a BRM using colon-26 carcinoma-bearing mice. This fraction enhanced the production of IL-12 and IFN-gamma by splenocytes in tumor-bearing mice and clearly showed an inhibitory effect on tumor cell growth.

  8. Variability in the fractionation of Cu, Ag, and Zn among cytosolic proteins in the bivalve Macoma balthica

    USGS Publications Warehouse

    Johansson, C.; Cain, Daniel J.; Luoma, Samuel N.

    1986-01-01

    Gel filtration chromatographs of cytosols from the clam Macorna balthica analysed from both field and laboratory treated specimens showed that uptake of Cu, Ag, and Zn in the metallothionein-like protein (MLP) pool follows exposure both in nature and in the laboratory. Specimens collected from San Francisco Bay over 18 mo showed strong temporal variability in the fractionation of the metals among cytosolic proteins. A marked increase in Cu, Ag, and Zn in a very low molecular weight pool occurred when concentrations were highest In the MLP pool. The correlation between total cytosollc metal and MLP-metal also appeared to approach a hyperbolic character at the highest concentrations.

  9. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    SciTech Connect

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  10. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    PubMed Central

    2009-01-01

    Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64%) consisted of both mitochondrial and cytosolic (non-mitochondrial) proteins (mt-cy families) while the remaining 33 (36%) were composed of mitochondrial proteins (mt-mt families). Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1) relocalization from mitochondria to cytosol, 2) from cytosol to mitochondria and 3) multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on the genomic scale was

  11. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    DOE PAGES

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; ...

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test thismore » strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.« less

  12. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    PubMed Central

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-01-01

    Reengineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. PMID:25313039

  13. Distribution of Animal Drugs among Curd, Whey, and Milk Protein Fractions in Spiked Skim Milk and Whey.

    PubMed

    Shappell, Nancy W; Shelver, Weilin L; Lupton, Sara J; Fanaselle, Wendy; Van Doren, Jane M; Hakk, Heldur

    2017-02-01

    It is important to understand the partitioning of drugs in processed milk and milk products, when drugs are present in raw milk, in order to estimate the potential consumer exposure. Radioisotopically labeled erythromycin, ivermectin, ketoprofen, oxytetracycline, penicillin G, sulfadimethoxine, and thiabendazole were used to evaluate the distribution of animal drugs among rennet curd, whey, and protein fractions from skim cow milk. Our previous work reported the distribution of these same drugs between skim and fat fractions of milk. Drug distribution between curd and whey was significantly correlated (R(2) = 0.70) to the drug's lipophilicity (log P), with improved correlation using log D (R(2) = 0.95). Distribution of drugs was concentration independent over the range tested (20-2000 nM). With the exception of thiabendazole and ivermectin, more drug was associated with whey protein than casein on a nmol/g protein basis (oxytetracycline experiment not performed). These results provide insights into the distribution of animal drug residues, if present in cow milk, among milk fractions, with possible extrapolation to milk products.

  14. Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels.

    PubMed

    Tome, Margaret E; Schaefer, Charles P; Jacobs, Leigh M; Zhang, Yifeng; Herndon, Joseph M; Matty, Fabian O; Davis, Thomas P

    2015-07-01

    Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.

  15. A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties

    PubMed Central

    Unverdorben, Felix; Hutt, Meike; Seifert, Oliver; Kontermann, Roland E.

    2015-01-01

    Background Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. Methodology/Principal Findings Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. Conclusions/Significance The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity. PMID:26430884

  16. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

    PubMed

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří; Wilson, Daniel N

    2016-12-15

    Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

  17. Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly.

    PubMed

    Gamalinda, Michael; Woolford, John L

    2014-11-01

    Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function. © 2014 Gamalinda and Woolford; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Assessing the reproducibility of fractional rates of protein synthesis in muscle tissue measured using the flooding dose technique.

    PubMed

    McCarthy, Ian D; Brown, James

    2016-07-01

    The flooding dose technique of Garlick et al. (1980) has become the main method for measuring tissue and whole-animal rates of protein synthesis in ectotherms. However, single tissue samples are used to determine rates of protein synthesis and no studies have examined the pattern of flooding in large tissues such as the white muscle in fishes, which can comprise up to 55% of the wet body mass of a fish and which is poorly perfused. The present study has examined, for the first time, the patterns of flooding and measured rates of protein synthesis in five different regions of the white muscle in the Arctic charr Salvelinus alpinus ranging in size from 25g to 1.6kg following a flooding dose injection of L-[(3)H]-phenylalanine. The results indicate that the degree of flooding (i.e. free pool specific radioactivity relative to that of the injection solution) and elevation in free phenylalanine concentrations can vary between regions but the calculated fractional rates of protein synthesis were similar in four of the five regions studied. The variability in rates of protein synthesis increased with body size with greater variability observed between regions for fish >1kg in body mass. For consistency between studies, it is recommended that samples are taken from the epaxial muscle in the region below the dorsal fin when measuring fractional rates of white muscle synthesis in fishes. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Forage Management Effects on Protein and Fiber Fractions, Protein Degradability, and Dry Matter Yield of Red Clover Conserved as Silage

    USDA-ARS?s Scientific Manuscript database

    Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover (Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein...

  20. Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

    PubMed Central

    Nick Pace, C; Huyghues-Despointes, Beatrice M P; Fu, Hailong; Takano, Kazufumi; Scholtz, J Martin; Grimsley, Gerald R

    2010-01-01

    The goal of this article is to gain a better understanding of the denatured state ensemble (DSE) of proteins through an experimental and computational study of their denaturation by urea. Proteins unfold to different extents in urea and the most hydrophobic proteins have the most compact DSE and contain almost as much secondary structure as folded proteins. Proteins that unfold to the greatest extent near pH 7 still contain substantial amounts of secondary structure. At low pH, the DSE expands due to charge–charge interactions and when the net charge per residue is high, most of the secondary structure is disrupted. The proteins in the DSE appear to contain substantial amounts of polyproline II conformation at high urea concentrations. In all cases considered, including staph nuclease, the extent of unfolding by urea can be accounted for using the data and approach developed in the laboratory of Wayne Bolen (Auton et al., Proc Natl Acad Sci 2007; 104:15317–15323). PMID:20198681

  1. An Extensive Targeted Proteomic Analysis of Disease-Related Protein Biomarkers in Urine from Healthy Donors

    PubMed Central

    Nolen, Brian M.; Orlichenko, Lidiya S.; Marrangoni, Adele; Velikokhatnaya, Liudmila; Prosser, Denise; Grizzle, William E.; Ho, Kevin; Jenkins, Frank J.; Bovbjerg, Dana H.; Lokshin, Anna E.

    2013-01-01

    The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development. PMID:23723977

  2. Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

    PubMed Central

    Labunskyy, Vyacheslav M.; Gerashchenko, Maxim V.; Delaney, Joe R.; Kaya, Alaattin; Kennedy, Brian K.; Kaeberlein, Matt; Gladyshev, Vadim N.

    2014-01-01

    Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. PMID:24391512

  3. Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance.

    PubMed

    Boll, Björn; Bessa, Juliana; Folzer, Emilien; Ríos Quiroz, Anacelia; Schmidt, Roland; Bulau, Patrick; Finkler, Christof; Mahler, Hanns-Christian; Huwyler, Jörg; Iglesias, Antonio; Koulov, Atanas V

    2017-04-03

    A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.

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

    PubMed Central

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

    2009-01-01

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

  5. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk.

    PubMed

    Manjunath, Puttaswamy; Nauc, Veronica; Bergeron, Annick; Ménard, Martin

    2002-10-01

    Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.

  6. Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension.

    PubMed

    Stelter, Philipp; Huber, Ferdinand M; Kunze, Ruth; Flemming, Dirk; Hoelz, André; Hurt, Ed

    2015-06-04

    Eukaryotic ribosome biogenesis requires nuclear import and hierarchical incorporation of ∼80 ribosomal proteins (RPs) into the ribosomal RNA core. In contrast to prokaryotes, many eukaryotic RPs possess long extensions that interdigitate in the mature ribosome. RpL4 is a prime example, with an ∼80-residue-long surface extension of unknown function. Here, we identify assembly chaperone Acl4 that initially binds the universally conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain, thereby restricting RpL4 loop insertion at its cognate nascent rRNA site. RpL4 release from Acl4 is orchestrated with pre-ribosome assembly, during which the eukaryote-specific RpL4 extension makes several distinct interactions with the 60S surface, including a co-evolved site on neighboring RpL18. Consequently, mutational inactivation of this contact site, on either RpL4 or RpL18, impairs RpL4-Acl4 disassembly and RpL4 pre-ribosome incorporation. We propose that hierarchical ribosome assembly can be achieved by eukaryotic RP extensions and dedicated assembly chaperones. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer.

    PubMed

    Podust, Vladimir N; Sim, Bee-Cheng; Kothari, Dharti; Henthorn, Lana; Gu, Chen; Wang, Chia-wei; McLaughlin, Bryant; Schellenberger, Volker

    2013-11-01

    XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.

  8. Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein A.

    PubMed

    Unverdorben, Felix; Färber-Schwarz, Aline; Richter, Fabian; Hutt, Meike; Kontermann, Roland E

    2012-02-01

    Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

  9. Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

    PubMed Central

    Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

    2012-01-01

    A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

  10. Amino acid sequences of peptides from a tryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool

    PubMed Central

    Corfield, M. C.; Fletcher, J. C.; Robson, A.

    1967-01-01

    1. A tryptic digest of the protein fraction U.S.3 from oxidized wool has been separated into 32 peptide fractions by cation-exchange resin chromatography. 2. Most of these fractions have been resolved into their component peptides by a combination of the techniques of cation-exchange resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid compositions of 58 of the peptides in the digest present in the largest amounts have been determined. 4. The amino acid sequences of 38 of these have been completely elucidated and those of six others partially derived. 5. These findings indicate that the parent protein in wool from which the protein fraction U.S.3 is derived has a minimum molecular weight of 74000. 6. The structures of wool proteins are discussed in the light of the peptide sequences determined, and, in particular, of those sequences in fraction U.S.3 that could not be elucidated. PMID:16742497

  11. The nanoparticle protein corona formed in human blood or human blood fractions

    PubMed Central

    Augustsson, Cecilia; Lilja, Malin; Lundkvist, Kristoffer; Dahlbäck, Björn; Linse, Sara; Cedervall, Tommy

    2017-01-01

    The protein corona formed around nanoparticles in protein-rich fluids plays an important role for nanoparticle biocompatibility, as found in several studies during the last decade. Biological fluids have complex compositions and the molecular components interact and function together in intricate networks. Therefore, the process to isolate blood or the preparation of blood derivatives may lead to differences in the composition of the identified protein corona around nanoparticles. Here, we show distinct differences in the protein corona formed in whole blood, whole blood with EDTA, plasma, or serum. Furthermore, the ratio between particle surface area to protein concentration influences the detected corona. We also show that the nanoparticle size per se influences the formed protein corona due to curvature effects. These results emphasize the need of investigating the formation and biological importance of the protein corona in the same environment as the nanoparticles are intended for or released into. PMID:28414772

  12. Identification of proteins from a cell wall fraction of the diatom Thalassiosira pseudonana: insights into silica structure formation.

    PubMed

    Frigeri, Luciano G; Radabaugh, Timothy R; Haynes, Paul A; Hildebrand, Mark

    2006-01-01

    Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and

  13. Kinetics, aggregation behavior and optimization of the fractionation of whey protein isolate with hydrochloric acid

    USDA-ARS?s Scientific Manuscript database

    Concentrated WPI solutions (10% (w/w)) containing approximately 30% alpha-lactalbumin (alpha-LA) and 60% beta-lactoglobulin (beta-LG) were fractionated with HCl at acidic pH and moderate temperatures to denature alpha-LA and recover the alpha-LA aggregates via centrifugation. Aggregation behavior an...

  14. Multiscale enhanced sampling for protein systems: An extension via adiabatic separation

    NASA Astrophysics Data System (ADS)

    Moritsugu, Kei; Terada, Tohru; Kidera, Akinori

    2016-09-01

    Multiscale enhanced sampling (MSES) calculates the configurational ensemble of all-atom (MM) protein systems with the help of coupling to a coarse-grained (CG) model. Here, for further improvement of the sampling efficiency, the approximation of adiabatic separation was introduced to the original MSES, by adopting a high CG temperature limit. An application to the folding of chignolin in explicit solvent demonstrated that the MSES formula based on adiabatic separation correctly sampled the canonical ensemble with excellent efficiency and robustness against the parameter selection, and thus MSES successfully achieved the scalability for applications to large protein systems.

  15. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed

    Gianazza, E; Arnaud, P

    1982-01-01

    The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.

  16. Automated 2D-HPLC method for characterization of protein aggregation with in-line fraction collection device.

    PubMed

    Williams, Abasha; Read, Erik K; Agarabi, Cyrus D; Lute, Scott; Brorson, Kurt A

    2017-03-01

    Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module. Published by Elsevier B.V.

  17. Proteomic characterization of specific minor proteins in the human milk casein fraction.

    PubMed

    Liao, Yalin; Alvarado, Rudy; Phinney, Brett; Lönnerdal, Bo

    2011-12-02

    Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

  18. The significance of the incorporation of [14C]leucine into different protein fractions by isolated ox heart mitochondria

    PubMed Central

    Krymkiewicz, Norberto; González-Cadavid, Néstor

    1970-01-01

    The problem of whether isolated mitochondria are able to synthesize specific proteins was investigated, particular consideration being paid to the possible contribution of micro-organisms to this activity. With ox heart mitochondria it was shown that: (1) The medium used for the incubations inhibits the exponential phase of bacterial growth for at least 8h either in the absence or the presence of fresh mitochondria, but the inhibition disappears after 4h when mitochondria damaged by freezing and thawing are used. (2) The incorporation of [14C]leucine into total proteins is linear up to at least 8h, although part of the radioactivity at the later periods might be due to some incorporation by resting-phase bacteria. (3) A contamination by as little as 800 cells/mg of mitochondrial protein is enough to contribute substantially to the total radioactivity incorporated by the mitochondrial preparations. (4) Purified cytochrome b and cytochrome oxidase are labelled even under conditions of minimal contamination by micro-organisms (less than 60 cells/mg of mitochondrial protein) and the contribution of bacterial proteins to the radioactivity found in cytochromes is negligible, as shown by double-labelling experiments. (5) At 4h the specific radioactivities of cytochrome b and cytochrome oxidase are seven- and 16-fold lower respectively than that of a structural protein-rich fraction, suggesting that the labelling of cytochromes is due to a residual contamination by these proteins. PMID:5414100

  19. Thermal and physicochemical properties and nutritional value of the protein fraction of Mexican chia seed (Salvia hispanica L.).

    PubMed

    Olivos-Lugo, B L; Valdivia-López, M Á; Tecante, A

    2010-02-01

    Thermal, functional and nutritional properties of the main protein fractions and a protein isolate of chia seed from the state of Jalisco, Mexico, were studied by differential scanning calorimetry, gelling, foaming, water-holding capacity (WHC) and oil-holding capacity, amino acid profile, chemical score and in vitro digestibility tests. The protein isolate showed good WHC (4.06 g/g) and excellent oil-retention capacities (4.04 g/g), making it attractive as an additive in bakery products and food emulsions. It also contained high amounts of glutamic acid (123 g/kg raw protein), arginine (80.6 g/kg raw protein) and aspartic acid (61.3 g/kg raw protein). However, its essential amino acid profile showed deficiencies with respect to the 1985 standard of the FAO/WHO/UNU for pre-school children. Therefore, its use as a sole protein source is not recommended; supplementation with a lysine-rich source would be necessary, as this was the limiting amino acid.

  20. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    USDA-ARS?s Scientific Manuscript database

    In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. C...

  1. Structuprint: a scalable and extensible tool for two-dimensional representation of protein surfaces.

    PubMed

    Kontopoulos, Dimitrios Georgios; Vlachakis, Dimitrios; Tsiliki, Georgia; Kossida, Sofia

    2016-02-24

    The term 'molecular cartography' encompasses a family of computational methods for two-dimensional transformation of protein structures and analysis of their physicochemical properties. The underlying algorithms comprise multiple manual steps, whereas the few existing implementations typically restrict the user to a very limited set of molecular descriptors. We present Structuprint, a free standalone software that fully automates the rendering of protein surface maps, given - at the very least - a directory with a PDB file and an amino acid property. The tool comes with a default database of 328 descriptors, which can be extended or substituted by user-provided ones. The core algorithm comprises the generation of a mould of the protein surface, which is subsequently converted to a sphere and mapped to two dimensions, using the Miller cylindrical projection. Structuprint is partly optimized for multicore computers, making the rendering of animations of entire molecular dynamics simulations feasible. Structuprint is an efficient application, implementing a molecular cartography algorithm for protein surfaces. According to the results of a benchmark, its memory requirements and execution time are reasonable, allowing it to run even on low-end personal computers. We believe that it will be of use - primarily but not exclusively - to structural biologists and computational biochemists.

  2. Six Subgroups and Extensive Recent Duplications Characterize the Evolution of the Eukaryotic Tubulin Protein Family

    PubMed Central

    Findeisen, Peggy; Mühlhausen, Stefanie; Dempewolf, Silke; Hertzog, Jonny; Zietlow, Alexander; Carlomagno, Teresa; Kollmar, Martin

    2014-01-01

    Tubulins belong to the most abundant proteins in eukaryotes providing the backbone for many cellular substructures like the mitotic and meiotic spindles, the intracellular cytoskeletal network, and the axonemes of cilia and flagella. Homologs have even been reported for archaea and bacteria. However, a taxonomically broad and whole-genome-based analysis of the tubulin protein family has never been performed, and thus, the number of subfamilies, their taxonomic distribution, and the exact grouping of the supposed archaeal and bacterial homologs are unknown. Here, we present the analysis of 3,524 tubulins from 504 species. The tubulins formed six major subfamilies, α to ζ. Species of all major kingdoms of the eukaryotes encode members of these subfamilies implying that they must have already been present in the last common eukaryotic ancestor. The proposed archaeal homologs grouped together with the bacterial TubZ proteins as sister clade to the FtsZ proteins indicating that tubulins are unique to eukaryotes. Most species contained α- and/or β-tubulin gene duplicates resulting from recent branch- and species-specific duplication events. This shows that tubulins cannot be used for constructing species phylogenies without resolving their ortholog–paralog relationships. The many gene duplicates and also the independent loss of the δ-, ε-, or ζ-tubulins, which have been shown to be part of the triplet microtubules in basal bodies, suggest that tubulins can functionally substitute each other. PMID:25169981

  3. Antioxidant and Antihypertensive Potential of Protein Fractions from Flour and Milk Substitutes from Canary Seeds (Phalaris canariensis L.).

    PubMed

    Valverde, María Elena; Orona-Tamayo, Domancar; Nieto-Rendón, Blanca; Paredes-López, Octavio

    2017-03-01

    Canary seed (Phalaris canariensis) is used to feed birds but it has been recently considered a promising cereal with nutraceutical potential for humans. The aim of this work was to analyze the protein fractions from canary seed flour and from milk substitutes (prepared by soaking the seeds in water 12 and 24 h), and to evaluate antioxidant and antihypertensive capacity of peptides obtained after in vitro digestion. Prolamins were the major protein fraction, followed by glutelins. After digestion, albumins and prolamins fractions from milks presented higher levels of peptides than flour, globulins showed more peptides in flour and glutelins were found in similar concentrations in all samples; 24 h milk prolamins had the highest concentration of peptides. Purification by high performance liquid chromatography (HPLC), sequencing of peptides, in vitro antioxidant ABTS (2,2'-azino-bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, and antihypertensive capacity (angiotensin converting enzyme (ACE) assay), indicated that peptides from canary seed prolamins were the most efficient compounds with antioxidant and antihypertensive activity. Canary seeds may be considered an accessible and cheap source to prepare milk substitutes with high contents of bioactive peptides with remarkable functional properties to promote better human health and healthy ageing.

  4. [Effect of graded dietary protein rations on the amino acid content of crude protein in various parts of the gastrointestinal tract and blood fractions of laying hens].

    PubMed

    Gruhn, K; Wiefel, P

    1985-03-01

    Four groups of four colostomized laying hens each received rations only consisting of wheat, vitamins plus a mineral mixture and a graded daily feed supply of 110 g, 88 g, 66 g and 44 g. The determination of amino acids from faeces was carried out after hydrolysis from a 6-day sampling period from the 16 laying hybrids. At the end of the experiment the animals were slaughtered. The corpuscular fractions of the blood, the contents of crop and stomach as well as of the intestines were also hydrolysed and the amino acid content in the crude protein was determined. In addition, the content of free amino acids in the blood plasma was determined. The content of amino acids in the protein of the corpuscular blood fractions remained uninfluenced by the decreasing amino acid and energy supply. The content of free basic amino acids in the blood plasma decreased with the decreasing supply with amino acids and energy, whereas the content of free amino acids with branched chains and hydroxylized ones increased. The content of glutamic acid in the contents of crop and stomach changed considerably in comparison with feed protein. The amino acid values of the crude protein in the contents of the intestines and in faeces to a large extent differ considerably from those of the wheat fed and are approximate values of body protein. Deficient supply with amino acids and energy did not influence the apparent digestibility of the amino acids.

  5. STUDY OF THE BLOOD PROTEIN BY THE ELECTROPHORETIC FRACTIONIZATION METHOD OF CHRONIC DISEASES OF THE MONTH,

    DTIC Science & Technology

    BLOOD PROTEINS, *HISTAMINE, *MOUTH, ELECTROPHORESIS, BLOOD SERUM , DISEASES, GASTROINTESTINAL SYSTEM, HERPETIC VIRUSES, INFECTIOUS DISEASES, DIAGNOSIS(MEDICINE), DIAMINE OXIDASE, CAPILLARIES(ANATOMY), PERMEABILITY, USSR.

  6. Haematococcus pluvialis soluble proteins: Extraction, characterization, concentration/fractionation and emulsifying properties.

    PubMed

    Ba, Fatou; Ursu, Alina Violeta; Laroche, Céline; Djelveh, Gholamreza

    2016-01-01

    A water-soluble matrix was extracted from green vegetative Haematococcus pluvialis through high-pressure cell disruption either at native pH (5.7) or with pH shifting to neutral (7). The resulting supernatant is mainly composed of carbohydrates and proteins, with the highest yield of proteins obtained at neutral pH (73±2% of total biomass proteins). The key emulsification properties of the proteins isolated in neutral supernatant (emulsification capacity (EC): 534±41mLoilg(-1) protein, emulsification stability (ES): 94±3% and emulsification activity index (EAI): 80±1m(2)g(-1)) were comparable to the native supernatant values (EC: 589±21mLoilg(-1) protein, ES: 84±3% and EAI: 75±1m(2)g(-1)). Confronted to sodium caseinate (EC: 664±30mLoilg(-1) protein, ES: 63±4%, and EAI: 56±4m(2)g(-1)) these results highlighted the strong potential of proteins isolated from H. pluvialis as emulsifier agent. Moreover, experiments have shown that the stability of emulsions obtained from supernatants is due to the proteins rather than the carbohydrates.

  7. A chemical study of the protein fraction of Mediterranean sweet almond varieties (Prunus amygdalus).

    PubMed

    Calixto, F S; Cañellas, J; Martinez de Toda, F

    1982-01-01

    The protein contents and amino-acid compositions of five Mediterranean almond varieties were determined. The protein contents were between 18 and 24 g/100 g of almond, according to variety. The content of each amino-acid, expressed in g/100 g of protein, was similar in all varieties. The essential amino-acid composition was compared with the FAO pattern of amino-acid requirements and to the protein of milk and eggs. Percentages of albumins, globulins, glutelins and prolamines were also determined.

  8. Mitochondrial genome evolution in Alismatales: Size reduction and extensive loss of ribosomal protein genes.

    PubMed

    Petersen, Gitte; Cuenca, Argelia; Zervas, Athanasios; Ross, Gregory T; Graham, Sean W; Barrett, Craig F; Davis, Jerrold I; Seberg, Ole

    2017-01-01

    The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus.

  9. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters.

    PubMed

    Strohl, William R

    2015-08-01

    The purpose of making a "biobetter" biologic is to improve on the salient characteristics of a known biologic for which there is, minimally, clinical proof of concept or, maximally, marketed product data. There already are several examples in which second-generation or biobetter biologics have been generated by improving the pharmacokinetic properties of an innovative drug, including Neulasta(®) [a PEGylated, longer-half-life version of Neupogen(®) (filgrastim)] and Aranesp(®) [a longer-half-life version of Epogen(®) (epoetin-α)]. This review describes the use of protein fusion technologies such as Fc fusion proteins, fusion to human serum albumin, fusion to carboxy-terminal peptide, and other polypeptide fusion approaches to make biobetter drugs with more desirable pharmacokinetic profiles.

  10. Mitochondrial genome evolution in Alismatales: Size reduction and extensive loss of ribosomal protein genes

    PubMed Central

    Cuenca, Argelia; Zervas, Athanasios; Ross, Gregory T.; Graham, Sean W.; Barrett, Craig F.; Davis, Jerrold I.; Seberg, Ole

    2017-01-01

    The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus. PMID:28545148

  11. Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes.

    PubMed

    Travers, Timothy S; Harlow, Lisa; Rosas, Ivan O; Gochuico, Bernadette R; Mikuls, Ted R; Bhattacharya, Sanjoy K; Camacho, Carlos J; Ascherman, Dana P

    2016-09-01

    Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context. Copyright © 2016 by The American Association of Immunologists, Inc.

  12. Protective Effect of High Molecular Weight Protein Sub-fraction of Calotropis procera Latex in Monoarthritic Rats

    PubMed Central

    Chaudhary, Priyanka; Ramos, Marcio V.; Vasconcelos, Mirele da Silveira; Kumar, Vijay L.

    2016-01-01

    Background: Proteins present in the latex of Calotropis procera have been shown to produce anti-inflammatory effect and to afford protection in various disease models. Objectives: To determine the efficacy of high molecular weight protein sub-fraction (LPPI) of latex of C. procera in ameliorating joint inflammation and hyperalgesia in a preclinical model of arthritis. Materials and Methods: Monoarthritis was induced in rats by intra-articular injection of Freund's complete adjuvant (FCA) and the effect of two doses of LPPI (5 and 25 mg/kg) and diclofenac (5 mg/kg) was evaluated on joint swelling, stair climbing ability, motility, and dorsal flexion pain on day 3. The rats were sacrificed on day 3 to measure tissue levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). Evaluation of joint histology was also made. Results: Intra-articular injection of FCA produced joint swelling and difficulty in stair climbing ability, motility, and pain on flexion of the joint as revealed by scores obtained for these functional parameters. LPPI produced a dose-dependent decrease in joint swelling and improved joint functions. Arthritic rats also revealed altered oxidative homeostasis where joint tissue GSH levels were decreased and TBARS levels were increased as compared to normal rats. The levels of these oxidative stress markers were near normal in arthritic rats treated with LPPI. Moreover, treatment with LPPI also maintained the structural integrity of the joint. The protective effect of LPPI was comparable to the standard anti-inflammatory drug, diclofenac. Conclusion: The findings of the present study show that LPPI fraction comprising high molecular weight proteins could be used for the alleviation of arthritic symptoms. SUMMARY High molecular weight protein sub-fraction of latex of Calotropis procera (LPPI) reduced joint swelling and hyperalgesia in arthritic ratsLPPI produced a significant improvement in stair climbing ability and motility

  13. Protective Effect of High Molecular Weight Protein Sub-fraction of Calotropis procera Latex in Monoarthritic Rats.

    PubMed

    Chaudhary, Priyanka; Ramos, Marcio V; Vasconcelos, Mirele da Silveira; Kumar, Vijay L

    2016-05-01

    Proteins present in the latex of Calotropis procera have been shown to produce anti-inflammatory effect and to afford protection in various disease models. To determine the efficacy of high molecular weight protein sub-fraction (LPPI) of latex of C. procera in ameliorating joint inflammation and hyperalgesia in a preclinical model of arthritis. Monoarthritis was induced in rats by intra-articular injection of Freund's complete adjuvant (FCA) and the effect of two doses of LPPI (5 and 25 mg/kg) and diclofenac (5 mg/kg) was evaluated on joint swelling, stair climbing ability, motility, and dorsal flexion pain on day 3. The rats were sacrificed on day 3 to measure tissue levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). Evaluation of joint histology was also made. Intra-articular injection of FCA produced joint swelling and difficulty in stair climbing ability, motility, and pain on flexion of the joint as revealed by scores obtained for these functional parameters. LPPI produced a dose-dependent decrease in joint swelling and improved joint functions. Arthritic rats also revealed altered oxidative homeostasis where joint tissue GSH levels were decreased and TBARS levels were increased as compared to normal rats. The levels of these oxidative stress markers were near normal in arthritic rats treated with LPPI. Moreover, treatment with LPPI also maintained the structural integrity of the joint. The protective effect of LPPI was comparable to the standard anti-inflammatory drug, diclofenac. The findings of the present study show that LPPI fraction comprising high molecular weight proteins could be used for the alleviation of arthritic symptoms. High molecular weight protein sub-fraction of latex of Calotropis procera (LPPI) reduced joint swelling and hyperalgesia in arthritic ratsLPPI produced a significant improvement in stair climbing ability and motility in arthritic ratsLPPI normalized the levels of oxidative stress markers in

  14. A study of low level selenium determination by hydride generation atomic fluorescence spectrometry in water soluble protein and peptide fractions.

    PubMed

    Stibilj, V; Mazej, D; Falnoga, I

    2003-12-01

    Development of a method for very low level selenium determination in water soluble protein and peptide fractions, obtained after various separation procedures, is presented. A hydride generation atomic fluorescence spectrometry (HG-AFS) detection system was optimised and the influence of Cu(II), Sb(V), As(III) and HNO3 interferences in the measurement of Se by HG-AFS was investigated. A destruction procedure using HNO3 and H2O2 was also optimised and the average recovery of the digestion of a solution of selenomethioneine was 92 +/- 4% (n=14). Combination of this digestion with the detection system gave reliable results. Accuracy was tested by comparison with two independent methods. A very low detection limit (DL) of 0.2 ng/g of measuring solution was achieved. The whole procedure from weighing to measuring was performed in the same Teflon tube. The addition of HNO3 to the fractions before long term storage at -20 degrees C was necessary to prevent adsorption on the test tubes. Selenium was measured in water soluble protein and peptide fractions obtained after extraction, and Sephadex G-75 chromatography performed on liver samples from: i) hens exposed to As2O3, ii) hens fed with a high fat feed and iii) the certified reference material dogfish liver (CRM DOLT-2). Because of the very low DL we were able to observe the Se distribution in chromatographic fractions of samples of organisms which were not exposed to excess amounts of Se. The presence of selenium associated with metallothioneins was observed.

  15. Protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves: enzymatic characterization and procoagulant and fibrinogenolytic activities.

    PubMed

    de Menezes, Yamara A S; Félix-Silva, Juliana; da Silva-Júnior, Arnóbio A; Rebecchi, Ivanise M M; de Oliveira, Adeliana S; Uchoa, Adriana F; Fernandes-Pedrosa, Matheus de F

    2014-03-21

    Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  16. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system.

    PubMed

    Hogan, Daniel J; Riordan, Daniel P; Gerber, André P; Herschlag, Daniel; Brown, Patrick O

    2008-10-28

    RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.

  17. Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.

    PubMed

    Pal, Manoj; Moffa, Allison; Sreekumar, Arun; Ethier, Stephen P; Barder, Timothy J; Chinnaiyan, Arul; Lubman, David M

    2006-02-01

    A combination of protein microarrays and two-dimensional liquid-phase separation of proteins has been used for global profiling of the phosphoproteome in human breast cancer cells. This method has been applied to study changes in phosphorylation profile resulting from treatment of the cancer cells with PD173074, a known receptor tyrosine kinase inhibitor. The proteins separated by 2-D liquid-phase separation were arrayed on epoxy-coated glass slides and first screened for phosphorylation using fluorescent Pro-Q Diamond stain. The candidate proteins were then identified using MALDI/ESI MS/MS analysis. Further, validation was achieved by immunoblot analysis using anti-phosphotyrosine antibodies. A dynamic range of approximately 100 was achieved on the microarray when beta-casein was used as a standard protein for obtaining quantitative data. Importantly, the power of this method lies in its ability to identify a large group of proteins in a single experiment that are coregulated in their posttranslational modifications, upon treatment with the inhibitor. Since proteins are known to form interacting circuits that eventually lead to various signaling events, detection of such global phosphorylation profiles might enable delineation of functional pathways that play an important role during cancer initiation and progression.

  18. Protein Molecular Structures and Protein Fraction Profiles of New Co-Products of BioEthanol Production: A Novel Approach

    SciTech Connect

    Yu, P.; Niu, Z; Damiran, D

    2010-01-01

    The objectives of this study were to determine the protein molecular structures of the new coproducts from bioethanol production, quantify protein structure amide I to II and {alpha}-helix to {beta}-sheet spectral peak intensity ratio, and illustrate multivariate molecular spectral analyses as a novel research tool for rapid characterization of protein molecular structures in bioethonal bioproducts. The study demonstrated that the grains had a significantly higher ratio of {alpha}-helix to {beta}-sheet in the protein structure than their coproducts produced from bioethanol processing (1.38 vs 1.03, P < 0.05). There were significant differences between wheat and corn (1.47 vs 1.29, P < 0.05) but no difference between wheat dried distiller grains with solubles (DDGS) and corn DDGS (1.04 vs 1.03, P > 0.05). The grains had a significantly higher ratio of protein amide I to II in the protein structure than their coproducts produced from bioethanol processing (4.58 vs 2.84, P < 0.05). There were no significant differences between wheat and corn (4.61 vs 4.56, P > 0.05), but there were significant differences between wheat DDGS and corn DDGS (3.08 vs 2.21, P < 0.05). This preliminary study indicated that bioethanol processing changes protein molecular structures, compared with original grains. Further study is needed with a large set of the new bioethanol coproducts to quantify protein molecular structures ({alpha}-helix to {beta}-sheet ratio; amide I to II ratio) of the bioethanol coproducts in relation to nutrient supply and availability in animals.

  19. Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

    PubMed Central

    Ng, Julian

    2012-01-01

    Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus. PMID:22147954

  20. Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ${\\Phi}$6 Major Capsid Protein

    SciTech Connect

    Nemecek, Daniel; Plevka, Pavel; Boura, Evzen

    2013-11-29

    Bacteriophage ${\\Phi}$6 is a double-stranded RNA virus that has been extensively studied as a model organism. In this paper we describe structure determination of ${\\Phi}$6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ${\\Phi}$6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. Lastly, the averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.

  1. Extensive Positive Selection Drives the Evolution of Nonstructural Proteins in Lineage C Betacoronaviruses

    PubMed Central

    Cagliani, Rachele; Mozzi, Alessandra; Pozzoli, Uberto; Al-Daghri, Nasser; Clerici, Mario; Sironi, Manuela

    2016-01-01

    ABSTRACT Middle East respiratory syndrome-related coronavirus (MERS-CoV) spreads to humans via zoonotic transmission from camels. MERS-CoV belongs to lineage C of betacoronaviruses (betaCoVs), which also includes viruses isolated from bats and hedgehogs. A large portion of the betaCoV genome consists of two open reading frames (ORF1a and ORF1b) that are translated into polyproteins. These are cleaved by viral proteases to generate 16 nonstructural proteins (nsp1 to nsp16) which compose the viral replication-transcription complex. We investigated the evolution of ORF1a and ORF1b in lineage C betaCoVs. Results indicated widespread positive selection, acting mostly on ORF1a. The proportion of positively selected sites in ORF1a was much higher than that previously reported for the surface-exposed spike protein. Selected sites were unevenly distributed, with nsp3 representing the preferential target. Several pairs of coevolving sites were also detected, possibly indicating epistatic interactions; most of these were located in nsp3. Adaptive evolution at nsp3 is ongoing in MERS-CoV strains, and two selected sites (G720 and R911) were detected in the protease domain. While position 720 is variable in camel-derived viruses, suggesting that the selective event does not represent a specific adaptation to humans, the R911C substitution was observed only in human-derived MERS-CoV isolates, including the viral strain responsible for the recent South Korean outbreak. It will be extremely important to assess whether these changes affect host range or other viral phenotypes. More generally, data herein indicate that CoV nsp3 represents a major selection target and that nsp3 sequencing should be envisaged in monitoring programs and field surveys. IMPORTANCE Both severe acute respiratory syndrome coronavirus (SARS-CoV) and MERS-CoV originated in bats and spread to humans via an intermediate host. This clearly highlights the potential for coronavirus host shifting and the relevance

  2. Ebony (Phitecellobium flexicaule Benth) and proteins fractionation, solubilization, characterization and production of an isolate.

    PubMed

    González-Quijada, Mario R; Alanís-Guzman, María Guadalupe; Serna-Saldivar, Sergio O

    2003-03-01

    Different combinations of pHs (2 to 12) and temperatures (25, 30 and 35 degrees C) were tested to obtain a protein isolate from ebony (Pithecellobium flexicaule, Benth) seeds. Seed proteins contained 54.6% albumins, 32% globulins, 5.7% glutelins and 1.3% prolamins. The isoelectric points for albumins, globulins and glutelins were in the pH range of 2.3-2.7. The average molecular weight of albumins ranged from 92 to 100 kDa and for the four globulin subunits in the range of 28.4 to 57.3 kDa. For isolate production, proteins were sequentially extracted with distilled water and a 5% NaCl solution. The resulting supernatants were mixed. The best extraction was achieved at pH 11 and 25 degrees C. 45.6% of the total seed protein was precipitated at pH 2.6 yielding an isolate with 90% protein (N x 6.25). The isolate contained high quantities of lysine, leucine, threonine and phenylalanine but were low in sulfur containing amino acids methionine and cysteine. The extraction process reduced tannins, phytates and trypsin inhibitor in 53, 70 and 70%, respectively. In vivo protein digestibility of the protein isolate was 85.4% and the corrected digestibility essential amino acid score was of 44% due to the lack of sulfur containing amino acids. In order to upgrade the protein quality of ebony isolate it is recommend to supplement with methionine or sulfur containing rich foods.

  3. Development and characterization of biodegradable films made from wheat gluten protein fractions.

    PubMed

    Hernández-Muñoz, Pilar; Kanavouras, Antonis; Ng, Perry K W; Gavara, Rafael

    2003-12-17

    Gliadins and glutenins were extracted from commercial wheat gluten on the basis of their extractability in ethanol and used to produce film-forming solutions. Films cast using these gliadin- and glutenin-rich solutions were characterized. Glycerol was used as a plasticizer, and its effect on the films was also studied. Films obtained from the glutenin fraction presented higher tensile strength values and lower elongation at break and water vapor permeability values than gliadin films. Gliadin films disintegrated when immersed in water. The GAB isotherm model was used to describe the equilibrium moisture sorption of the films. The glycerol concentration largely modified mechanical and water vapor barrier properties of both film types.

  4. An extensively hydrolysed rice protein-based formula in the management of infants with cow's milk protein allergy: preliminary results after 1 month.

    PubMed

    Vandenplas, Yvan; De Greef, Elisabeth; Hauser, Bruno

    2014-10-01

    Guidelines recommend extensively hydrolysed cow's milk protein formulas (eHF) in the treatment of infants diagnosed with cow's milk protein allergy (CMPA). Extensively hydrolysed rice protein infant formulas (eRHFs) have recently become available, and could offer a valid alternative. A prospective trial was performed to evaluate the clinical tolerance of a new eRHF in infants with a confirmed CMPA. Patients were followed for 1 month. Clinical tolerance of the eRHF was evaluated with a symptom-based score (SBS) and growth (weight and length) was monitored. Thirty-nine infants (mean age 3.4 months, range 0.5-6 months) diagnosed with CMPA were enrolled. All infants tolerated the eRHF and experienced a normal growth. In accordance with current guidelines, this eRHF is tolerated by more than 90% of children with proven CMPA with a 95% CI, and is an adequate alternative to cow's milk-based eHF. ClinicalTrials.gov NCT01998074. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  5. Safety and tolerance of a new extensively hydrolyzed rice protein-based formula in the management of infants with cow's milk protein allergy.

    PubMed

    Vandenplas, Yvan; De Greef, Elisabeth; Hauser, Bruno

    2014-09-01

    Guidelines recommend the use of extensively hydrolyzed cow's milk protein-based formulas (eHF) in the treatment of infants with cow's milk protein allergy (CMPA). Extensively hydrolyzed rice protein infant formula (eRHF) has recently become available and could offer a valid alternative. A prospective trial was performed to evaluate the hypo-allergenicity and safety of a new eRHF in infants with a confirmed CMPA. Patients were fed the study formula for 6 months. Clinical tolerance of the eRHF was evaluated with a symptom-based score (SBS) and growth (weight and length) was monitored. Forty infants (mean age, 3.4 months; range, 1-6 months) with CMPA confirmed by a food challenge were enrolled. All infants tolerated the eRHF and the SBS significantly decreased as of the first month of intervention. Moreover, the eRHF allowed a catch-up to normal weight gain as of the first month as well as a normalization of the weight-for-age, weight-for length, and BMI z-scores within the 6-month study period. In accordance with current guidelines, this eRHF was tolerated by more than 90 % of children with proven CMPA with a 95 % confidence interval. This eRHF is an adequate and safe alternative to cow milk-based eHF.

  6. Optimization of protein fractionation by skim milk microfiltration: Choice of ceramic membrane pore size and filtration temperature.

    PubMed

    Jørgensen, Camilla Elise; Abrahamsen, Roger K; Rukke, Elling-Olav; Johansen, Anne-Grethe; Schüller, Reidar B; Skeie, Siv B

    2016-08-01

    The objective of this study was to investigate how ceramic membrane pore size and filtration temperature influence the protein fractionation of skim milk by cross flow microfiltration (MF). Microfiltration was performed at a uniform transmembrane pressure with constant permeate flux to a volume concentration factor of 2.5. Three different membrane pore sizes, 0.05, 0.10, and 0.20µm, were used at a filtration temperature of 50°C. Furthermore, at pore size 0.10µm, 2 different filtration temperatures were investigated: 50 and 60°C. The transmission of proteins increased with increasing pore size, giving the permeate from MF with the 0.20-µm membrane a significantly higher concentration of native whey proteins compared with the permeates from the 0.05- and 0.10-µm membranes (0.50, 0.24, and 0.39%, respectively). Significant amounts of caseins permeated the 0.20-µm membrane (1.4%), giving a permeate with a whitish appearance and a casein distribution (αS2-CN: αS1-CN: κ-CN: β-CN) similar to that of skim milk. The 0.05- and 0.10-µm membranes were able to retain all caseins (only negligible amounts were detected). A permeate free from casein is beneficial in the production of native whey protein concentrates and in applications where transparency is an important functional characteristic. Microfiltration of skim milk at 50°C with the 0.10-µm membrane resulted in a permeate containing significantly more native whey proteins than the permeate from MF at 60°C. The more rapid increase in transmembrane pressure and the significantly lower concentration of caseins in the retentate at 60°C indicated that a higher concentration of caseins deposited on the membrane, and consequently reduced the native whey protein transmission. Optimal protein fractionation of skim milk into a casein-rich retentate and a permeate with native whey proteins were obtained by 0.10-µm MF at 50°C.

  7. Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.

    PubMed

    Wang, Wei; Lu, Joann J; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-07-16

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

  8. Overexpression of Pa_1_10620 encoding a mitochondrial Podospora anserina protein with homology to superoxide dismutases and ribosomal proteins leads to lifespan extension.

    PubMed

    Grimm, Carolin; Böhl, Lena; Osiewacz, Heinz D

    2015-02-01

    In biological systems, reactive oxygen species (ROS) represent 'double edged swords': as signaling molecules they are essential for proper development, as reactive agents they cause molecular damage and adverse effects like degeneration and aging. A well-coordinated control of ROS is therefore of key importance. Superoxide dismutases (SODs) are enzymes active in the detoxification of superoxide. The number of isoforms of these proteins varies among species. Here we report the characterization of the putative protein encoded by Pa_1_10620 that has been previously annotated to code for a mitochondrial ribosomal protein but shares also sequence domains with SODs. We report that the gene is transcribed in P. anserina cultures of all ages and that the encoded protein localizes to mitochondria. In strains overexpressing Pa_1_10620 in a genetic background in which PaSod3, the mitochondrial MnSOD of P. anserina, is deleted, no SOD activity could be identified in isolated mitochondria. However, overexpression of the gene leads to lifespan extension suggesting a pro-survival function of the protein in P. anserina.

  9. Arginine depletion by arginine deiminase does not affect whole protein metabolism or muscle fractional protein synthesis rate in mice

    USDA-ARS?s Scientific Manuscript database

    Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depl...

  10. Evidence of protein-free homology recognition in magnetic bead force-extension experiments

    NASA Astrophysics Data System (ADS)

    O'Lee, D. J.; Danilowicz, C.; Rochester, C.; Kornyshev, A. A.; Prentiss, M.

    2016-07-01

    Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data.

  11. Evidence of protein-free homology recognition in magnetic bead force–extension experiments

    PubMed Central

    (O’) Lee, D. J.; Danilowicz, C.; Rochester, C.; Prentiss, M.

    2016-01-01

    Earlier theoretical studies have proposed that the homology-dependent pairing of large tracts of dsDNA may be due to physical interactions between homologous regions. Such interactions could contribute to the sequence-dependent pairing of chromosome regions that may occur in the presence or the absence of double-strand breaks. Several experiments have indicated the recognition of homologous sequences in pure electrolytic solutions without proteins. Here, we report single-molecule force experiments with a designed 60 kb long dsDNA construct; one end attached to a solid surface and the other end to a magnetic bead. The 60 kb constructs contain two 10 kb long homologous tracts oriented head to head, so that their sequences match if the two tracts fold on each other. The distance between the bead and the surface is measured as a function of the force applied to the bead. At low forces, the construct molecules extend substantially less than normal, control dsDNA, indicating the existence of preferential interaction between the homologous regions. The force increase causes no abrupt but continuous unfolding of the paired homologous regions. Simple semi-phenomenological models of the unfolding mechanics are proposed, and their predictions are compared with the data. PMID:27493568

  12. Monitoring the fractionation of a whey protein isolate during dead-end membrane filtration using fluorescence and chemometric methods.

    PubMed

    Elshereef, Rand; Budman, Hector; Moresoli, Christine; Legge, Raymond L

    2010-01-01

    During membrane-based separation of proteins, changes in protein concentration of the permeate and retentate streams occurs over time. The current work proposes a new approach for monitoring the changes in concentrations of proteins in both permeate and retentate by making use of data collected using fluorescence spectroscopy and intrinsic protein fluorescence analyzed by multivariate statistical techniques. Whey protein isolate consists mainly of alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), and small proportion of bovine serum albumin (BSA) and was used as a model system in this study. A fiber optic probe (FOP) was used to acquire multiwavelength fluorescence spectra for permeate and retentate streams at different times during UF-based separation of the components from a multicomponent solution. Multivariate regression models were developed for predicting the concentrations of alpha-LA, beta-LG, and BSA by establishing a calibration model between data acquired using the FOP and the corresponding protein concentration levels measured by size-exclusion chromatography. The model was validated using FOP data that were not previously used for calibration of the regression models. This comparison showed that concentrations of alpha-LA, beta-LG, and BSA could be predicted directly from FOP data within reasonable accuracy by making use of multivariate calibration tools. This approach has several attractive features including that it is nondestructive, fast, and relatively simple to perform. This technique has potential practical applications as it could offer the opportunity for in situ monitoring of membrane filtration processes by tracking individual protein transmission and selectivity of fractionation.

  13. Dry Fractionation Methods to Produce Barley Meals Varying in Protein, Beta-Glucan and Starch Contents

    USDA-ARS?s Scientific Manuscript database

    Barley contains several valuable nutrients including beta-glucan (BG), protein and starch. Each has additional value when concentrated. Dehulled and hulless barleys were sequentially pearled for 1 to 6 cycles, each with 8% removal. The 6 pearled kernels plus the initial kernel were subjected to imp...

  14. Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing

    USDA-ARS?s Scientific Manuscript database

    Capillary electrophoresis (CE) and reversed-phase high performance liquid chromatography (RP-HPLC) analysis were utilized to detect differences in the sarcoplasmic protein profiles of beef strip loins subjected to aging and hydrodynamic pressure processing (HDP) treatments. At 48 h postmortem, stri...

  15. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions.

    PubMed

    McReynolds, Jayme R; Donowho, Kyle; Abdi, Amin; McGaugh, James L; Roozendaal, Benno; McIntyre, Christa K

    2010-03-01

    Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a beta-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of beta-adrenoceptors with propranolol (0.5 microg/0.2 microL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA beta-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.

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

    USDA-ARS?s Scientific Manuscript database

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

  17. FRACTIONATION OF DAIRY PROTEINS USING HIGH-PRESSURE AND SUPERCRITICAL CARBON DIOXIDE

    USDA-ARS?s Scientific Manuscript database

    While several methods have been proposed for isolating dairy proteins from milk or aqueous solution, one of the most promising uses high pressure or supercritical carbon dioxide. In our laboratory, high pressure carbon dioxide has been used to precipitate casein from milk in a continuous pilot plant...

  18. Subproteomic analysis of basic proteins in aged skeletal muscle following offgel pre-fractionation.

    PubMed

    Gannon, Joan; Ohlendieck, Kay

    2012-04-01

    The progressive loss of skeletal muscle mass is a serious pathophysiological problem in the elderly, which warrants detailed biochemical studies into the underlying mechanism of age-related fiber degeneration. Over the last few years, mass spectrometry (MS)-based proteomics has identified a considerable number of new biomarkers of muscle aging in humans and animal models of sarcopenia. However, interpretation of the proteomic findings is often complicated by technical and biological limitations. Although gel electrophoresis-based approaches represent a highly sensitive analytical way for the large-scale and high-throughput survey of global changes in skeletal muscle proteins during aging, often the presence of components with an isoelectric point in the basic range is underestimated. We, therefore, carried out a comparative subproteomic study of young versus aged rat muscle focusing on potential changes in muscle proteins with an alkaline isoelectric point, using a combination of offgel electrophoresis and two-dimensional (2D) slab gel electrophoresis. Offgel electrophoresis was successfully applied as a prefractionation step to enrich basic protein species from crude tissue extracts representing young adult versus senescent muscle specimens. Proteomics has demonstrated alterations in a small cohort of basic proteins during muscle aging. The mass spectrometric identification of altered proteins and immunoblotting revealed a decrease in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a concomitant increase in mitochondrial creatine kinase (CK) and ubiquinol cytochrome‑c reductase. This agrees with the idea of a glycolytic-to-oxidative shift during muscle aging, which is indicative of an overall fast-to-slow transition process in senescent rat muscle. Thus, alterations in the abundance of metabolic enzymes appear to play a central role in the molecular pathogenesis of age‑dependent muscle wasting.

  19. A novel mass spectrometric strategy “BEMAP” reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli

    PubMed Central

    Boysen, Anders; Palmisano, Giuseppe; Krogh, Thøger Jensen; Duggin, Iain G.; Larsen, Martin R.; Møller-Jensen, Jakob

    2016-01-01

    The attachment of sugars to proteins via side-chain oxygen atoms (O-linked glycosylation) is seen in all three domains of life. However, a lack of widely-applicable analytical tools has restricted the study of this process, particularly in bacteria. In E. coli, only four O-linked glycoproteins have previously been characterized. Here we present a glycoproteomics technique, termed BEMAP, which is based on the beta-elimination of O-linked glycans followed by Michael-addition of a phosphonic acid derivative, and subsequent titanium dioxide enrichment. This strategy allows site-specific mass-spectrometric identification of proteins with O-linked glycan modifications in a complex biological sample. Using BEMAP we identified cell surface-associated and membrane vesicle glycoproteins from Enterotoxigenic E. coli (ETEC) and non-pathogenic E. coli K-12. We identified 618 glycosylated Serine and Threonine residues mapping to 140 proteins in ETEC, including several known virulence factors, and 34 in E. coli K-12. The two strains had 32 glycoproteins in common. Remarkably, the majority of the ETEC glycoproteins were conserved in both strains but nevertheless were only glycosylated in the pathogen. Therefore, bacterial O-linked glycosylation is much more extensive than previously thought, and is especially important to the pathogen. PMID:27562176

  20. A novel mass spectrometric strategy "BEMAP" reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli.

    PubMed

    Boysen, Anders; Palmisano, Giuseppe; Krogh, Thøger Jensen; Duggin, Iain G; Larsen, Martin R; Møller-Jensen, Jakob

    2016-08-26

    The attachment of sugars to proteins via side-chain oxygen atoms (O-linked glycosylation) is seen in all three domains of life. However, a lack of widely-applicable analytical tools has restricted the study of this process, particularly in bacteria. In E. coli, only four O-linked glycoproteins have previously been characterized. Here we present a glycoproteomics technique, termed BEMAP, which is based on the beta-elimination of O-linked glycans followed by Michael-addition of a phosphonic acid derivative, and subsequent titanium dioxide enrichment. This strategy allows site-specific mass-spectrometric identification of proteins with O-linked glycan modifications in a complex biological sample. Using BEMAP we identified cell surface-associated and membrane vesicle glycoproteins from Enterotoxigenic E. coli (ETEC) and non-pathogenic E. coli K-12. We identified 618 glycosylated Serine and Threonine residues mapping to 140 proteins in ETEC, including several known virulence factors, and 34 in E. coli K-12. The two strains had 32 glycoproteins in common. Remarkably, the majority of the ETEC glycoproteins were conserved in both strains but nevertheless were only glycosylated in the pathogen. Therefore, bacterial O-linked glycosylation is much more extensive than previously thought, and is especially important to the pathogen.

  1. Molecular spectroscopic investigation on fractionation-induced changes on biomacromolecule of co-products from bioethanol processing to explore protein metabolism in ruminants.

    PubMed

    Zhang, Xuewei; Yan, Xiaogang; Beltranena, Eduardo; Yu, Peiqiang

    2014-03-25

    Fractionation processing is an efficient technology which is capable to redesign/redevelop a new food or feed product with a specified chemical and nutrient profile. This processing technique was able to produce four different fractions (called "A", "B", "C", "D" fractions/treatments) with different nutrient profile form a co-product of bioethanol processing [wheat dried distillers grains with soluble (DDGS)]. To date, there is no study on the effect of fractionation processing on inherent molecular structure of different fractions and how the processing-induced structural change affect the metabolic characteristics of protein and nutrient availability. The objectives of this experiment were to: (1) investigate the effect of fractionation processing on changes of protein functional groups (amide I, amide II, and their ratio) and molecular structure (modeled α-helix, β-sheet, and their ratio), and (2) study the relationship between the fractionation processing-induced changes of protein molecular structure and nutrients availability as well as the metabolic characteristics of protein. The hypothesis of this study was that the fractionation processing changes the molecular structure and such changes affect the metabolic characteristics of protein. The protein molecular structure spectral profile of the fractions A, B, C and D were identified by Fourier-transform infrared attenuated total reflection spectroscopy (FT/IR-ATR). The results showed that the fractionation processing significantly affected the protein molecular spectral profiles. The differences in amide I to amide II peak area and height ratios were strongly significant (P<0.01) among the treatment fractions, ranging from 4.98 to 6.33 and 3.28 to 4.00, respectively. The difference in the modeled protein α-helix to β-sheet ratio was also strongly significant (P<0.01) among the treatment fractions. Multivariate molecular spectral analysis with cluster (CLA) and principal component analyses (PCA) showed

  2. Molecular spectroscopic investigation on fractionation-induced changes on biomacromolecule of co-products from bioethanol processing to explore protein metabolism in ruminants

    NASA Astrophysics Data System (ADS)

    Zhang, Xuewei; Yan, Xiaogang; Beltranena, Eduardo; Yu, Peiqiang

    2014-03-01

    Fractionation processing is an efficient technology which is capable to redesign/redevelop a new food or feed product with a specified chemical and nutrient profile. This processing technique was able to produce four different fractions (called "A", "B", "C", "D" fractions/treatments) with different nutrient profile form a co-product of bioethanol processing [wheat dried distillers grains with soluble (DDGS)]. To date, there is no study on the effect of fractionation processing on inherent molecular structure of different fractions and how the processing-induced structural change affect the metabolic characteristics of protein and nutrient availability. The objectives of this experiment were to: (1) investigate the effect of fractionation processing on changes of protein functional groups (amide I, amide II, and their ratio) and molecular structure (modeled α-helix, β-sheet, and their ratio), and (2) study the relationship between the fractionation processing-induced changes of protein molecular structure and nutrients availability as well as the metabolic characteristics of protein. The hypothesis of this study was that the fractionation processing changes the molecular structure and such changes affect the metabolic characteristics of protein. The protein molecular structure spectral profile of the fractions A, B, C and D were identified by Fourier-transform infrared attenuated total reflection spectroscopy (FT/IR-ATR). The results showed that the fractionation processing significantly affected the protein molecular spectral profiles. The differences in amide I to amide II peak area and height ratios were strongly significant (P < 0.01) among the treatment fractions, ranging from 4.98 to 6.33 and 3.28 to 4.00, respectively. The difference in the modeled protein α-helix to β-sheet ratio was also strongly significant (P < 0.01) among the treatment fractions. Multivariate molecular spectral analysis with cluster (CLA) and principal component analyses (PCA

  3. IMMUNOLOGICAL STUDIES ON CRYPTOCOSSOSIS. REPORT III. STUDIES ON THE IMMUNOLOGICAL SPECIFICITY OF THE PROTEIN FRACTION AS ANTIGEN, AND ON THE DOSES OF THE POLYSACCHARIDE FRACTION FOR IMMUNOLOGICAL DIAGNOSIS

    DTIC Science & Technology

    polysaccharide fraction as far as the precipitin or skin reaction was concerned. Not a single non- cryptococcosis patient demonstrated a positive reaction when...examined with the antigen, of 500 mcg./ml. or less in concentration. So far, the investigation was not extended to clinical cryptococcosis . However, should a...clinical cryptococcosis patient reveal positive skin or precipitin reaction in which the polysaccharide fraction is used as its antigen at an

  4. A rapid and convenient method for measuring the fractional rate of protein synthesis in ectothermic animal tissues using a stable isotope tracer.

    PubMed

    Lamarre, S G; Saulnier, R J; Blier, P U; Driedzic, W R

    2015-04-01

    A method was devised to measure the fractional rate of protein synthesis in fish using a stable isotope labelled tracer (ring-D5-phenylalanine) instead of radioactive phenylalanine. This modified flooding dose technique utilizes gas chromatography with mass spectrometry detection (GC-MS). The technique was validated by measuring the fractional rate of protein synthesis in the liver and white muscle of Arctic charr (Salvelinus alpinus) and then tested by comparing the fractional rate of protein synthesis of fed and starved Arctic charr. The modified technique met the assumptions of the flooding dose technique and was successfully used to detect alterations in the rate of protein synthesis in fed and starved fish. This modified technique allows for studies on protein metabolism to be carried out in situations where the use of radioactivity is difficult, if not impossible.

  5. Dependence of the hormonal stimulation of adenylate cyclase on the fraction of the plasma membrane accessible for lateral displacement of proteins of the adenylate cyclase complex

    SciTech Connect

    Kazarov, A.R.; Rozenkrants, A.A.; Sobolev, A.S.

    1986-09-10

    Hormonal activation of the adenylate cyclase complex is associated with lateral displacement in the membrane of the proteins that constitute this complex. In this work an experimental investigation was made of the changes in the interaction of the proteins of the adenylate cyclase complex with the changing fraction of fluid lipids in the cell membrane. A decrease in the fraction of fluid lipids of rat reticulocyte membranes led to a decrease (all the way down to a total suppression) of the interaction of the ..beta..-adrenoreceptors with the regulatory N-proteins. The interaction of the N-proteins with the catalytic proteins was also suppressed. On the other hand, an increase in the fraction of fluid lipids led to more effective interaction. It was shown that in this case the functional intactness of the interacting proteins is unimpaired. An analysis of the results obtained, performed on the basis of the percolation theory, suggests the conclusion that the hormonal stimulation of adenylate cyclase depends on the fraction of fluid lipids in the membrane, and the proteins are displaced during interaction over distances comparable with the size of the membrane itself. It was also shown that characteristic activity of the ..beta..-agonist 1-isoproterenol varies from 1.0 to 0, depending on the fraction of fluid lipids in the membrane. The data obtained suggest that in the absence of guanylic nucleotides in the membrane in vitro there are no preexisting complexes with a high affinity for the agonist.

  6. Absorbed aluminium is found with two cytosolic protein fractions, other than ferritin, in the rat duodenum.

    PubMed Central

    Cochran, M; Goddard, G; Ramm, G; Ludwigson, N; Marshall, J; Halliday, J

    1993-01-01

    After in vivo perfusion of the upper intestine of the rat with a range of concentrations of aluminium chloride, entry of the metal into the portal system was only detected when the perfusate exceeded 400 mumol/l, suggesting a mucosal block. Using gel filtration of a mucosal cytosol extract, two consistently appearing aluminium peaks were identified which may represent aluminium binding proteins. Both were heat stable at 60 degrees C and had molecular sizes of about 700 (kilo daltons) (kD) and 17 kD respectively. The larger molecule was distinct from ferritin. Neither molecule associated with 59Fe nor 45Ca. It is suggested that the aluminium peaks are relatively specific aluminium binding proteins that have a scavenging role, reducing entry of the metal from the intestinal contents into the portal blood. PMID:8504964

  7. Study of the protein-bound fraction of calcium, iron, magnesium and zinc in bovine milk

    NASA Astrophysics Data System (ADS)

    Silva, Fernando V.; Lopes, Gisele S.; Nóbrega, Joaquim A.; Souza, Gilberto B.; Nogueira, Ana Rita A.

    2001-10-01

    Two approaches were used to study the interaction of Ca, Fe, Mg and Zn with bovine milk proteins by inductively coupled plasma optical emission spectrometry (ICPOES). Selective separations in bovine milk samples were accomplished employing an acid protein precipitation using 100 g l -1 trichloroacetic acid (TCA), and an enzymatic protein hydrolysis using 50 g l -1 pepsin (PEP) solution, respectively. The results were compared with total mineral contents determined after microwave-assisted acid digestion. The results obtained by enzymatic and acid precipitation evidenced the different interaction forms of Ca, Fe, Mg and Zn in the system formed by milk components. Iron was not solubilized by the TCA treatment, but was recovered completely after the enzymatic treatment. Quantitative recoveries of Ca, Mg and Zn were obtained using both approaches, showing that these analytes were bound to milk compounds affected by either treatment. Calcium, Mg and Zn are mainly associated with colloidal calcium phosphate and Fe is bound to the backbone of the casein polypeptide chain, cleaved by pepsin enzyme. The proposed approaches could be used to assess the complexity of these chemical interactions.

  8. The developmentally regulated expression of Menkes protein ATP7A suggests a role in axon extension and synaptogenesis.

    PubMed

    El Meskini, Rajaâ; Cline, Laura B; Eipper, Betty A; Ronnett, Gabriele V

    2005-01-01

    Menkes disease (MD) is a neurodegenerative disorder caused by mutation of the copper transporter ATP7A. While several enzymes expressed in mature neurons require copper, MD neurodegenerative changes cannot be explained by known requirements for ATP7A in neuronal development. To investigate additional roles for ATP7A during development, we characterized its pattern of expression using the olfactory system as a neurodevelopmental model. ATP7A expression in neurons was developmentally regulated rather than constitutively. Initially expressed in the cell bodies of developing neurons, ATP7A protein later shifted to extending axons, peaking prior to synaptogenesis. Similarly, after injury-stimulated neurogenesis, ATP7A expression increased in neurons and axons preceding synaptogenesis. Interestingly, copper-transport-deficient ATP7A still exhibits axonal localization. These results support a role for ATP7A in axon extension, which may contribute to the severe neurodegeneration characteristic of MD.

  9. Multidimensional Separation Using HILIC and SCX Pre-fractionation for RP LC-MS/MS Platform with Automated Exclusion List-based MS Data Acquisition with Increased Protein Quantification

    PubMed Central

    Zhou, Yu; Meng, Zhen; Edman-Woolcott, Maria; Hamm-Alvarez, Sarah F; Zandi, Ebrahim

    2015-01-01

    Liquid chromatography–mass spectrometry (LC-MS) based proteomics is one of the most widely used analytical platforms for global protein discovery and quantification. One of the challenges is the difficulty of identifying low abundance biomarker proteins from limited biological samples. Extensive fractionation could expand proteomics dynamic range, however, at the cost of high sample and time consumption. Extensive fractionation would increase the sample need and the labeling cost. Also quantitative proteomics depending on high resolution MS have the limitation of spectral acquisition speed. Those practical problems hinder the in-depth quantitative proteomics analysis such as tandem mass tag (TMT) experiments. We found the joint use of hydrophilic interaction liquid chromatography (HILIC) and strong cation exchange Chromatography (SCX) prefractionation at medium level could improve MS/MS efficiency, increase proteome coverage, shorten analysis time and save valuable samples. In addition, we scripted a program, Exclusion List Convertor (ELC), which automates and streamlines data acquisition workflow using the precursor ion exclusion (PIE) method. PIE reduces redundancy of high abundance MS/MS analyses by running replicates of the sample. The precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. We compared PIE methods with standard data dependent acquisition (DDA) methods running replicates without PIE for their effectiveness in quantifying TMT-tagged peptides and proteins in mouse tears. We quantified a total of 845 proteins and 1401 peptides using the PIE workflow, while the DDA method only resulted in 347 proteins and 731 peptides. This represents a 144% increase of protein identifications as a result of PIE analysis. PMID:26807013

  10. Unwinding protein specific for mRNA translation fractionated together with rabbit reticulocyte initiation factor 3 complex

    PubMed Central

    Ilan, Joseph; Ilan, Judith

    1977-01-01

    Experiments with a rabbit reticulocyte cell-free system dependent on the addition of initiation factor 3 (eIF-3) and mRNA were carried out. In this system, using ribosomal subunits, AUG(U)n can direct polyphenylalanine synthesis in the absence of eIF-3 at 3 mM MgCl2. Globin mRNA was not translated under similar conditions; its translation requires the addition of eIF-3. Moreover, the maximal rate of globin synthesis was achieved when the molar ratio of eIF-3 to ribosomes was approximately 1. This was taken to indicate that some ribosomal proteins were fractionated with eIF-3 and functioned in reconstitution of salt-washed ribosomes. In our system, almost all ribosomes were active, as evident from the fact that all were found in polysomes when analyzed at the time of linear incorporation, and the molar ratio of ribosomes to mRNA was maintained at 4:1. When AUG(U)n was hybridized with poly(A), it could not direct polyphenylalanine synthesis with or without eIF-3 and was a potent inhibitor of the translation of globin mRNA in the presence of eIF-3. When poly(A) containing 10% U was hybridized with AUG(U)n and added to the cell-free system, addition of eIF-3 promoted polyphenylalanine synthesis to about 80% of control. Moreover, eIF-3 was seen to shift significantly the melting temperature of globin and synthetic double-stranded RNA. These observations suggest that extraction of ribosomes with 0.5 M KCl may release a ribosomal protein that fractionates with eIF-3. This protein may function in unwinding or melting the secondary structure of mRNA and thus facilitate translation. PMID:267926

  11. Toxic peptides in Frazer's fraction interact with the actin cytoskeleton and affect the targeting and function of intestinal proteins.

    PubMed

    Reinke, Yvonne; Zimmer, Klaus-Peter; Naim, Hassan Y

    2009-11-15

    Celiac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in HLA-DQ2/8 positive individuals. An essential role in the pathogenesis of CD is played by a fraction of the peptic-tryptic digest of gluten, Frazer's Fraction (FF). Here, we investigate the effects of FF on the integrity of intestinal cells with particular emphasis on brush border membrane (BBM) components, their subsequent trafficking and endocytosis. Caco-2 cells were incubated with FF at different concentrations. Thereafter, several protein and lipid components of treated and untreated cells were analysed at the molecular, functional and cellular levels. The control employed tryptic-peptic digests of ovalbumin. Our results show that FF directly interacts with actin in an alternating manner eliciting substantial alterations in its integrity and extent in the BBM. These alterations lead to an impaired trafficking of SI to the apical membrane and reduction in its enzymatic function. ApN and DPPIV follow a transcytotic pathway and are only partly affected by FF. By contrast, the trafficking of LPH remains unaffected concomitant with its actin-independent trafficking pattern. Finally, the endocytic pathway is substantially blocked in FF-treated cells leading to an accumulation of cholesterol, and sphingolipids in the BBM. We conclude that FF deteriorates the actin cytoskeleton in Caco-2 leading to reduced protein sorting and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest a potential pathomechanism of carbohydrate malabsorption in CD.

  12. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

    PubMed

    Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-07-08

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases

    PubMed Central

    Floden, Evan W.; Tommaso, Paolo D.; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-01-01

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. PMID:27106060

  14. Extensive protein hydrolysate formula effectively reduces regurgitation in infants with positive and negative challenge tests for cow's milk allergy.

    PubMed

    Vandenplas, Y; De Greef, E

    2014-06-01

    Cow's milk protein allergy (CMPA) is treated using an elimination diet with an extensive protein hydrolysate. We explored whether a thickened or nonthickened version was best for infants with suspected CMPA, which commonly causes regurgitation/vomiting. Diagnosis of CMPA was based on a positive challenge test. We compared the efficacy of two casein extensive hydrolysates (eCH), a nonthickened version (NT-eCH) and a thickened version (T-eCH), using a symptom-based score covering regurgitation, crying, stool consistency, eczema, urticarial and respiratory symptoms. A challenge was performed in 52/72 infants with suspected CMPA and was positive in 65.4%. All confirmed CMPA cases tolerated eCH. The symptom-based score decreased significantly in all infants within a month, and the highest reduction was in those with confirmed CMPA. Regurgitation was reduced in all infants (6.4 ± 3.2-2.8 ± 2.9, p < 0.001), but fell more with the T-eCH (-4.2 ± 3.2 regurgitations/day vs. -3.0 ± 4.5, ns), especially in infants with a negative challenge (-3.9 ± 4.0 vs. -1.9 ± 3.4, ns). eCH fulfilled the criteria for a hypoallergenic formula, and the NT-eCH and T-eCH formulas both reduced CMPA symptoms. The symptom-based score is useful for evaluating how effective dietary treatments are for CMPA. ©2014 The Authors. Acta Paediatrica published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.

  15. Isotopic fractionation in proteins as a measure of hydrogen bond length

    SciTech Connect

    McKenzie, Ross H.; Athokpam, Bijyalaxmi; Ramesh, Sai G.

    2015-07-28

    If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent, it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor Φ is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds, we calculate Φ as a function of the proton donor-acceptor distance R. For numerical results, we use a parameterization of the model for symmetric O–H⋯O bonds [R. H. McKenzie, Chem. Phys. Lett. 535, 196 (2012)]. We consider the relative contributions of the O–H stretch vibration, O–H bend vibrations (both in plane and out of plane), tunneling splitting effects at finite temperature, and the secondary geometric isotope effect. We compare our total Φ as a function of R with NMR experimental results for enzymes, and in particular with an earlier model parametrization Φ(R), used previously to determine bond lengths.

  16. Isotopic fractionation in proteins as a measure of hydrogen bond length

    NASA Astrophysics Data System (ADS)

    McKenzie, Ross H.; Athokpam, Bijyalaxmi; Ramesh, Sai G.

    2015-07-01

    If a deuterated molecule containing strong intramolecular hydrogen bonds is placed in a hydrogenated solvent, it may preferentially exchange deuterium for hydrogen. This preference is due to the difference between the vibrational zero-point energy for hydrogen and deuterium. It is found that the associated fractionation factor Φ is correlated with the strength of the intramolecular hydrogen bonds. This correlation has been used to determine the length of the H-bonds (donor-acceptor separation) in a diverse range of enzymes and has been argued to support the existence of short low-barrier H-bonds. Starting with a potential energy surface based on a simple diabatic state model for H-bonds, we calculate Φ as a function of the proton donor-acceptor distance R. For numerical results, we use a parameterization of the model for symmetric O-H⋯O bonds [R. H. McKenzie, Chem. Phys. Lett. 535, 196 (2012)]. We consider the relative contributions of the O-H stretch vibration, O-H bend vibrations (both in plane and out of plane), tunneling splitting effects at finite temperature, and the secondary geometric isotope effect. We compare our total Φ as a function of R with NMR experimental results for enzymes, and in particular with an earlier model parametrization Φ(R), used previously to determine bond lengths.

  17. The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

    PubMed Central

    Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan; Gao, Ning

    2016-01-01

    Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo—that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling. PMID:27390266

  18. Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.

    PubMed

    Zauber, Henrik; Szymanski, Witold; Schulze, Waltraud X

    2013-12-01

    During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.

  19. OSMOTIC PRESSURE STUDY OF PROTEIN FRACTIONS IN NORMAL AND IN NEPHROTIC SUBJECTS

    PubMed Central

    Bourdillon, Jaques

    1939-01-01

    In serum of patients with nephrosis both albumin and globulin showed by osmotic pressure nearly double the molecular weights of normal albumin and globulin. In the urines of such patients, on the other hand, both proteins showed molecular weights lower even than in normal serum. The colloidal osmotic pressures were measured by the author's method at such dilutions that the van't Hoff law relating pressures to molecular concentrations could be directly applied. For the albumin and globulin of normal serum the molecular weights found were 72,000 and 164,000 respectively, in agreement with the weights obtained by other methods. PMID:19870879

  20. Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins.

    PubMed

    Fatima, Aiman; Husain, Qayyum

    2007-06-01

    Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

  1. Effects of Red Bean (Vigna angularis) Protein Isolates on Rheological Properties of Microbial Transglutaminase Mediated Pork Myofibrillar Protein Gels as Affected by Fractioning and Preheat Treatment

    PubMed Central

    Lee, Hong Chul

    2016-01-01

    Fractioning and/or preheating treatment on the rheological properties of myofibrillar protein (MP) gels induced by microbial transglutaminase (MTG) has been reported that they may improve the functional properties. However, the optimum condition was varied depending on the experimental factors. This study was to evaluate the effect of red bean protein isolate (RBPI) on the rheological properties of MP gels mediated by MTG as affected by modifications (fractioning: 7S-globulin of RBPI and/or preheat treatment (pre-heating; 95℃/30 min): pre-heating RBPI or pre-heating/7S-globulin). Cooking yields (CY, %) of MP gels was increased with RBPI (p<0.05), while 7S-globulin decreased the effect of RBPI (p<0.05); however, preheating treatments did not affect the CY (p>0.05). Gel strength of MP was decreased when RBPI or 7S-globulin added, while preheat treatments compensated for the negative effects of those in MP. This effect was entirely reversed by MTG treatment. Although the major band of RBPI disappeared, the preheated 7S globulin band was remained. In scanning electron microscopic (SEM) technique, the appearance of more cross-linked structures were observed when RBPI was prepared with preheating at 95℃ to improve the protein-protein interaction during gel setting of MP mixtures. Thus, the effects of RBPI and 7S-globulin as a substrate, and water and meat binder for MTG-mediated MP gels were confirmed to improve the rheological properties. However, preheat treatment of RBPI should be optimized. PMID:27857544

  2. Multi-dimensional fractionation and characterization of crude protein mixtures: toward establishment of a database of protein purification process development parameters.

    PubMed

    Nfor, Beckley K; Ahamed, Tangir; Pinkse, Martijn W H; van der Wielen, Luuk A M; Verhaert, Peter D E M; van Dedem, Gijs W K; Eppink, Michel H M; van de Sandt, Emile J A X; Ottens, Marcel

    2012-12-01

    A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation. Copyright © 2012 Wiley Periodicals, Inc.

  3. Spectroscopic characterization by photodiode array detection of human urinary and amniotic protein HC subpopulations fractionated by anion-exchange and size-exclusion high-performance liquid chromatography.

    PubMed

    Calero, M; Escribano, J; Soriano, F; Grubb, A; Brew, K; Méndez, E

    1996-01-05

    A procedure for spectroscopic characterization and partial fractionation of human protein HC populations by high-performance liquid chromatography-photodiode array ultraviolet-visible detection is reported. Human protein HC from urine or amniotic fluid fractionated by anion-exchange HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as judged by the asymmetric elution pattern, consisting of a continuous irregular broad peak with several shoulders distributed along the whole chromatogram. Selected fractions containing shoulders were rechromatographed and finally six symmetrical homogeneous peaks with different retention times were obtained from each protein HC preparation. The direct automatic absorption spectra analyses at each peak maximum, indicated that all of the homogeneous peaks seemed to be protein HC, all of them associated to the same chromophore although with different stoichiometry ratios. Isoelectric focusing showed that each peak was composed of a limited number of subpopulations of protein HC with different isoelectric points. Size microheterogeneity has been also demonstrated in both urinary and amniotic protein HC preparations by a combination of size-exclusion HPLC on a TSK 3000 SW6 column and photodiode array detection. Partial fractionation of human albumin on an analytical anion-exchange Mono-Q PC 1.6/5 column, has allowed the identification of heterogeneous chromophore-containing populations displaying significant absorption in the visible region in resemblance to that of protein HC.

  4. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  5. Evaluation of an Amino Acid-Based Formula in Infants Not Responding to Extensively Hydrolyzed Protein Formula.

    PubMed

    Vanderhoof, Jon; Moore, Nancy; de Boissieu, Delphine

    2016-11-01

    Nearly 2% to 3% of infants and children younger than 3 years have confirmed cow's milk protein allergy with multiple clinical presentations including atopic dermatitis (AD), diarrhea, and vomiting/spitting up. Although most infants with cow's milk protein allergy experience clinical improvement with the use of an extensively hydrolyzed (EH) formula, highly sensitive infants may require an amino acid-based formula. In this observational, prospective study, 30 infants (1-12 months of age) with a history of weight loss and persistent allergic manifestations while on an EH formula were provided an amino acid-based formula for 12 weeks. Mean weight gain (z score change) improved +0.43 ± 0.28 (mean ± standard deviation) after the 12-week feeding period. Improvement was observed for many allergic symptoms including significant decreases in AD severity (P = 0.02). These results indicate the new amino acid-based infant formula supported healthy weight gain and improvement in allergic manifestations in infants not responding to EH formulas.

  6. Evaluation of an Amino Acid−Based Formula in Infants Not Responding to Extensively Hydrolyzed Protein Formula

    PubMed Central

    Vanderhoof, Jon; Moore, Nancy; de Boissieu, Delphine

    2016-01-01

    ABSTRACT Nearly 2% to 3% of infants and children younger than 3 years have confirmed cow's milk protein allergy with multiple clinical presentations including atopic dermatitis (AD), diarrhea, and vomiting/spitting up. Although most infants with cow's milk protein allergy experience clinical improvement with the use of an extensively hydrolyzed (EH) formula, highly sensitive infants may require an amino acid−based formula. In this observational, prospective study, 30 infants (1–12 months of age) with a history of weight loss and persistent allergic manifestations while on an EH formula were provided an amino acid−based formula for 12 weeks. Mean weight gain (z score change) improved +0.43 ± 0.28 (mean ± standard deviation) after the 12-week feeding period. Improvement was observed for many allergic symptoms including significant decreases in AD severity (P = 0.02). These results indicate the new amino acid–based infant formula supported healthy weight gain and improvement in allergic manifestations in infants not responding to EH formulas. PMID:27526059

  7. Short communication: Potential of Fresco-style cheese whey as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme inhibitory activities.

    PubMed

    Tarango-Hernández, S; Alarcón-Rojo, A D; Robles-Sánchez, M; Gutiérrez-Méndez, N; Rodríguez-Figueroa, J C

    2015-11-01

    Recently, traditional Mexican Fresco-style cheese production has been increasing, and the volume of cheese whey generated represents a problem. In this study, we investigated the chemical composition of Fresco-style cheese wheys and their potential as a source of protein fractions with antioxidant and angiotensin-I-converting enzyme (ACE)-inhibitory activities. Three samples from Fresco, Panela, and Ranchero cheeses whey were physicochemically characterized. Water-soluble extracts were fractionated to obtain whey fractions with different molecular weights: 10-5, 5-3, 3-1 and <1 kDa. The results indicated differences in the lactose, protein, ash, and dry matter contents (% wt/wt) in the different Fresco-style cheese wheys. All whey fractions had antioxidant and ACE-inhibitory activities. The 10-5 kDa whey fraction of Ranchero cheese had the highest Trolox equivalent antioxidant capacity (0.62 ± 0.00 mM), and the 3-1 kDa Panela and Fresco cheese whey fractions showed the highest ACE-inhibitory activity (0.57 ± 0.02 and 0.59 ± 0.04 μg/mL 50%-inhibitory concentration values, respectively). These results suggest that Fresco-style cheese wheys may be a source of protein fractions with bioactivity, and thus could be useful ingredients in the manufacture of functional foods with increased nutritional value.

  8. Trace elements and their distribution in protein fractions of camel milk in comparison to other commonly consumed milks.

    PubMed

    Al-Awadi, F M; Srikumar, T S

    2001-08-01

    Studies on camels' milk, whether with respect to concentration or bioavailability of trace elements from this milk, are limited and warrant further investigation. The object of this study was to analyse the concentration and distribution of zinc, copper, selenium, manganese and iron in camel milk compared to those in human milk, cows' milk and infant formula under similar experimental conditions. Camels' milk and cows' milk were collected from local farms, human milk samples were obtained from healthy donors in Kuwait and infant formula was purchased locally. Milk fractionation was performed by ultra-centrifugation and gelcolumn chromatography. The concentration of trace elements was analysed by atomic absorption spectrometry and that of protein was determined spectrophotometrically. The concentration of manganese and iron in camels' milk was remarkably higher (7-20-fold and 4-10-fold, respectively) than in human milk, cows' milk and infant formula. The zinc content of camels' milk was higher than that of human milk but slightly lower than in cows' milk and infant formula. The concentration of copper in camels' milk was similar to that of cows' milk but lower than in human milk and infant formula. The selenium content of camels' milk was comparable to those of other types of milk, Approximately 50-80% of zinc, copper and manganese in camels' milk were associated with the casein fraction, similar to that of cows' milk, The majority of selenium and iron in camels' milk was in association with the low molecular weight fraction, It is recommended that camels' milk be considered as a potential source of manganese, selenium and iron, perhaps not only for infants, but also for other groups suspected of mild deficiency of these elements. Further investigations are required to confirm this proposal.

  9. Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

    NASA Technical Reports Server (NTRS)

    Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

    1980-01-01

    Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

  10. Process steps for the preparation of purified fractions of alpha-lactalbumin and beta-lactoglobulin from whey protein concentrates.

    PubMed

    Gésan-Guiziou, G; Daufin, G; Timmer, M; Allersma, D; van der Horst, C

    1999-05-01

    Fractions enriched with alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were produced by a process comprising the following successive steps: clarification-defatting of whey protein concentrate, precipitation of alpha-lactalbumin, separation of soluble beta-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of alpha-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble beta-lg was separated from the precipitate using diafiltration or microfiltration and the purities of alpha-la and beta-lg were in the range 52-83 and 85-94% respectively. The purity of the beta-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for alpha-la; 51% for beta-lg) were disappointing, but ways of improving each step in the process are discussed.

  11. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

    PubMed Central

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-01-01

    The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. PMID:27422873

  12. Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension

    PubMed Central

    Lima, Luís; Azevedo, Rita; Soares, Janine; Cotton, Sofia; Parreira, Beatriz; Neves, Manuel; Amaro, Teresina; Tavares, Ana; Teixeira, Filipe; Palmeira, Carlos; Rangel, Maria; Silva, André M.N.; Reis, Celso A.; Santos, Lúcio Lara; Oliveira, Maria José; Ferreira, José Alexandre

    2016-01-01

    Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may

  13. Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension.

    PubMed

    Peixoto, Andreia; Fernandes, Elisabete; Gaiteiro, Cristiana; Lima, Luís; Azevedo, Rita; Soares, Janine; Cotton, Sofia; Parreira, Beatriz; Neves, Manuel; Amaro, Teresina; Tavares, Ana; Teixeira, Filipe; Palmeira, Carlos; Rangel, Maria; Silva, André M N; Reis, Celso A; Santos, Lúcio Lara; Oliveira, Maria José; Ferreira, José Alexandre

    2016-09-27

    Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may

  14. Field-flow fractionation of nucleic acids and proteins under large-scale gradient magnetic fields

    NASA Astrophysics Data System (ADS)

    Iwasaka, M.

    2007-05-01

    For the purpose of developing techniques for separating biological macromolecules, the present study reports a magnetic chromatography system employing high performance liquid chromatography and superconducting magnets of 14 and 5T. We observed chromatograms of catalase and albumin, which were eluded from columns that were exposed to magnetic fields of up to 14T with a maximum gradient of 90T/m. Without the magnetic fields, the chromatograms of the macromolecules showed a clear peak, while the chromatograms changed to have separated peaks for the same molecules after exposure to gradient magnetic fields. When the chromatocolumn was placed so the magnetic forces were opposite to the direction of flow, the albumin molecules separated into two groups. In addition, the chromatograms of catalase exposed to the magnetic fields indicated that the retention times of the two kinds of magnetically separated catalase were relatively changed if the column-field configuration was changed. Probably, the balance of paramagnetism in the heme and diamagnetism in the protein controlled the transport velocity under the influence of the gradient magnetic fields. In addition, the transport velocity of DNA molecules in the flow with a high gradient magnetic field was observed using a time-resolved spectrophotometric system.

  15. The role of continuous versus fractionated physical training on muscle oxidative stress parameters and calcium-handling proteins in aged rats.

    PubMed

    Tromm, Camila B; Pozzi, Bruna G; Paganini, Carla S; Marques, Scherolin O; Pedroso, Giulia S; Souza, Priscila S; Silveira, Paulo C L; Silva, Luciano A; De Souza, Claudio T; Pinho, Ricardo A

    2016-10-01

    Age-associated decline in skeletal muscle mass and strength is associated with oxidative stress and Ca(2+) homeostasis disturbance. Exercise should be considered a viable modality to combat aging of skeletal muscle. This study aimed to investigate whether continuous and fractionated training could be useful tools to attenuate oxidative damage and retain calcium-handling proteins. We conducted the study using 24-month-old male Wistar rats, divided into control, continuous, and fractionated groups. Animals ran at 13 m min(-1) for five consecutive days (except weekends) for 6 weeks, for a total period of 42 days. Each session comprised 45 min of exercise, either continuous or divided into three daily sessions of 15 min each. Metabolic and oxidative stress markers, protein levels of mitochondrial transcription factors, and calcium-handling proteins were analyzed. Continuous exercise resulted in reduced ROS production as well as showed a decrease in TBARS levels and carbonyl content. On the other hand, fractionated training increased the antioxidant enzyme activities. The ryanodine receptor and phospholamban protein were regulated by continuous training while sodium calcium exchange protein was increased by the fractionated training. These data suggest that intracellular Ca(2+) can be modulated by various training stimuli. In addition, the modulation of oxidative stress by continuous and fractionated training may play an important regulatory role in the muscular contraction mechanism of aged rats, due to changes in calcium metabolism.

  16. Cholesteryl Ester Transfer Protein Inhibition With Anacetrapib Decreases Fractional Clearance Rates of High-Density Lipoprotein Apolipoprotein A-I and Plasma Cholesteryl Ester Transfer Protein.

    PubMed

    Reyes-Soffer, Gissette; Millar, John S; Ngai, Colleen; Jumes, Patricia; Coromilas, Ellie; Asztalos, Bela; Johnson-Levonas, Amy O; Wagner, John A; Donovan, Daniel S; Karmally, Wahida; Ramakrishnan, Rajasekhar; Holleran, Stephen; Thomas, Tiffany; Dunbar, Richard L; deGoma, Emil M; Rafeek, Hashmi; Baer, Amanda L; Liu, Yang; Lassman, Michael E; Gutstein, David E; Rader, Daniel J; Ginsberg, Henry N

    2016-05-01

    Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA)-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP. Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II, and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, ANA treatment increased plasma HDL-C (63.0%; P<0.001) and apoA-I levels (29.5%; P<0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate. ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein. © 2016 American Heart Association, Inc.

  17. Antioxidant properties of salmon (Salmo salar L.) protein fraction hydrolysates revealed following their ex vivo digestion and in vitro hydrolysis.

    PubMed

    Borawska, Justyna; Darewicz, Małgorzata; Pliszka, Monika; Vegarud, Gerd E

    2016-06-01

    Salmon (Salmo salar L.) myofibryllar protein (MP) and sarcoplasmic protein (SP) were digested with human gastric and duodenal juices and hydrolysed in vitro with commercial pepsin and Corolase PP. The digestion after duodenal juice/Corolase PP caused almost complete breakdown of peptide bonds in MP and SP. The DPPH(•) scavenging activity of proteins decreased during both ex vivo digestion and in vitro hydrolysis. The highest value of DPPH(•) scavenging activity was shown for the gastric digest of SP (8.88 ± 0.87%). The ABTS(+•) scavenging activity of MP and SP increased during digestion/hydrolysis. The duodenal digest of SP was characterised by the highest value of ABTS(+•) scavenging activity (72.7 ± 1.2%). In turn, the highest value of ferric-reducing power was determined for the gastric digest of SP (84.8 ± 0.2%). Salmon antioxidant peptides Phe-Ile-Lys-Lys, His-Leu, Ile-Tyr, Pro-His-Leu, Pro-Trp, Val-Pro-Trp were identified in both ex vivo digested and in vitro hydrolysed MP and SP. An antioxidant peptide, Val-Tyr, was additionally detected in the in vitro hydrolysate of SP. The results indicate the salmon myofibrillar and sarcoplasmic protein fractions as potential sources of antioxidant peptides that could be released in the gastrointestinal tract but their amino acid sequence and quantification vary. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  18. Chicken-based formula is better tolerated than extensively hydrolyzed casein formula for the management of cow milk protein allergy in infants.

    PubMed

    Jirapinyo, Pipop; Densupsoontorn, Narumon; Kangwanpornsiri, Channagan; Wongarn, Renu

    2012-01-01

    The effective treatment of cow milk allergy in infants consists of elimination of cow milk protein and the introduction of formulas based on an extensively hydrolyzed protein formula or an amino acid-based formula. However, about 10% of these infants are still allergic to an extensively hydrolyzed protein formula and an amino acid-based formula is very expensive. We conducted a study to verify whether the new chicken-based formula will be better tolerated than an extensively hydrolyzed protein formula for the treatment of cow milk allergy in infants. One hundred infants, diagnosed with cow milk allergy by double-blind, placebo-controlled food challenge tests, were enrolled in a double-blind, randomized, cross-over study to compare a response to an extensively hydrolyzed protein formula and the chicken-based formula. Subjects were randomly given one of the two formulas for 2 weeks. There was a 2-week washout period of taking an amino acid-based formula before being switched to the other formula for another 2 weeks. If the subjects showed allergic symptoms during the 2 weeks of test formula, they would be announced as intolerance or allergic to that formula. Sixty seven of 80 confirmed subjects agreed to enroll their infants. Fifty-eight subjects completed the study. Twenty and 33 infants were tolerant whereas and 38 and 25 infants were intolerant to an extensively hydrolyzed protein formula and the chicken-based formula, respectively. The chicken-based formula showed significantly better tolerance than an extensively hydrolyzed protein formula in the management of cow milk allergy in infants.

  19. Comparing serum responses to acute feedings of an extensively hydrolyzed whey protein concentrate versus a native whey protein concentrate in rats: a metabolomics approach.

    PubMed

    Roberts, Michael D; Cruthirds, Clayton L; Lockwood, Christopher M; Pappan, Kirk; Childs, Thomas E; Company, Joseph M; Brown, Jacob D; Toedebusch, Ryan G; Booth, Frank W

    2014-02-01

    We examined how gavage feeding extensively hydrolyzed whey protein (WPH) versus a native whey protein concentrate (WPC) transiently affected serum biochemical profiles in rodents. Male Wistar rats (250-300 g) were 8 h fasted and subsequently fed isonitrogenous amounts of WPH or WPC, or remained unfed (control). Animals were sacrificed 15 min, 30 min, and 60 min post-gavage for serum extraction, and serum was analyzed using untargeted global metabolic profiling via gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS platforms. We detected 333 serum metabolites amongst the experimental and control groups. Both WPH and WPC generally increased amino acids (1.2-2.8-fold), branched-chain amino acids (1.2-1.7-fold), and serum di- and oligo-peptides (1.1-2.7-fold) over the 60 min time course compared with control (q < 0.05). However, WPH increased lysine (false discovery rate using a q-value <0.05) and tended to increase isoleucine and valine 15 min post-feeding (q < 0.10) as well as aspartylleucine 30 min post-feeding compared with WPC (q < 0.05). While both protein sources led to a dramatic increase in free fatty acids compared with control (up to 6-fold increases, q < 0.05), WPH also uniquely resulted in a 30 min post-feeding elevation in free fatty acids compared with WPC (q < 0.05), an effect which may be due to the robust 30 min postprandial increase in epinephrine in the WPH cohort. These data provide a unique postprandial time-course perspective on how WPH versus WPC feedings affect circulating biochemicals and will guide future research comparing these 2 protein sources.

  20. Protein pre-fractionation with a mixed-bed ion exchange column in 3D LC-MS/MS proteome analysis.

    PubMed

    Zhang, Luofu; Yao, Ling; Zhang, Yan; Xue, Ting; Dai, Guangchen; Chen, Keying; Hu, Xiaofang; Xu, Lisa X

    2012-09-15

    The fractionation of complex samples at the protein level prior to shotgun proteomics analysis is an efficient means to more comprehensive analysis of samples. A mixed-bed ion-exchange (IEX) column, packed with both weak anion exchange (WAX) and weak cation exchange (WCX) materials, was used for the first dimensional separation of complex samples at the protein level using volatile solvents. The peptides from digestion of each fraction were then identified by 2D SCX-RP-LC-MS/MS. We applied this 3D strategy to mouse mammary tumor 4T1 cell lysate and identified a total of 3084 proteins in a typical experiment. The moderate separation performance of the mixed-bed IEX column facilitated the in-depth identification of the proteins in the complex sample. There were some acceptable inter-fraction overlaps. Nearly half (45.8%) of the proteins were only identified in single fractions, while 82.3% were identified in no more than 3 fractions. The identified proteins covered a broad range of pI, size and grand average hydrophobicity (GRAVY) values. Detailed analysis of proteins identified in each fraction elucidated the separation characteristics of mixed-bed IEX. Retention on mixed-bed IEX was associated, but not restricted to the extreme pI values (pI<5, pI>10) and to the percentage of charged residues of both signs. In conclusion, we have exploited the mixed-bed IEX column to establish an efficient and comprehensive identification method for complex samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    SciTech Connect

    Yu,P.

    2007-01-01

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

  2. Distribution of carbon isotopes in amino acids of protein fraction of micro-organisms as a means of studying the mechanisms of their biosynthesis in the cell

    SciTech Connect

    Ivlev, A.A.

    1986-04-10

    The intramolecular distribution of carbon isotopes in the amino acids of the protein fraction of a number of photosynthesizing microorganisms was analyzed using the previously proposed model of carbon isotope fractionation in the cell. A correlation was found between the distributions of the isotopes in the amino acids and the pathways and sequence of their synthesis in the cell cycle. The feasibility of using the isotopic distributions of metabolites for a study of the temporal organization of metabolism in the cell is illustrated.

  3. Subcellular fractionation and localization studies reveal a direct interaction of the fragile X mental retardation protein (FMRP) with nucleolin.

    PubMed

    Taha, Mohamed S; Nouri, Kazem; Milroy, Lech G; Moll, Jens M; Herrmann, Christian; Brunsveld, Luc; Piekorz, Roland P; Ahmadian, Mohammad R

    2014-01-01

    Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs.

  4. Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

    PubMed

    Piry, A; Heino, A; Kühnl, W; Grein, T; Ripperger, S; Kulozik, U

    2012-04-01

    We investigated the fractionation of casein micelles and the whey protein β-lactoglobulin (β-LG) of skim milk by crossflow microfiltration (0.1 μm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and β-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for β-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

  5. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin

    PubMed Central

    Elleman, T. C.

    1972-01-01

    1. The amino acid sequence of a protein from the reduced and carboxymethylated high-sulphur fraction of wool has been determined. 2. The sequence of this S-carboxymethylkerateine (SCMK-B2C) of 151 amino acid residues displays much internal homology and an unusual residue distribution. Thus a ten-residue sequence occurs four times near the N-terminus and five times near the C-terminus with few changes. These regions contain much of the molecule's half-cystine, whereas between them there is a region of 19 residues that are mainly small and devoid of cystine and proline. 3. Certain models of the wool fibre based on its mechanical and physical properties propose a matrix of small compact globular units linked together to form beaded chains. The unusual distribution of the component residues of protein SCMK-B2C suggests structures in the wool-fibre matrix compatible with certain features of the proposed models. PMID:4678578

  6. Portraying the Effect of Calcium-Binding Proteins on Cytosolic Calcium Concentration Distribution Fractionally in Nerve Cells.

    PubMed

    Jha, Brajesh Kumar; Joshi, Hardik; Dave, Devanshi D

    2016-11-23

    Nerve cells like neurons and astrocytes in central nervous system (CNS) take part in the signaling process which means the transformation of the information from one cell to another via signals. The signaling process is affected by various external parameters like buffers calcium-binding proteins, voltage-gated calcium channel. In the present paper, the role of buffers in the cytoplasmic calcium concentration distribution is shown. The elicitation in calcium concentration is due to the presence of lower amount calcium-binding proteins which can be shown graphically. The mathematical model is designed by keeping in mind the physiological condition taking place in CNS of mammalian brain. The thing to be noted here is that the more elicitation in the calcium concentration distribution results in the cell death which finally give neurodegenerative disease to the mammalian brain. The present paper gives a glimpse of Parkinson's diseases in particular. Computational results are performed in Wolfram Mathematica 9.0 and simulated on core(TM) i5-3210M CPU @ 2.50 GHz processing speed and 4 GB memory. It is found that the different types of buffer like ethylene glycol-bis([Formula: see text]-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calmodulin have noteworthy effect at different fractions of time.

  7. Purif ied Protein Fraction of Garlic Extract Modulates Cellular Immune Response against Breast Transplanted Tumors in BALB/c Mice Model

    PubMed Central

    Ebrahimi, Marzieh; Mohammad Hassan, Zuhair; Mostafaie, Ali; Zare Mehrjardi, Narges; Ghazanfari, Tooba

    2013-01-01

    Objective: Garlic (Allium sativum) has anti-inflammatory, anti-mutagenesis, and immunomodulatory properties that modulate anti-tumor immunity and inhibit tumor growth. In this study we have examined the effect of a protein fraction isolated from fresh garlic on anti-tumor response and intra-tumor lymphocyte infiltration. Materials and Methods: In this experimental study a protein fraction was purified from fresh garlic bulbs using ultra-filtration, followed by chromatofocusing, and SDS-PAGE analysis. Anti-tumor activity was assessed by intra-tumor injection of the protein fraction and garlic extract, itself, into groups of 5 mice each. The percentage of peripheral blood and intra-tumor CD4+ and CD8+ cells were assessed by flow cytometry. Unpaired student’s t test using the SPSS program was applied for all statistical analyses. Results: Garlic extract included different type of proteins with different molecular weight. One of protein’s fraction was immunomodeulator and was composed of three single polypeptides, with molecular masses of ~10-13 kDa and different isoelectric points (pI). These molecules augmented the delayed type hypersensitivity (DTH) response compared to the control group. Intra-tumor injection of the fraction provoked a significant increase in the CD8+ subpopulation of T-lymphocytes, as well as a decrease in tumor size. The fraction increased peripheral blood CD8+ T-lymphocytes in treated animals. Conclusion: The data confirms that protein fractions purified from fresh garlic bulbs augment CD8+ T-cell infiltration into the tumor site, inhibiting tumor growth more efficiently than garlic extract. These findings provide a basis for further investigations on the purified polypeptide as a useful candidate for immunomodulation and tumor treatment. PMID:23700562

  8. Fractionation of complex protein mixture by virtual three-dimensional liquid chromatography based on combined pH and salt steps.

    PubMed

    Ning, Zhi-Bin; Li, Qing-Run; Dai, Jie; Li, Rong-Xia; Shieh, Chia-Hui; Zeng, Rong

    2008-10-01

    The complexity and diversity of biological samples in proteomics require intensive fractionation ahead of mass spectrometry identification. This work developed a chromatographic method called virtual three-dimensional chromatography to fractionate complex protein mixtures. By alternate elution with different pHs and salt concentrations, we implemented pH and salt steps by turns on a single strong cation exchange column to fully exploit its chromatographic ability. Given standard proteins that were not resolved solely by pH or salt gradient elution could be successfully separated using this combined mode. With a reversed phase column tandem connected behind, we further fractionated as well as desalted proteins as the third dimension. This present strategy could readily be adapted with respect to special complexity of biological samples. Crude plasma without depleting high abundance proteins were fractionated by this three-dimensional mode and then analyzed by reversed phase liquid chromatography coupled with LTQ mass spectrometry. In total, 1933 protein groups with wide dynamic ranges were identified from a single experiment. Some characteristics that correlated to the behavior of proteins on strong cation exchange columns are also discussed.

  9. Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase by Raman crystallography

    PubMed Central

    Espinoza-Herrera, Shirly J.; Gaur, Vineet; Suo, Zucai; Carey, Paul R.

    2013-01-01

    Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. 13C, 15N labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ or Mn2+, nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of C* modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following single d*CTP addition. Adding these two bases caused visible damage to the crystal possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction did not occur; instead a ternary protein/template/d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features appear due to ring modes from the template/primer bases being perturbed and from the DNA backbone, as well as from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary than in the binary complex. Using standardized Raman intensities followed as a function of time C(*C)TP population in the crystal maximized at about 20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred. PMID:23855392

  10. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    PubMed

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process.

  11. A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS

    PubMed Central

    2010-01-01

    Background Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS. Results Based on the reported Borax/PVPP/Phenol (BPP) protocol, we developed an efficient method for protein preparation from different latex subcellular fractions and constructed high-resolution reference 2-DE maps. The obtained proteins from both total latex and C-serum fraction with this protocol generate more than one thousand protein spots and several hundreds of protein spots from rubber particles as well as lutoid fraction and its membranes on the CBB stained 2-DE gels. The identification of 13 representative proteins on 2-DE gels by MALDI TOF/TOF MS/MS suggested that this method is compatible with MS. Conclusion The proteins extracted by this method are compatible with 2-DE and MS. This protein preparation protocol is expected to be used in future comparative proteomic analysis for natural rubber latex. PMID:20565811

  12. Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis.

    PubMed

    Wedholm, A; Møller, H S; Stensballe, A; Lindmark-Månsson, H; Karlsson, A H; Andersson, R; Andrén, A; Larsen, L B

    2008-10-01

    The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of beta-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of beta-, alpha(s1)-, and alpha(s2)-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.

  13. Treating cow's milk protein allergy: a double-blind randomized trial comparing two extensively hydrolysed formulas with probiotics.

    PubMed

    Vandenplas, Yvan; Steenhout, Philippe; Planoudis, Yannis; Grathwohl, Dominik

    2013-10-01

    The treatment for cow's milk protein allergy (CMPA) is a diet with an extensive hydrolysate. This study aimed to determine whether a whey (eWH) or casein hydrolysate (eCH) is the best option. Infants with suspected CMPA were treated with an eWH or eCH, and efficacy was assessed with a symptom-based score developed by the authors. Diagnosis of CMPA was based on a positive challenge. If positive, the same eHF/eCH was continued. If negative, a standard starter and follow-up formula were given up to the age of 12 months. An open challenge was performed on 85/116 (73%) infants suspected of CMPA on clinical grounds and was positive in 59/85 (69%). After 1 month, the symptom-based scores in both groups showed significant statistical and clinical reductions, and total and specific IgE and skin prick test results were similar. Both hydrolysates were enriched with probiotics, which were recovered in the gastrointestinal flora. The eWH-Standard Formula sequence led to better growth at the age of 1 year than the other three feeding regimens tested. The eWH and eCH are equally effective. The symptom-based score is a useful tool to evaluate the efficacy of dietary treatment in infants with CMPA. ©2013 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  14. Total salivary gland proteins of female Culex pipiens and Aedes caspius (Diptera: Culicidae) and their fractionation during adult development and after blood sucking.

    PubMed

    Soliman, M A; Abdel-Hamid, M E; Mansour, M M; Seif, A I; Kamel, K I; el Hamshary, E M

    1999-08-01

    Salivary glands of Culex pipiens and Aedes caspius were analyzed to determine total protein content and its fractionation during adult female development and after blood sucking. In both species, the molecular weight of proteins ranged between 26.000 and 84.000 Daltons. These proteins were not identical in the two species. In Cx. pipiens, the total protein level increased during the first 3 days of adult development from 4.94 +/- 0.84 to 6.6 +/- 0.37 micrograms/gland. During this period, the salivary gland proteins were separated into 35, 34 and 37 fractions respectively. Cx. pipiens released in the human host 64% of the total proteins while taking a blood meal compared to unfed females. This decrease in protein level was proportional to protein fractions. Over the next 6 days, the protein level increased again to attain values comparable to those obtained prior to blood sucking. In Ae. caspius, the total protein level of the salivary glands did not change during the first 4 days of adult development (range between 3.13 +/- 0.27 and 3.91 +/- 0.36 micrograms gland), but on the fifth day, 2-fold increase was observed. The total salivary gland protein increased during the next 3 days after blood sucking to reach 15.5 +/- 0.98 micrograms/gland. During this period, a tremendous change in protein patterns was observed. After oviposition, on the fourth day, a significant reduction in the total protein level was observed (4.13 +/- 0.56 micrograms/gland), but over the next 3 days the level increased again (range between 4.13 +/- 0.66 and 7.13 +/- 0.66 micrograms/gland).

  15. Ecl1 is a zinc-binding protein involved in the zinc-limitation-dependent extension of chronological life span in fission yeast.

    PubMed

    Shimasaki, Takafumi; Ohtsuka, Hokuto; Naito, Chikako; Azuma, Kenko; Tenno, Takeshi; Hiroaki, Hidekazu; Murakami, Hiroshi; Aiba, Hirofumi

    2017-04-01

    Overexpression of Ecl1-family genes (ecl1 (+), ecl2 (+), and ecl3 (+)) results in the extension of the chronological life span in Schizosaccharomyces pombe. However, the mechanism for this extension has not been defined clearly. Ecl1-family proteins consist of approximately 80 amino acids, and four cysteine residues are conserved in their N-terminal domains. This study focused on the Ecl1 protein, mutating its cysteine residues sequentially to confirm their importance. As a result, all mutated Ecl1 proteins nearly lost the function to extend the chronological life span, suggesting that these four cysteine residues are essential for the Ecl1 protein. Utilizing ICP-AES (inductively coupled plasma atomic emission spectroscopy) analysis, we found that wild-type Ecl1 proteins contain zinc, while cysteine-mutated Ecl1 proteins do not. We also analyzed the effect of environmental zinc on the chronological life span. We found that zinc limitation extends the chronological life span, and this extension depends on the Ecl1-family proteins.

  16. Distribution of chromium species in a Cr-polluted soil: presence of Cr(III) in glomalin related protein fraction.

    PubMed

    Gil-Cardeza, María L; Ferri, Alejandro; Cornejo, Pablo; Gomez, Elena

    2014-09-15

    The accumulation of Cr in soil could be highly toxic to human health; therefore Cr soil distribution was studied in rhizosphere soils from Ricinus communis and Conium maculatum and bare soil (BS) from an industrial and urban area in Argentina. Total Cr, Cr(VI) and Cr(III) concentrations were determined in 3 soil fractions: total, extractable and associated to total-glomalin-related protein (T-GRSP). BS had the highest total Cr and total Cr(VI) concentrations. Total Cr(VI) concentration from both rhizosphere soils did not differ from the allowed value for residential area in Argentina (8 μg Cr(VI) g(-1) soil), while total Cr(VI) in BS was 1.8 times higher. Total Cr concentration in all the soils was higher than the allowed value (250 μg Cr g(-1) soil). Extractable and associated to T-GRSP Cr(VI) concentrations were below the detection limit. Cr(III) bound to T-GRSP was the highest in the BS. These findings are in agreement with a long term effect of glomalin in sequestrating Cr. In both plant species, total Cr was higher in root than in shoot and both species presented arbuscular mycorrhizal fungi (AMF). As far as we know, this is the first study that reports the presence of Cr in T-GRSP fraction of soil organic matter. These findings suggest that Cr mycorrhizostabilization could be a predominant mechanism used by R. communis and C. maculatum to diminish Cr soil concentration. Nevertheless, further research is needed to clarify the contribution of native AMF isolated from R. communis and C. maculatum rhizosphere to the Cr phytoremediation process.

  17. Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation

    PubMed Central

    Beckhove, Philipp; Warta, Rolf; Lemke, Britt; Stoycheva, Diana; Momburg, Frank; Schnölzer, Martina; Warnken, Uwe; Schmitz-Winnenthal, Hubertus; Ahmadi, Rezvan; Dyckhoff, Gerhard; Bucur, Mariana; Jünger, Simone; Schueler, Thomas; Lennerz, Volker; Woelfel, Thomas; Unterberg, Andreas; Herold-Mende, Christel

    2010-01-01

    Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele. PMID:20458140

  18. Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model.

    PubMed

    Ramos, Márcio V; Oliveira, Jefferson S; Figueiredo, Jozy G; Figueiredo, Ingrid S T; Kumar, Vijay L; Bitencourt, Flávio S; Cunha, F Q; Oliveira, Raquel S B; Bomfim, Liezelotte R; Vitor Lima-Filho, José; Alencar, Nylane M N

    2009-09-25

    The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII) and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production.

  19. Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen.

    PubMed

    Pumford, N R; Roberts, D W; Benson, R W; Hinson, J A

    1990-08-01

    The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1 hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellet indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl)acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.

  20. High speed two-dimensional protein separation without gel by isoelectric focusing-asymmetrical flow field flow fractionation: application to urinary proteome.

    PubMed

    Kim, Ki Hun; Moon, Myeong Hee

    2009-09-01

    An online multilane channel system for isoelectric focusing and asymmetrical flow field-flow fractionation (IEF-AF4) is utilized for the two-dimensional separation (2D: isoelectric point, pI, and hydrodynamic diameter, d(s)) of a human proteome sample followed by the shotgun proteomic analysis using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). IEF-AF4 was recently developed to carry out nongel-based high speed two-dimensional protein separation [ Kim , K. , et al. Anal. Chem. 2009, 81 , 1715 ]. In IEF-AF4, proteins are separated according to pI along an IEF channel located at the head of six AF4 channels, and then the fractionated protein bands are directed to multilane AF4 channels for size-based separation. In this report, the original IEF-AF4 system has been modified to avoid the possible adsorption of proteins onto the membrane wall of IEF segments during isoelectric focusing by isolating the IEF channel segments from the multilane AF4 channels. The performance of the modified IEF-AF4 system was tested with protein standards and was further applied for the 2D fractionation of the human urinary proteome sample under two ampholyte solutions with different pH ranges (pH 3-10 and 3-6). The entire 2D separation was achieved in less than 30 min. The collected protein fractions were digested for peptide analysis using nLC-ESI-MS-MS, resulting in the identification of 245 total urinary proteins, including 110 unique proteins that are not yet reported in literature. Our experiments also showed a higher efficiency in the identification of urine proteins using ampholyte solution in the narrower pH range.

  1. Synergistic skin heat shock protein expression in response to combined laser treatment with a diode laser and ablative fractional lasers.

    PubMed

    Paasch, Uwe; Sonja, Grunewald; Haedersdal, Merete

    2014-06-01

    Diode laser-based skin heating has been shown to minimise scars by interfering with wound healing responses through the induction of heat shock proteins (HSP). HSP are also induced after ablative fractional laser (AFXL) wound healing. AFXL itself is highly recommended for scar treatment. Therefore, the sequential combination of both modalities may produce superior outcomes. The aim of this study was to examine the pretreatment effects of a diode laser before AFXL on wound healing responses in terms of HSP up-regulation in an in vitro model. Immediate responses and responses on days 1, 3 or 6 post-procedure were studied in an in vitro porcine skin model (n = 240). Untreated samples served as control. Immunohistochemical investigation (Hsp70) was performed in all untreated controls, diode laser-, AFXL-, and in diode laser + AFXL-treated samples. Hsp70 was shown to be up-regulated by all interventions between days 1 and 6 after interventions. The largest effect was caused by the combination of a diode laser and an AFXL procedure. Diode laser exposure induces a skin HSP response that can be further enhanced by sequential AFXL treatment. Clinical studies are necessary to investigate the dose response of HSP on scar formation and refine suitable laser exposure settings.

  2. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    PubMed

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  3. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3*

    PubMed Central

    Floor, Stephen N.; Condon, Kendall J.; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A.

    2016-01-01

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins. PMID:26598523

  4. [Quantitative determination of blood-plasma protein fractions using micro-agar gel electrophoresis in highly pregnant dairy cows close to the day of delivery].

    PubMed

    Blauärmel, H; Krüger, I

    1976-01-01

    Ten clinically intact Jersey dairy cows in advanced pregnancy were examined and tested over a span of three hours before to seven hours after parturition. Blood was sampled from them in intervals of ten tp 20 minutes, and the blood plasma was checked total protein and the "classical" fractions. The average total protein value of the plasma samples was 6.5 g/100 ml. The albumin and alpha 1-fractions were inversely proportional the psi-globulin concentrations before, during, and after parturition. The albumin values were on a declining trend three hours prior to parturition and recommenced to rise ten minutes prior to parturition. They were relatively constant after parturition. The pri-globulins went up slightly prior to parturition, but that rise remained statistically unsecured. It is assumed that in a certain period of time from before to somewhen after parturition change of the fractions is caused by the corticoids and sexual hormones.

  5. Storage Stability of Keratinocyte Growth Factor-2 in Lyophilized Formulations: Effects of Formulation Physical Properties and Protein Fraction at the Solid-Air Interface

    PubMed Central

    Devineni, Dilip; Gonschorek, Christoph; Cicerone, Marcus T; Xu, Yemin; Carpenter, John F.; Randolph, Theodore W.

    2014-01-01

    Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60 °C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements −1 for hydrogen atoms (fast β relaxation), and the relaxation time τβ, which correlates with relaxation due to fast Johari-Goldstein motions in the glass[1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer-Emmet-Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid-air interface. KGF-2 degradation rates were highest in formulations wherein the protein’s structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid-air interface. PMID:24859390

  6. Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction.

    PubMed

    Lamberts, Regis R; Lingam, Shivanjali J; Wang, Heng-Yu; Bollen, Ilse A E; Hughes, Gillian; Galvin, Ivor F; Bunton, Richard W; Bahn, Andrew; Katare, Rajesh; Baldi, J Chris; Williams, Michael J A; Saxena, Pankaj; Coffey, Sean; Jones, Peter P

    2014-04-05

    Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels. Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes

  7. Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction

    PubMed Central

    2014-01-01

    Background Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. Methods Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. Results In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels. Conclusions Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory

  8. Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

    PubMed Central

    Di Cagno, Raffaella; De Angelis, Maria; Lavermicocca, Paola; De Vincenzi, Massimo; Giovannini, Claudio; Faccia, Michele; Gobbetti, Marco

    2002-01-01

    Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination

  9. Proteolysis by sourdough lactic acid bacteria: effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance.

    PubMed

    Di Cagno, Raffaella; De Angelis, Maria; Lavermicocca, Paola; De Vincenzi, Massimo; Giovannini, Claudio; Faccia, Michele; Gobbetti, Marco

    2002-02-01

    Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination

  10. Protein Stretching III: Force-Extension Curves of Tethered Bovine Carbonic Anhydrase B to the Silicon Substrate under Native, Intermediate and Denaturing Conditions

    NASA Astrophysics Data System (ADS)

    Wang, Tong; Ikai, Atsushi

    1999-06-01

    Atomic force microscope (AFM) was used to measure theforce-extension relationship of the globular protein, carbonic anhydrase B, having 259 amino acid residues, under native, denaturing and intermediate solution conditions. For thispurpose, the protein was genetically engineered in order toendow it with -SH groups at its N- and C- termini and was fixed tothe silanized surface of a silicon wafer using a covalent cross-linker with the reactive end to -SH and with a long spacer of polyethylene glycol. The silicon nitride tip of the AFM was covered with a cross-linker with the reactive end to -SH and was brought into contact with the protein on the silicon surface. Subsequent force curve measurements showed occasional stable bond formation, and the force extension relationship obtained from such curves showed distinct characteristics of the native, denatured and intermediate forms of carbonic anhydrase B.

  11. Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses.

    PubMed

    Srivastava, K; Chaves, J M; Srivastava, O P; Kirk, M

    2008-10-01

    The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate-PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M(r) between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of alpha-, beta-, and gamma-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but

  12. Contribution of the starch, protein, and lipid fractions to the physical, thermal, and structural properties of amaranth (Amaranthus caudatus) flour films.

    PubMed

    Tapia-Blácido, D; Mauri, A N; Menegalli, F C; Sobral, P J A; Añón, M C

    2007-06-01

    Amaranth protein-lipid (PL) and protein (P) films were elaborated and compared with amaranth flour films in order to determine the contribution of the interactions between the biopolymer (starch and protein) and the lipids to the film properties. The films were made by the casting method, using the same glycerol concentration (0.9 g glycerol/100 g solution). A separation of the lipid fraction in the PL films and a polymorphic transformation of the corresponding fatty acids were observed by differential scanning calorimetry (DSC) and verified by an analysis of the microstructure by scanning electron microscopy (SEM). The flour films showed no separation of the lipid fraction, evidence that the lipids were strongly associated with the proteins and homogenously distributed throughout the starch network, contributing to the good mechanical properties when compared to the PL films and to the excellent barrier properties when compared to both the PL and P films. The protein-protein interactions also contributed to the mechanical properties of the flour films. The presence of proteins and lipids in the flour films had an important effect on film solubility, and also on the color and opacity of the films. This study showed that the flour film properties depended on the interactions formed by their polymers (starches and proteins) and by the lipid, on the distribution of these interactions within the film matrix and on the concentrations of each component in the film.

  13. aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism.

    PubMed

    Chu, Yindi; Zhu, Yanping; Chen, Yuling; Li, Wei; Zhang, Zhenfeng; Liu, Di; Wang, Tongkun; Ma, Juncai; Deng, Haiteng; Liu, Zhi-Jie; Ouyang, Songying; Huang, Li

    2016-09-01

    Protein methylation is believed to occur extensively in creanarchaea. Recently, aKMT, a highly conserved crenarchaeal protein lysine methyltransferase, was identified and shown to exhibit broad substrate specificity in vitro Here, we have constructed an aKMT deletion mutant of the hyperthermophilic crenarchaeon Sulfolobus islandicus The mutant was viable but showed a moderately slower growth rate than the parental strain under non-optimal growth conditions. Consistent with the moderate effect of the lack of aKMT on the growth of the cell, expression of a small number of genes, which encode putative functions in substrate transportation, energy metabolism, transcriptional regulation, stress response proteins, etc, was differentially regulated by more than twofold in the mutant strain, as compared with that in the parental strain. Analysis of the methylation of total cellular protein by mass spectrometry revealed that methylated proteins accounted for ∼2/3 (1,158/1,751) and ∼1/3 (591/1,757) of the identified proteins in the parental and the mutant strains, respectively, indicating that there is extensive protein methylation in S. islandicus and that aKMT is a major protein methyltransferase in this organism. No significant sequence preference was detected at the sites of methylation by aKMT. Methylated lysine residues, when visible in the structure, are all located on the surface of the proteins. The crystal structure of aKMT in complex with S-adenosyl-l-methionine (SAM) or S-adenosyl homocysteine (SAH) reveals that the protein consists of four α helices and seven β sheets, lacking a substrate recognition domain found in PrmA, a bacterial homolog of aKMT, in agreement with the broad substrate specificity of aKMT. Our results suggest that aKMT may serve a role in maintaining the methylation status of cellular proteins required for the efficient growth of the organism under certain non-optimal conditions.

  14. Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

    PubMed Central

    Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

    2016-01-01

    Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522

  15. Isolation of CA1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins.

    PubMed

    Yuanxiang, Pingan; Bera, Sujoy; Karpova, Anna; Kreutz, Michael R; Mikhaylova, Marina

    2014-08-10

    Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

  16. Composition of the non-protein nitrogen fraction of goat whole milk powder and goat milk-based infant and follow-on formulae.

    PubMed

    Prosser, Colin G; Mclaren, Robert D; Frost, Deborah; Agnew, Michael; Lowry, Dianne J

    2008-03-01

    The non-protein nitrogen fraction of goat whole milk powder and of infant and follow-on formulae made from goat milk was characterized and compared with cow milk powder and formulae. Goat milk infant formula contained 10% non-protein nitrogen, expressed as a proportion of total nitrogen, compared with 7.1% for cow milk formula. Goat follow-on formula contained 9.3% and cow 7.4% non-protein nitrogen. Urea, at 30%, was quantitatively the most abundant component of the non-protein nitrogen fraction of goat milk and formulae, followed by free amino acids at 7%. Taurine, glycine and glutamic acid were the most abundant free amino acids in goat milk powders. Goat milk infant formula contained 4 mg/100 ml total nucleotide monophosphates, all derived from the goat milk itself. Goat milk has a very different profile of the non-protein nitrogen fraction to cow milk, with several constituents such as nucleotides at concentrations approaching those in human breast milk.

  17. Thermoase-derived flaxseed protein hydrolysates and membrane ultrafiltration peptide fractions have systolic blood pressure-lowering effects in spontaneously hypertensive rats.

    PubMed

    Nwachukwu, Ifeanyi D; Girgih, Abraham T; Malomo, Sunday A; Onuh, John O; Aluko, Rotimi E

    2014-10-09

    Thermoase-digested flaxseed protein hydrolysate (FPH) samples and ultrafiltration membrane-separated peptide fractions were initially evaluated for in vitro inhibition of angiotensin I-converting enzyme (ACE) and renin activities. The two most active FPH samples and their corresponding peptide fractions were subsequently tested for in vivo antihypertensive activity in spontaneously hypertensive rats (SHR). The FPH produced with 3% thermoase digestion showed the highest ACE- and renin-inhibitory activities. Whereas membrane ultrafiltration resulted in significant (p < 0.05) increases in ACE inhibition by the <1 and 1-3 kDa peptides, only a marginal improvement in renin-inhibitory activity was observed for virtually all the samples after membrane ultrafiltration. The FPH samples and membrane fractions were also effective in lowering systolic blood pressure (SBP) in SHR with the largest effect occurring after oral administration (200 mg/kg body weight) of the 1-3 kDa peptide fraction of the 2.5% FPH and the 3-5 kDa fraction of the 3% FPH. Such potent SBP-lowering capacity indicates the potential of flaxseed protein-derived bioactive peptides as ingredients for the formulation of antihypertensive functional foods and nutraceuticals.

  18. Selective fermentation of carbohydrate and protein fractions of Scenedesmus, and biohydrogenation of its lipid fraction for enhanced recovery of saturated fatty acids.

    PubMed

    Lai, YenJung Sean; Parameswaran, Prathap; Li, Ang; Aguinaga, Alyssa; Rittmann, Bruce E

    2016-02-01

    Biofuels derived from microalgae have promise as carbon-neutral replacements for petroleum. However, difficulty extracting microalgae-derived lipids and the co-extraction of non-lipid components add major costs that detract from the benefits of microalgae-based biofuel. Selective fermentation could alleviate these problems by managing microbial degradation so that carbohydrates and proteins are hydrolyzed and fermented, but lipids remain intact. We evaluated selective fermentation of Scenedesmus biomass in batch experiments buffered at pH 5.5, 7, or 9. Carbohydrates were fermented up to 45% within the first 6 days, protein fermentation followed after about 20 days, and lipids (measured as fatty acid methyl esters, FAME) were conserved. Fermentation of the non-lipid components generated volatile fatty acids, with acetate, butyrate, and propionate being the dominant products. Selective fermentation of Scenedesmus biomass increased the amount of extractable FAME and the ratio of FAME to crude lipids. It also led to biohydrogenation of unsaturated FAME to more desirable saturated FAME (especially to C16:0 and C18:0), and the degree of saturation was inversely related to the accumulation of hydrogen gas after fermentation. Moreover, the microbial communities after selective fermentation were enriched in bacteria from families known to perform biohydrogenation, i.e., Porphyromonadaceae and Ruminococcaceae. Thus, this study provides proof-of-concept that selective fermentation can improve the quantity and quality of lipids that can be extracted from Scenedesmus.

  19. Hard-to-cook bean (Phaseolus vulgaris L.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress.

    PubMed

    Oseguera-Toledo, Miguel E; Gonzalez de Mejia, Elvira; Amaya-Llano, Silvia L

    2015-10-01

    The objective was to evaluate the effect of bioactive peptide fractions from de-hulled hard-to-cook (HTC) bean on enzyme targets of type-2 diabetes and oxidative stress. Protein isolates from Pinto Durango and Negro 8025 beans were hydrolyzed (120min) with either alcalase® or bromelain and separated into five peptide fractions (<1, 1-3.5, 3.5-5, 5-10, and >10kDa) using an ultrafiltration membrane system. The <1kDa pinto Durango-bromelain fraction showed the best inhibition of α-amylase (49.9±1.4%), and the <1kDa pinto Durango-alcalase fraction inhibited both, α-glucosidase (76.4±0.5%), and dipeptidyl peptidase-IV (DPP-IV, 55.3±1.6%). Peptides LLSL, QQEG and NEGEAH were present in the most potent fractions. Hydrolysates and peptide fractions showed antioxidant capacity (ORAC: 159.6±2.9 to 932.6±1.1mmolTE/g) and nitric oxide inhibition (57.5±0.9 to 68.3±4.2%). Hydrolysates and fractions <1 and 1-3kDa were able to increase glucose-stimulated insulin secretion from iNS-1E cells up to 57% compared to glucose control. Hydrolysates from HTC beans inhibited enzymes related to diabetes management, being the smallest peptides (<1kDa) the most potent. HTC bean could be a source of protein to produce bioactive peptides with potential antidiabetic properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells

    SciTech Connect

    Tai, Cheng-Jeng; Shen, Shing-Chuan; Lee, Woan-Ruoh; Liao, Ching-Fong; Deng, Win-Ping; Chiou, Hung-Yi; Hsieh, Cheng-I; Tung, Jai-Nien; Chen, Ching-Shyang; Chiou, Jeng-Fong; Li, Li-Tzu; Lin, Chuang-Yu; Hsu, Chung-Huei; Jiang, Ming-Chung

    2010-10-15

    Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of {alpha}-tubulin with {beta}-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with {alpha}-tubulin and {beta}-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of {alpha}-tubulin and {beta}-tubulin, increased {alpha}-tubulin and {beta}-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

  1. Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells.

    PubMed

    Tai, Cheng-Jeng; Shen, Shing-Chuan; Lee, Woan-Ruoh; Liao, Ching-Fong; Deng, Win-Ping; Chiou, Hung-Yi; Hsieh, Cheng-I; Tung, Jai-Nien; Chen, Ching-Shyang; Chiou, Jeng-Fong; Li, Li-Tzu; Lin, Chuang-Yu; Hsu, Chung-Huei; Jiang, Ming-Chung

    2010-10-15

    Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of α-tubulin with β-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with α-tubulin and β-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of α-tubulin and β-tubulin, increased α-tubulin and β-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Controlled intra- and transdermal protein delivery using a minimally invasive Erbium:YAG fractional laser ablation technology.

    PubMed

    Bachhav, Y G; Heinrich, A; Kalia, Y N

    2013-06-01

    The aim of the study was (i) to investigate the feasibility of using fractional laser ablation to create micropore arrays in order to deliver proteins into and across the skin and (ii) to demonstrate how transport rates could be controlled by variation of poration and formulation conditions. Four proteins with very different structures and properties were investigated - equine heart cytochrome c (Cyt c; 12.4 kDa), recombinant human growth hormone expressed in Escherichia coli (hGH; 22 kDa), urinary follicle stimulating hormone (FSH; 30 kDa) and FITC-labelled bovine serum albumin (FITC-BSA; 70 kDa). The transport experiments were performed using a scanning Er:YAG diode pumped laser (P.L.E.A.S.E.®; Precise Laser Epidermal System). The distribution of FITC-BSA in the micropores following P.L.E.A.S.E.® poration was visualised by using confocal laser scanning microscopy (CLSM). Porcine skin was used for the device parameter and CLSM studies; its validity as a model was confirmed by subsequent comparison with transport of Cyt c and FITC-BSA across P.L.E.A.S.E.® porated human skin. No protein transport (deposition or permeation) was observed across intact skin; however, P.L.E.A.S.E.® poration enabled total delivery after 24h of 48.2±8.9, 8.1±4.2, 0.2±0.1 and 273.3±30.6 μg/cm(2) for Cyt c, hGH, FSH and FITC-BSA, respectively, using 900 pores/135.9 cm(2). Calculation of permeability coefficients showed that there was no linear dependence of transport on molecular weight ((1.6±0.3), (0.1±0.05), (0.08±0.03) and (0.9±0.1)×10(-3) cm/h, for Cyt c, hGH, FSH and FITC-BSA, respectively); indeed, a U-shaped curve was observed. This suggested that molecular weight was not a sufficiently sensitive descriptor and that transport was more likely to be determined by the surface properties of the respective proteins since these would govern interactions with the local microenvironment. Increasing pore density (i.e. the number of micropores per unit area) had a statistically

  3. Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

    PubMed Central

    Sockolosky, Jonathan T.; Tiffany, Matthew R.; Szoka, Francis C.

    2012-01-01

    The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337–2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins. PMID:22991460

  4. Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue.

    PubMed

    Sajic, Tatjana; Varesio, Emmanuel; Szanto, Ildiko; Hopfgartner, Gérard

    2015-09-01

    In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K(+) as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Effects of nitrogen fertilization and harvest date on yield, digestibility, fiber, and protein fractions of tropical grasses.

    PubMed

    Johnson, C R; Reiling, B A; Mislevy, P; Hall, M B

    2001-09-01

    To evaluate the response of three tropical forage species to varying rates of nitrogen (N) fertilization [0, 39, 78, 118, 157 kg of N/(ha x cutting)] and five summer harvests, forage DM mass and nutritive value were evaluated in a randomized complete block design with a split-split plot arrangement of treatments. Plots (n = 60) were established in 1996, and five harvests were conducted every 28 d from June through September in 1997 and 1998, with fertilizer applications occuring after each harvest. Fertilization with 78 kg of N/(ha x cutting) increased forage mass in these grasses by 129% (P < 0.01) compared with no N fertilization. Additional N did not result in further increases of forage mass. Bermudagrass (Cynodon dactylon) produced more forage DM [P < 0.01; 1,536 +/- 43 kg/(ha x cutting)] than stargrass [Cynodon nlemfuensis; 1,403 +/- 43 kg/(ha x cutting)] or bahiagrass [Paspalum notatum; 1,297 +/- 43 kg/(ha x cutting)]. Peak forage mass for all species occurred in late June and July. In vitro organic matter digestibility (IVOMD) of stargrass increased (P < 0.01) linearly with fertilization. A quadratic response to N fertilization (P < 0.01) was noted in IVOMD of bermudagrass, whereas bahiagrass was not affected. Bermudagrass was more (P < 0.01) digestible (57.5 +/- 0.4) than stargrass (54.6 +/- 0.4) and bahiagrass (51.9 +/- 0.4%). As fertilization level increased, NDF decreased linearly (P < 0.01) in all three forages. Total N concentration increased (P < 0.01) linearly as N fertilization increased in all forages. Total N concentration was highest (P < 0.01) in stargrass (2.4%, DM basis) compared with bermudagrass (2.2%) and bahiagrass (2.0%). Total N concentration was depressed in all forages for late June and July harvests (P < 0.01). Fertilization increased (P < 0.05) the concentration (% of DM) of all protein fractions. In July and August, nonprotein N was reduced 11.8% (P < 0.01), whereas ADIN increased in July (P < 0.01). Bahiagrass had less N in cell

  6. Concentration and selective fractionation of an antihypertensive peptide from an alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition chromatography.

    PubMed

    Boudesocque, Leslie; Kapel, Romain; Paris, Cedric; Dhulster, Pascal; Marc, Ivan; Renault, Jean-Hugues

    2012-09-15

    This article reports a promising use of the mixed ion-exchange centrifugal partition chromatography (MIXCPC) technique in the field of downstream processes. A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate) showing anti-hypertensive properties was successfully fractionated by MIXCPC to yield a L-valyl-L-tryptophan (VW) enriched fraction in one run. This dipeptide shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An analytical method based on RP-LC/MS-MS was developed to quantify the target VW peptide in both the starting material and the enriched fractions. The best results for the MIXCPC fractionation were obtained by the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two displacers: calcium chloride and hydrochloric acid. The complexity of the starting material involved the selectivity optimization by splitting the stationary phase into two sections that differed by their triethylamine concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7 mg of a VW enriched fraction were recovered with a purity of 10.9%, corresponding to a purification factor of 41 and a recovery of 97%.

  7. Analysis of mRNA With Microsomal Fractionation Using a SAGE-Based DNA Microarray System Facilitates Identification of the Genes Encoding Secretory Proteins

    PubMed Central

    Toyoda , Nobuaki; Nagai, Shigenori; Terashima, Yuya; Motomura, Kazushi; Haino, Makoto; Hashimoto, Shin-ichi; Takizawa, Hajime; Matsushima, Kouji

    2003-01-01

    In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)–based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins. PMID:12805275

  8. Determination of the Mercury Fraction Linked to Protein of Muscle and Liver Tissue of Tucunaré (Cichla spp.) from the Amazon Region of Brazil.

    PubMed

    Vieira, José C S; Cavecci, Bruna; Queiroz, João V; Braga, Camila P; Padilha, Cilene C F; Leite, Aline L; Figueiredo, Wllyane S; Buzalaf, Marília A R; Zara, Luiz F; Padilha, Pedro M

    2015-11-01

    This study used metalloproteomic techniques to characterize mercury (Hg)-bound proteins in the muscle and liver tissue of Tucunaré (Cichla spp.) collected at the Jirau Hydroelectric Power Plant in Madeira River Basin, Brazil. The proteome of the muscle and liver tissue was obtained after two steps of fractional precipitation and separating the proteins by 2-D polyacrylamide gel electrophoresis. Hg was identified and quantified in the protein spots by graphite furnace atomic absorption spectrometry after acid mineralization in an ultrasound bath. Hg with a molecular weight <20 kDa and a concentration between 13.30 and 33.40 mg g(-1) was found in the protein spots. These protein spots were characterized by electrospray ionization tandem mass spectrometry after trypsin digestion. From a total of 12 analyzed spots, seven proteins showing Hg biomarker characteristics were identified: parvalbumin and its isoforms, ubiquitin-40S ribosomal protein S27a, zinc (Zn) finger and BTB domain-containing protein 24, and dual-specificity protein phosphatase 22-B.

  9. The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

    PubMed Central

    Graham, A. B.; Woodcock, B. G.; Wood, G. C.

    1974-01-01

    After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not signific