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Sample records for extensive protein fractionation

  1. Manufacture and use of dairy protein fractions.

    PubMed

    Etzel, Mark R

    2004-04-01

    Fractionation of the mixture of proteins found in milk and whey to form pure, individual dairy protein fractions might allow individuals with special nutritional needs to tailor their diet to improve health. Ion exchange process chromatography was examined for this purpose using selective elution to release separately the proteins bound from whey and produce several protein fractions. Alternatively, bound proteins were released all at once to make a whey protein isolate. Prototype beverages containing these proteins were examined for clarity before and after thermal processing. Beverages containing whey protein isolate were clear at pH 2-7 before heating, but only beverages at pH proteins formed during thermal processing. Alternatively, thermally processed clear beverages at pH 3-7 were possible using the whey protein glycomacropeptide. Because of the balance between sweetness and acidity, beverages with a pH greater than carbonated soft drinks and juices (pH 2.5-3) might remain palatable using less sugar. Development of high-protein low-carbohydrate beverages might provide health benefits for individuals suffering from diabetes, obesity, and hypercholesterolemia, especially when these beverages contain dairy protein fractions known to be high in essential amino acids and branched-chain amino acids. PMID:15051860

  2. Fractionation of the proteins of plant microbodies.

    PubMed

    Brown, R H; Lord, J M; Merrett, M J

    1974-12-01

    1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ;ghost' proteins). 3. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of ;ghost' fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000-28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ;ghosts' indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).

  3. 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.

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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)...

  11. 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)...

  12. 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)...

  13. 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)...

  14. 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.

  15. In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques.

    PubMed

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

    2012-09-01

    Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum. PMID:22848049

  16. Expressed protein ligation-mediated template protein extension.

    PubMed

    Kamei, Ayako; Hauser, Paul S; Beckstead, Jennifer A; Weers, Paul M M; Ryan, Robert O

    2012-06-01

    Expressed protein ligation (EPL) was performed to investigate sequence requirements for a variant human apolipoprotein A-I (apoA-I) to adopt a folded structure. A C-terminal truncated apoA-I, corresponding to residues 1-172, was expressed and isolated from Escherichia coli. Compared to full length apoA-I (243 amino acids), apoA-I(1-172) displayed less α-helix secondary structure and lower stability in solution. To determine if extension of this polypeptide would confer secondary structure content and/or stability, 20 residues were added to the C-terminus of apoA-I(1-172) by EPL, creating apoA-I(Milano)(1-192). The EPL product displayed biophysical properties similar to full-length apoA-I(Milano). The results provide a general protein engineering strategy to modify the length of a recombinant template polypeptide using synthetic peptides as well as a convenient, cost effective way to investigate the structure/function relations in apolipoprotein fragments or domains of different size.

  17. Method for voltage-gated protein fractionation

    DOEpatents

    Hatch, Anson; Singh, Anup K.

    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.

  18. Whey protein fractionation using supercritical carbon dioxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Whey protein fractionation using supercritical carbon dioxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sweet whey, the watery product of the cheesemaking process, is usually concentrated using ultrafiltration or ion-exchange to produce whey protein concentrates (WPC) and whey protein isolates (WPI), respectively. WPC are comprised mainly of beta-lactoglobulin (LG), alpha-lactalbumin (LA), proteose - ...

  20. Multielement analysis of metal-binding proteins in cytosol fractions.

    PubMed

    Bray, J T; Webb, L A; Reilly, F J

    1983-06-01

    The distribution of 24 elements among the cytosol protein fractions was determined for specimens of the bivalve mollusc Macoma balthica obtained from three estuarine locations subject to varying levels of metal pollution and on specimens of Rangia cuneata from three areas subject to varying degrees of thermal pollution. Of the 24 elements examined 15 occurred at levels above detection limits and in association with one or more of four distinct protein fractions. Levels of Ag and Cu associated with high molecular weight proteins and with "metallothionein-like" proteins permitted identification of those Macoma balthica specimens exposed to the greatest degree of metal stress.

  1. Structure characterization of protein fractions from lotus ( Nelumbo nucifera) seed

    NASA Astrophysics Data System (ADS)

    Zeng, Hong-Yan; Cai, Lian-Hui; Cai, Xi-Ling; Wang, Ya-Ju; Li, Yu-Qin

    2011-08-01

    Protein fractionation of lotus seed was carried out and the structures of the protein fractions were studied. Fourier transform infrared spectroscopy (FTIR) as well as ultraviolet visible spectroscopy (UV-vis) was used to investigate changes in molecular structures of the protein fractions. FTIR and UV-vis spectra showed the protein fractions had different protein molecular structures. FTIR spectra showed β-sheets and β-turns as the major secondary structures in the individual protein fractions, while the amounts of α-helix and random coil structures among the different fractions did not significantly change. The amounts of β-sheet structures of albumin and globulin were significantly higher than ones of prolamin and glutelin, implying albumin and globulin had high stabilities because of the high content in β-sheet structures. The observed similarity in the amounts of α-helix, random coil, β-sheet and β-turn structures shared by albumin and globulin indicated that their interior conformations were similar.

  2. Annotation extension through protein family annotation coherence metrics

    PubMed Central

    Bastos, Hugo P.; Clarke, Luka A.; Couto, Francisco M.

    2013-01-01

    Protein functional annotation consists in associating proteins with textual descriptors elucidating their biological roles. The bulk of annotation is done via automated procedures that ultimately rely on annotation transfer. Despite a large number of existing protein annotation procedures the ever growing protein space is never completely annotated. One of the facets of annotation incompleteness derives from annotation uncertainty. Often when protein function cannot be predicted with enough specificity it is instead conservatively annotated with more generic terms. In a scenario of protein families or functionally related (or even dissimilar) sets this leads to a more difficult task of using annotations to compare the extent of functional relatedness among all family or set members. However, we postulate that identifying sub-sets of functionally coherent proteins annotated at a very specific level, can help the annotation extension of other incompletely annotated proteins within the same family or functionally related set. As an example we analyse the status of annotation of a set of CAZy families belonging to the Polysaccharide Lyase class. We show that through the use of visualization methods and semantic similarity based metrics it is possible to identify families and respective annotation terms within them that are suitable for possible annotation extension. Based on our analysis we then propose a semi-automatic methodology leading to the extension of single annotation terms within these partially annotated protein sets or families. PMID:24130572

  3. 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.

  4. 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.

  5. [Fractional and amino acid composition of krill proteins and the potential for obtaining protein preparations].

    PubMed

    Orlova, T A; Churina, E E; Kuranova, L K

    1985-01-01

    Studies of the fractional composition of krill proteins demonstrated that the content of protein fractions changes depending on the time of krill catch. The highest amount of water-soluble proteins is contained by krill caught in December (64%), of salt-soluble by krill caught in June (12%), base-soluble by krill caught in May, September and February (34%). Krill protein contains from 50 to 60% of water- and salt-soluble fractions. Analysis of the amino acid composition of krill proteins showed that it does not differ essentially from that of adequate food proteins.

  6. Protein Solubility, Digestibility and Fractionation after Germination of Sorghum Varieties

    PubMed Central

    Afify, Abd El-Moneim M. R.; El-Beltagi, Hossam S.; Abd El-Salam, Samiha M.; Omran, Azza A.

    2012-01-01

    The changes in crude protein, free amino acids, amino acid composition, protein solubility, protein fractionation and protein digestibility after germination of sorghum were investigated. Sorghum varieties (Dorado, Shandaweel-6, Giza-15) were soaked for 20 h followed by germination for 72 h; the results revealed that crude protein and free amino acids in raw sorghum varieties ranged from 10.62 to 12.46% and 0.66 to 1.03 mg/g, respectively. Shandaweel-6 was the highest variety in crude protein and free amino acids content. After germination, crude protein was decreased and free amino acids were increased. There was an increase in content of valine and phenylalanine amino acids after germination. On the other hand, there was a decrease in most of amino acids after germination. After germination protein solubility was significantly increased. Regarding protein fractions, there was an increase in albumin, globulin and kafirin proteins and a decrease in cross linked kafirin and cross linked glutelin after germination. PMID:22319611

  7. Multifunctional cationic peptide fractions from flaxseed protein hydrolysates.

    PubMed

    Udenigwe, Chibuike C; Aluko, Rotimi E

    2012-03-01

    The aim of this work was to determine the multifunctional properties of flaxseed protein-derived cationic peptide fractions. Alcalase hydrolysis of flaxseed protein fractions liberated cationic peptides, which were separated into two major fractions (FI and FII) by chromatography using a cation-exchange column. Due to their cationic property, the peptide fractions bound and inactivated calmodulin (CaM, a negatively charged enzyme activator protein) with concomitant inhibition of CaM-dependent phosphodiesterase (CaMPDE); this activity was substantially reduced as CaM concentration increased. Enzyme kinetics studies showed competitive inhibition of CaMPDE by FI and FII with enzyme-inhibitor dissociation constants of 0.0202 and 0.0511 mg/ml, respectively. Only the FII peptides showed multifunctional activities by inhibiting CaMPDE, angiotensin converting enzyme (ACE) and renin. Separation of FII peptides by reverse phase HPLC resulted in eight fractions (FII-2 to FII-9) that inhibited the activities of CaMPDE, ACE, and renin but this multifunctional activity was more pronounced in FII-6. From LC-MS analysis, identified peptides present in FII fraction had molecular size range of 330-735 Da, which suggests potential for increased absorption. Potential peptide sequences were identified for each of the HPLC fractions and shown to contain either lysine or arginine as the positively charged amino acid residue. The multifunctional properties of the cationic peptide fractions can potentially enhance their use in targeting multiple symptoms of cardiovascular disease, considering that the excessive levels of CaM, CaMPDE, renin and ACE play important roles in enhancing progression and intensity of chronic human diseases. PMID:22327315

  8. Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2.

    PubMed

    Bricker, T M; Sherman, L A

    1984-11-15

    The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem. 256, 1604-1607, as adapted for photosynthetic membranes by T.M. Bricker and L.A. Sherman (1982) FEBS Lett. 149, 197-202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b6, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c550 and c554, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. 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.

  11. An Extension of the Hirshfeld Method to Open Shell Systems Using Fractional Occupations.

    PubMed

    Geldof, D; Krishtal, A; Blockhuys, F; Van Alsenoy, C

    2011-05-10

    In this work, a new partitioning method is presented which allows one to calculate properties of radicals, in particular, atomic spin populations. The method can be seen as an extension of the Hirshfeld-I method [ Bultinck , P. et al. J. Chem. Phys. 2007 , 126 , 144111 ], in which the atomic weight functions, defining the atoms-in-molecules, are constructed by means of an iterative scheme in which the charges of the atoms-in-molecules are altered but the spin remains fixed. The Hirshfeld-I method is therefore not suitable for the calculation of atomic spin populations of open-shell systems. The new fractional occupation Hirshfeld-I (FOHI) uses an iterative scheme in which both the atomic charge and spin are optimized, resulting in a self-consistent method for the calculation of atomic spin populations. The results obtained with the FOHI method are compared with experimental results obtained using polarized neutron diffraction, thus serving as a validation of the FOHI method as well as the Hirshfeld definition of atoms-in-molecules in general. PMID:26610127

  12. Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels.

    PubMed

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

    2014-01-28

    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.

  13. 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

  14. Morphology of molecular soy protein fractions in binary composite gels.

    PubMed

    Kasapis, Stefan; Tay, Sok Li

    2009-08-01

    We investigate the structural properties of gels of binary mixtures of the three major soy protein fractions: 11S, 7S, and 2S. Gels are formed at 25 degrees C in the presence of glucono-delta-lactone and studied using a combination of dynamic rheology and scanning electron microscopy. The theological data was then modeled using a blending-law approach that yields insights into the solvent distribution between the gelled protein fractions and first-order reaction kinetics that follow the gelation process of the single fractions and their mixtures. Gelled mixtures of 11S and 7S yielded enhanced network strength with increasing solid content; in these gels, 50% more solvent partitioned into the 11S phase as compared to that in the 7S phase. In contrast, the addition of small-molecular-weight counterpart 2S to either 11S or 7S results in a catastrophic drop in the values of the overall strength of the mixture. The unexpected phase behavior has been rationalized on the basis of the high water-holding capacity of 2S; 450% more solvent partitions preferentially into the 2S phase as compared to that in the 11S phase. As the concentration of 2S is increased relative to that of 11S or 7S, it becomes the dominant phase and entraps the polymeric segments of 11S (or 7S), thus preventing them from becoming the structural knots of the gel. In addition to the solvent distribution in the gel, the rates of gelation differ markedly between 11S and 2S (with the 11S rate of gelation being up to 2 orders of magnitude greater); a fixed 11S concentration, the rate of gelation decreases with increasing amounts of 2S, further confirming that the latter essentially becomes the dominant phase in the composite gel.

  15. 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.

  16. 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.

  17. Fractionation of proteins with two-sided electro-ultrafiltration.

    PubMed

    Käppler, Tobias; Posten, Clemens

    2007-03-10

    Downstream processing is a major challenge in bioprocess industry due to the high complexity of bio-suspensions itself, the low concentration of the product and the stress sensitivity of the valuable target molecules. A multitude of unit operations have to be joined together to achieve an acceptable purity and concentration of the product. Since each of the unit operations leads to a certain product loss, one important aim in downstream-research is the combination of different separation principles into one unit operation. In the current work a dead-end membrane process is combined with an electrophoresis operation. In the past this concept has proven successfully for the concentration of biopolymers. The present work shows that using different ultrafiltration membranes in a two-sided electro-filter apparatus with flushed electrodes brought significant enhancement of the protein fractionation process. Due to electrophoretic effects, the filtration velocity could be kept on a very high level for a long time, furthermore, the selectivity of a binary separation process carried out exemplarily for bovine serum albumin (BSA) and lysozyme (LZ) could be greatly increased; in the current case up to a value of more than 800. Thus the new two-sided electro-ultrafiltration technique achieves both high product purity and short separation times.

  18. 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.

  19. 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.

  20. 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

  1. Inhibition of thermal induced protein denaturation of extract/fractions of Withania somnifera and isolated withanolides.

    PubMed

    Khan, Murad Ali; Khan, Haroon; Rauf, Abdul; Ben Hadda, Taibi

    2015-01-01

    This study describes the in vitro inhibition of protein denaturation of extract/fractions of Withania somnifera and isolated withanolides including 20β hydroxy-1-oxo(22R)-witha-2,5,24 trienolide (1), (20R,22R-14α,20α)-dihydroxy-1-oxowitha-2,5,16,24 tetraenolide (2). The results showed that the extract/fractions of the plant evoked profound inhibitory effect on thermal-induced protein denaturation. The chloroform fraction caused the most dominant attenuation of 68% at 500 μg/mL. The bioactivity-guided isolation from chloroform fraction led to the isolation of compounds 1 and 2 that showed profound protein inhibition with 78.05% and 80.43% effect at 500 μg/mL and thus strongly complimented the activity of extract/fractions. In conclusion, extract/fractions of W. somnifera possessed strong inhibition of protein denaturation that can be attributed to these isolated withanolides.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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% ...

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

    PubMed

    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

  4. An extensive library of surrogate peptides for all human proteins.

    PubMed

    Mohammed, Yassene; Borchers, Christoph H

    2015-11-01

    Selecting the most appropriate surrogate peptides to represent a target protein is a major component of experimental design in Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its v-score remains distinctive in its approach of integrating information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM that is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our "best knowledge" for selecting candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it has previously been observed. Here we present an updated approach where we have already compiled a list of all possible surrogate peptides of the human proteome. Using our stringent selection criteria, the list includes 165k suitable MRM peptides covering 17k proteins of the human reviewed proteins in UniProtKB. Compared to average of 2-4min per protein for retrieving and integrating the information, the precompiled list includes all peptides available instantly. This allows a more cohesive and faster design of a multiplexed MRM experiment and provides insights into evidence for a protein's existence. We will keep this list up-to-date as proteomics data repositories continue to grow. This article is part of a Special Issue entitled: Computational Proteomics. PMID:26232110

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

    PubMed

    Vachon, V; Pouliot, J F; Laprade, R; Béliveau, R

    1991-01-01

    Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.

  8. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Protein synthesis rates in rat brain regions and subcellular fractions during aging

    SciTech Connect

    Avola, R.; Condorelli, D.F.; Ragusa, N.; Renis, M.; Alberghina, M.; Giuffrida Stella, A.M.; Lajtha, A.

    1988-04-01

    In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Fractional statistical theory of adsorption applied to protein adsorption.

    PubMed

    Quiroga, E; Centres, P M; Ochoa, N A; Ramirez-Pastor, A J

    2013-01-15

    Experimental adsorption isotherms of bovine serum albumin (BSA) adsorbed on sulfonated microspheres were described by means of two analytical models: the first is the well-known Langmuir-Freundlich model (LF), and the second, called fractional statistical theory of adsorption (FSTA), is a statistical thermodynamics model developed recently by Ramirez-Pastor et al. [Phys. Rev. Lett. 93 (2004) 186101]. The experimental data, obtained by Hu et al. [Biochem. Eng. J. 23 (2005) 259] for different concentrations of sulfonate group on the surface of the microspheres, were correlated by using a fitting algorithm based on least-squares statistics. The combination of LF and FSTA models, along with the choice of an adequate fitting procedure, allowed us to obtain several conclusions: (i) as previously reported in the literature, the maximum amount adsorbed increases as the amount of sulfonate group increases; (ii) the equilibrium constant does not appear as a sensitive parameter to the amount of sulfonate group on the surface of the microspheres; and (iii) the values of the fitting parameters obtained from FSTA may be indicative of a mismatch between the equilibrium separation of the intermolecular interaction and the distance between the adsorption sites. The exhaustive study presented here has shown that FSTA model is a good one considering the complexity of the physical situation, which is intended to be described and could be more useful in interpreting experimental data of adsorption of molecules with different sizes and shapes. PMID:23084559

  16. Dissecting the Subcellular Compartmentation of Proteins and Metabolites in Arabidopsis Leaves Using Non-aqueous Fractionation *

    PubMed Central

    Arrivault, Stéphanie; Guenther, Manuela; Florian, Alexandra; Encke, Beatrice; Feil, Regina; Vosloh, Daniel; Lunn, John E.; Sulpice, Ronan; Fernie, Alisdair R.; Stitt, Mark; Schulze, Waltraud X.

    2014-01-01

    Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin–Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite–protein analysis on a single tissue sample. PMID:24866124

  17. 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.

  18. An Economical High-Throughput Protocol for Multidimensional Fractionation of Proteins

    PubMed Central

    Tooth, David John; Gopala Krishna, Varun; Layfield, Robert

    2012-01-01

    A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells. Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins. Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps. Chromatographic fractions were shown to be readily compatible with 1- and 2-dimensional gel electrophoresis or with direct analysis by mass spectrometry using linear-MALDI-TOF-MS. Precision of extraction was confirmed by reproducible SDS-PAGE profiles, MALDI-TOF-MS spectra, and quantitation of trypsinolytic peptides using LC-MS/MS (MRM) analyses. Solid phases were immobilised in disposable cartridges and mobile-phase flow was achieved using a combination of centrifugation and vacuum pumping. These approaches yielded parallel sample handling which was limited only by the capacities of the employed devices and which enabled both high-throughput and experimentally precise procedures, as demonstrated by the processing of experimental replicates. Protocols were employed at 10 mg scale of extracted cell protein, but these approaches would be directly applicable to both smaller and larger quantities merely by adjusting the employed solid- and mobile-phase volumes. Additional potential applications of the fractionation protocol are briefly described. PMID:23008771

  19. 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. PMID:27156453

  20. 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

  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. PMID:26697039

  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. 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. PMID:16841690

  5. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

    PubMed

    Kuromitsu, Sadao; Yokota, Hiroyuki; Hiramoto, Masashi; Yuri, Masatoshi; Naitou, Masanori; Nakamura, Naoto; Kawabata, Shigeki; Kobori, Masato; Katoh, Masao; Furuchi, Kiyoshi; Mita, Haruhisa; Yamada, Tetsuo

    2009-06-01

    Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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

  8. The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains.

    PubMed

    Oliveira, Pedro M; Waters, Deborah M; Arendt, Elke K

    2013-03-01

    Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.

  9. Gluten protein composition in several fractions obtained by shear induced separation of wheat flour.

    PubMed

    van der Zalm, Elizabeth E J; Grabowska, Katarzyna J; Strubel, Maurice; van der Goot, Atze J; Hamer, Rob J; Boom, Remko M

    2010-10-13

    Recently, it was found that applying curvilinear shear flow in a cone-cone shearing device to wheat flour dough induces separation, resulting in a gluten-enriched fraction in the apex of the cone and gluten-depleted fraction at the outer part. This article describes whether fractionation of the various proteineous components occurs during and after separation of Soissons wheat flour. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high performance liquid chromatography (SE-HPLC) were found to be suitable techniques for this. It is concluded that all protein fractions migrate to the center of the cone as a result of which the composition of the gluten-enriched fraction remains rather similar to that in the original flour. However, the larger glutenin polymer fraction migrated faster, as a result of which the concentration of large polymers was increased with a factor 2.4 compared to that of Soissons flour. The concentration of monomers in the gluten-enriched fraction was decreased to 70% of the original concentration in the original wheat flour.

  10. 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.

  11. 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

  12. 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.

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

    PubMed

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

    2015-11-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', 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-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C 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-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ 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.

  14. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.

    PubMed

    Baghirova, Sabina; Hughes, Bryan G; Hendzel, Michael J; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C. PMID:26740924

  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.

  16. 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. PMID:26948602

  17. Coil fraction-dependent phase behaviour of a model globular protein-polymer diblock copolymer.

    PubMed

    Thomas, Carla S; Olsen, Bradley D

    2014-05-01

    The self-assembly of the model globular protein-polymer block copolymer mCherry-b-poly(N-isopropyl acrylamide) is explored across a range of polymer coil fractions from 0.21 to 0.82 to produce a phase diagram for these materials as a function of molecular composition. Overall, four types of morphologies were observed: hexagonally packed cylinders, perforated lamellae, lamellae, and disordered nanostructures. Across all coil fractions and morphologies, a lyotropic re-entrant order-disorder transition in water was observed, with disordered structures below 30 wt% and above 70 wt% and well-ordered morphologies at intermediate concentrations. Solid state samples prepared by solvent evaporation show moderately ordered structures similar to those observed in 60 wt% solutions, suggesting that bulk structures result from kinetic trapping of morphologies which appear at lower concentrations. While highly ordered cylindrical nanostructures are observed around a bioconjugate polymer volume fraction of 0.3 and well-ordered lamellae are seen near a volume fraction of 0.6, materials at lower or higher coil fractions become increasingly disordered. Notable differences between the phase behaviour of globular protein-polymer block copolymers and coil-coil diblock copolymers include the lack of spherical nanostructures at either high or low polymer coil fractions as well as shifted phase boundaries between morphologies which result in an asymmetric phase diagram. PMID:24695642

  18. Sarcocystis fusiformis: some protein metabolic enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis).

    PubMed

    Gupta, R S; Kushwah, H S; Kushwah, A

    1993-01-01

    An investigation on the relative presence of some protein metabolic enzymes, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), NAD+ and NADP+ dependent glutamate dehydrogenase (GLDH) and arginase in cyst wall (CW), cyst fluid (CF) and zoite (ZT) fractions of the sarcocysts of Sarcocystis fusiformis in the oesophageal muscles of Indian water buffalo was carried out. Both the transaminases were present in all the fractions of the cyst, although in variable amounts. There was a higher level of AST activity than of ALT activity. AST activity was the highest in ZT, whereas ALT activity was at a maximum in the CF fraction. The levels of activity of NAD+ and NADP+ dependent GLDH and arginase remained beyond detectable limits. The study revealed that the intermediates of carbohydrate metabolism are linked to protein metabolism by transaminases. The possibility of concomitant removal of ammonia and its subsequent incorporation into the urea cycle is ruled out in this parasitic protozoan.

  19. Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells.

    PubMed Central

    Chatterjee, P K; Flint, S J

    1986-01-01

    The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells. Images PMID:3023654

  20. Determination of Polycomb Group of Protein Compartmentalization Through Chromatin Fractionation Procedure.

    PubMed

    Marasca, Federica; Marullo, Fabrizia; Lanzuolo, Chiara

    2016-01-01

    Epigenetic mechanisms modulate and maintain the transcriptional state of the genome acting at various levels on chromatin. Emerging findings suggest that the position in the nuclear space and the cross talk between components of the nuclear architecture play a role in the regulation of epigenetic signatures. We recently described a cross talk between the Polycomb group of proteins (PcG) epigenetic repressors and the nuclear lamina. This interplay is important for the maintenance of transcriptional repression at muscle-specific genes and for the correct timing of muscle differentiation. To investigate the synergism between PcG factors and nuclear architecture we improved a chromatin fractionation protocol with the aim to analyze the PcG nuclear compartmentalization. We thus separated PcG proteins in different fractions depending on their solubility. We surprisingly found a consistent amount of PcG proteins in the matrix-associated fraction. In this chapter we describe the chromatin fractionation procedure, a method that can be used to study the nuclear compartmentalization of Polycomb group of proteins and/or PcG targets in murine and Drosophila cells. PMID:27659984

  1. 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.

  2. Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes.

    PubMed

    Liu, Yi; Lin, Yong; Yan, Yizhong; Li, Jianjun; He, Quanze; Chen, Ping; Wang, Xianchun; Liang, Songping

    2012-01-01

    SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins. PMID:22222976

  3. Effectiveness of esterified whey proteins fractions against Egyptian Lethal Avian Influenza A (H5N1)

    PubMed Central

    2010-01-01

    Background Avian influenza A (H5N1) virus is one of the most important public health concerns worldwide. The antiviral activity of native and esterified whey proteins fractions (α- lactalbumin, β- lactoglobulin, and lactoferrin) was evaluated against A/chicken/Egypt/086Q-NLQP/2008 HPAI (H5N1) strain of clade 2.2.1 (for multiplicity of infection (1 MOI) after 72 h of incubation at 37°C in the presence of 5% CO2) using MDCK cell lines. Result Both the native and esterified lactoferrin seem to be the most active antiviral protein among the tested samples, followed by β- lactoglobulin. α-Lactalbumin had less antiviral activity even after esterification. Conclusion Esterification of whey proteins fractions especially lactoferrin and β-lactoglobulin enhanced their antiviral activity against H5N1 in a concentration dependent manner. PMID:21092081

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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.

  6. Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

    PubMed Central

    Wessels, Hans J. C. T.; Vogel, Rutger O.; Lightowlers, Robert N.; Spelbrink, Johannes N.; Rodenburg, Richard J.; van den Heuvel, Lambert P.; van Gool, Alain J.; Gloerich, Jolein; Smeitink, Jan A. M.; Nijtmans, Leo G.

    2013-01-01

    Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP. PMID:23935861

  7. Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes

    PubMed Central

    1975-01-01

    The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented. PMID:166079

  8. Distinct effects of boar seminal plasma fractions exhibiting different protein profiles on the functionality of highly diluted boar spermatozoa.

    PubMed

    García, E M; Calvete, J J; Sanz, L; Roca, J; Martínez, E A; Vázquez, J M

    2009-04-01

    The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 x 10(6) spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4-6 contained low numbers of spermatozoa (<500 x 10(6)/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1-3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1-3 appear to promote sperm survival, whereas fractions 4-6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa. PMID:19323794

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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

    2014-01-01

    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. PMID:24927368

  11. 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

  12. 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

  13. 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

  14. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC.

  15. 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.

  16. Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris.

    PubMed

    Ursu, Alina-Violeta; Marcati, Alain; Sayd, Thierry; Sante-Lhoutellier, Véronique; Djelveh, Gholamreza; Michaud, Philippe

    2014-04-01

    This paper deals with the extraction and emulsifying properties of proteins from Chlorella vulgaris. Solubilisation of proteins has been achieved using high pressure cell disrupter under pH=7 or pH=12. The higher solubilisation yield (52±3%w/w) was obtained using a combination of alkaline conditions and mechanical treatments (2.7kbar). After solubilisation, proteins were recovered by two procedures: precipitation in acid media and concentration/fractionation by tangential ultrafiltration. Proteins were analysed for their molecular weights, isoelectric points and amino acids compositions and their emulsifying properties were quantified and compared to those of commercial ingredients. In spite of lower yield, better emulsifying capacity was obtained when protein solubilisation takes place at pH=7 and when using proteins from permeate of tangential ultrafiltration. In all cases, emulsifying capacity (1780±20 and 3090±50mLoil/g protein) and stability (72±1% and 79±1%) of microalgae proteins remained comparable or higher than the commercial ingredients such as sodium caseinate.

  17. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

    PubMed Central

    Kohansal-Nodehi, Mahdokht; Chua, John JE; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. DOI: http://dx.doi.org/10.7554/eLife.14530.001 PMID:27115346

  18. 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

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

    PubMed

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

    2015-01-01

    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.

  20. 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

  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.

  2. Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

    PubMed

    Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

    2016-09-01

    A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1]. PMID:27583342

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

    PubMed Central

    2014-01-01

    Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the

  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. Novel type of phorbol ester-dependent protein phosphorylation in the particulate fraction of mouse epidermis

    SciTech Connect

    Gschwendt, M.; Kittstein, W.; Marks, F.

    1986-06-13

    In a Triton X100-extract from the particulate fraction of mouse epidermis but also of other murine tissues, the phosphorylation of a protein with the relative molecular mass of 82,000 (p82) is found to be dependent on phosphatidyl serine and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Unlike protein kinase C-catalyzed phosphorylation, p82 phosphorylation is neither observed in the presence of high concentrations of Ca/sup 2 +/ and phosphatidyl serine alone nor after addition of exogenous protein kinase C. Dioctanoylglycerol and the incomplete promoter 12-O-retinoylphorbol-13-acetate are also capable of stimulating p82 phosphorylation, whereas the non-promoting phorbol ester 4-O-methyl-TPA is at least 100-fold less active in this respect.

  6. 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.

  7. 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

  8. 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.

  9. 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. PMID:21129427

  10. Chemical characteristics and fractionation of proteins from Moringa oleifera Lam. leaves.

    PubMed

    Teixeira, Estelamar Maria Borges; Carvalho, Maria Regina Barbieri; Neves, Valdir Augusto; Silva, Maraíza Apareci; Arantes-Pereira, Lucas

    2014-03-15

    Moringa oleifera Lam. is a leguminous plant, originally from Asia, which is cultivated in Brazil because of its low production cost. Although some people have used this plant as food, there is little information about its chemical and nutritional characteristics. The objective of this study was to characterise the leaves of M. oleifera in terms of their chemical composition, protein fractions obtained by solubility in different systems and also to assess their nutritional quality and presence of bioactive substances. The whole leaf flour contained 28.7% crude protein, 7.1% fat, 10.9% ashes, 44.4% carbohydrate and 3.0mg 100g(-1) calcium and 103.1mg 100g(-1) iron. The protein profile revealed levels of 3.1% albumin, 0.3% globulins, 2.2% prolamin, 3.5% glutelin and 70.1% insoluble proteins. The hydrolysis of the protein from leaf flour employing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (ME) resulted in 39.5% and 29.5%, respectively. The total protein showed low in vitro digestibility (31.8%). The antinutritional substances tested were tannins (20.7 mg g(-1)), trypsin inhibitor (1.45TIU mg g(-1)), nitrate (17 mg g(-1)) and oxalic acid (10.5 mg g(-1)), besides the absence of cyanogenic compounds. β-Carotene and lutein stood out as major carotenoids, with concentrations of 161.0 and 47.0 μg g(-1) leaf, respectively. Although M. oleifera leaves contain considerable amount of crude protein, this is mostly insoluble and has low in vitro digestibility, even after heat treatment and chemical attack. In vivo studies are needed to better assess the use of this leaf as a protein source in human feed.

  11. 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.

  12. 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.

  13. Metabolic and cellular profile of wether goats: protein fractions and lactate dehydrogenase isoenzymes- reference values.

    PubMed

    Blackwell, J G; Libby, D W

    1982-06-01

    Blood serum concentrations of electrolytes; mineral, serum protein, and biochemical components; serum lactate dehydrogenase (LDH) activity; and blood cellular components were determined in 8 wether goats over a 59-day feeding period. The blood was collected on 6 sampling days, the blood cellular components were analyzed on collection days. The biochemical components were analyzed from frozen samples approximately 3 weeks after collection. The serum protein fractions and LDH isoenzymes were separated electrophoretically from frozen samples. The purpose was to determine the variability and changes in these values, using sample-day intervals over the feeding period. The blood serum concentrations of many of the biochemical and blood cellular components that were measured or calculated varied considerably, showing fluctuations over some portion of the period and ending with an erratic peak at period's end. The WBC count, neutrophil, and band neutrophil values were sharply decreased on sampling days 24 and 35, followed by fluctuations over the last half of the period.

  14. 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. PMID:27132821

  15. Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

    PubMed

    Surmacz, L; Wiejak, J; Wyroba, E

    2001-01-01

    SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

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

    PubMed

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

    2014-09-01

    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.

  17. 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.

  18. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC. PMID:23122071

  19. Nanospray FAIMS fractionation provides significant increases in proteome coverage of unfractionated complex protein digests.

    PubMed

    Swearingen, Kristian E; Hoopmann, Michael R; Johnson, Richard S; Saleem, Ramsey A; Aitchison, John D; Moritz, Robert L

    2012-04-01

    High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments. PMID:22186714

  20. 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

  1. 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

  2. Mass-spectrometric identification of binding proteins of Mr 25,000 protein, a part of vitellogenin B1, detected in particulate fraction of Xenopus laevis oocytes.

    PubMed

    Sugimoto, Isamu; Li, Zhijun; Yoshitome, Satoshi; Ito, Susumu; Hashimoto, Eikichi

    2004-10-01

    A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.

  3. 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.

  4. 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.

  5. Safety and efficacy of a new extensively hydrolyzed formula for infants with cow's milk protein allergy.

    PubMed

    Niggemann, B; von Berg, A; Bollrath, C; Berdel, D; Schauer, U; Rieger, C; Haschke-Becher, E; Wahn, U

    2008-06-01

    Cow's milk protein allergy (CMPA) is best treated by complete elimination of cow's milk from the diet. For infants with CMPA who cannot be breast-fed, formulas based on extensively hydrolyzed proteins or on amino acids are the preferred substitutes for cow's milk-based formulas. In this study, we compared the tolerance and growth of infants with CMPA who were fed a new extensively hydrolyzed formula containing lactose (eHF) with those who were fed an amino acid formula (AAF). This was a prospective, multi-center, randomized, reference-controlled study. Seventy-seven infants <12 months old with suspected CMPA were enrolled. In 66 of these, CMPA was confirmed by oral challenge in a double-blind, placebo-controlled food challenge (DBPCFC) or by a medical history of severe allergic reaction to cow's milk and a positive skin prick test. These infants were then tested for their reaction to eHF and AAF in a DBPCFC. All infants tolerated both formulas and were randomized to receive either eHF (n = 34) or AAF (n = 32) for 180 days. Growth (weight, length, and head circumference) and tolerance [skin, gastro-intestinal, and respiratory tract symptoms of allergy] were evaluated after 30, 60, 90, and 180 days. There were no significant differences between the two groups in any of the growth measurements. Length and head circumference were similar to Euro-growth standards, but weight was slightly lower. Gastro-intestinal and respiratory tract symptoms of allergy were also similar in the two groups. However, whereas SCORAD scores for atopic dermatitis remained constant throughout the study in infants-fed eHF, there was a slight decrease in those fed AAF. Infants-fed eHF had significantly fewer incidents of vomiting than infants-fed AAF and a significantly higher frequency of soft stools. The new eHF is safe and well tolerated in infants diagnosed with CMPA. PMID:18167160

  6. 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}.

  7. Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors.

    PubMed

    Li, Geoffrey C; Srivastava, Atul K; Kim, Jonggul; Taylor, Susan S; Veglia, Gianluigi

    2015-07-01

    Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme's conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI5-24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme's fold. PMID:26030372

  8. 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

  9. 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. PMID:27664660

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. 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

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

    PubMed

    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, (99m)Tc-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

  13. 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.

  14. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  15. 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

  16. Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa.

    PubMed

    Fang, Hejian; Zhang, Wenming; Niu, Xin; Liu, Zhonghua; Lu, Changmei; Wei, Hua; Yuan, Sheng

    2014-01-01

    Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.

  17. Stipe wall extension of Flammulina velutipes could be induced by an expansin-like protein from Helix aspersa.

    PubMed

    Fang, Hejian; Zhang, Wenming; Niu, Xin; Liu, Zhonghua; Lu, Changmei; Wei, Hua; Yuan, Sheng

    2014-01-01

    Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains. PMID:24433673

  18. 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

  19. 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.

  20. 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. PMID:24433585

  1. [THE SCREENING OF DIAGNOSTIC POTENTIAL OF NATIVE PROTEIN FRACTIONS OF MYCOBACTERIUM TUBERCULOSIS USING TECHNIQUE OF IMMUNE BLOTTING].

    PubMed

    Tsibulkin, A P; Khaertinova, I M; Urazov, N G; Khaertinov, K S

    2016-02-01

    The technique of immune blotting was used to analyze surface proteins obtained from cells M Tuberculosis exposed to partialmode of delipidization. At that, there were applied serums of patients with tuberculosis of lungs; HIV agents and patients with concomitant infection tuberculosis-AIDS and also HIV-negative patients without clinical signs of disease of lungs and with chronic diseases of lungs of other etiology The fractions oflow-molecular antigens with molecular mass 6.5-30 kilodaltons became diagnostically significant. To this fraction of antigens reacted serums of all patients with tuberculosis of lungs and serums of 91% of patients with concomitant tuberculosis-AIDS infection. The antigens of protein fractions with high (70-100 kilodaltons) and interim (30-69 kilodaltons) molecular mass became diagnostically insignificant. PMID:27455562

  2. 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.

  3. 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

  4. Expression of heat shock protein 70 in the liver of extensively and intensively kept heavy pigs.

    PubMed

    Negrato, E; Di Martino, G; Vascellari, M; Radaelli, G; Capello, K; Pascoli, F; Bertotto, D; Bonfanti, L

    2013-08-01

    The objective of this work was to investigate the expression of heat shock protein 70 (HSP70) by Western blot (WB) in swine liver. Subsequently, the study aimed to apply this method to two experimental groups of heavy pigs raised in different confinement systems: intensive/indoor (Group A) and extensive/outdoor (Group B). Thirty-six crossbred commercial heavy pigs were divided as follows: Group A (eight castrated males and eight females) was equally distributed into two single-sex indoor pens (1.02 m²/pig); Group B (11 castrated males and nine females) was kept in one single (partially grassy and partially wooded) open area of about 6000 m². Group A was slaughtered at 41 weeks of age (170 ± 9 kg) and Group B at 48 weeks of age (172 ± 13 kg). At the abattoir the livers of all the animals were collected and analyzed by WB assay in order to quantify the levels of HSP70. Moreover, a further liver sample was taken from the same animals in order to investigate the cellular localization of HSP70 by immunohistochemistry (IHC). The interaction between sex and group resulted statistically significant (P = 0.001). When stratified by sex, Group A showed significantly higher HSP70 values compared with Group B for both male and female subjects (P < 0.001). Stratifying by group, males showed significantly higher HSP70 values than females in Group A (P < 0.001), whereas no statistical differences were observed between sexes for Group B (P = 0.653). The IHC results evidenced cytoplasmic immunoreactivity in a granular pattern in both groups. The different expression pattern observed by WB could prove to be a useful tool in the assessment of pig health and welfare.

  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. Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats.

    PubMed

    Muthenna, Puppala; Raghu, Ganugula; Akileshwari, Chandrasekhar; Sinha, Sukesh Narayana; Suryanarayana, Palla; Reddy, Geereddy Bhanuprakash

    2013-11-01

    Accumulation of advanced glycation endproducts (AGE) from nonenzymatic glycation of proteins has been implicated in several diabetic complications including diabetic cataract. Previously, we have reported that extracts of dietary agents such as cinnamon have the potential to inhibit AGE formation. In this study, we have shown procyanidin-B2 as the active component of cinnamon that is involved in AGE inhibition using bioassay-guided fractionation of eye lens proteins under in vitro conditions. The data indicate that procyanidin-B2 enriched fraction scavenges dicarbonyls. Further, procyanidin-B2 fraction of cinnamon inhibited the formation of glycosylated hemoglobin in human blood under ex vivo conditions. We have also demonstrated the physiological significance of procyanidin-B2 fraction in terms of delay of diabetic cataract through inhibition of AGE in diabetic rats. These findings establish the antiglycating potential of procyanidin-B2 fraction of cinnamon which suggests a scope for controlling AGE-mediated diabetic complications by food sources that are rich in proanthocyanidins like procyanidin-B2.

  7. 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.

  8. Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

    PubMed Central

    Geisler, N; Kaufmann, E; Fischer, S; Plessmann, U; Weber, K

    1983-01-01

    Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha-helical coiled-coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino-terminal region built analogously to non-neuronal intermediate filament proteins should allow a co-polymerization process via the interaction of coiled-coil domains. The extra mass of all triplet proteins is allocated to carboxy-terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence. Images Fig. 1. PMID:10872323

  9. Impact of asymmetrical flow field-flow fractionation on protein aggregates stability.

    PubMed

    Bria, Carmen R M; Williams, S Kim Ratanathanawongs

    2016-09-23

    The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (<∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes. PMID

  10. The 45-kilodalton protein of cytomegalovirus (Colburn) B-capsids is an amino-terminal extension form of the assembly protein.

    PubMed Central

    Schenk, P; Woods, A S; Gibson, W

    1991-01-01

    Intranuclear B-capsids from cytomegalovirus (strain Colburn)-infected cells contain an abundant 37-kDa assembly protein, thought to be involved in capsid formation, and three minor protein constituents (i.e., 45, 39, and 38 kDa) that are immunologically and structurally related to the assembly protein. In the experiments reported here, antisera produced against synthetic peptides were used in conjunction with chemical protein cleavage to examine the structural relationship of these proteins in more detail. Results of these experiments verify that the carboxyl end of the 39-kDa assembly protein precursor is lost during maturation and suggest that the 38-kDa protein may be a processing intermediate. It is shown that the 45-kDa protein is coterminal with the mature assembly protein at its carboxyl end but differs by a predicted 115-amino-acid extension at its amino terminus. In addition, evidence is presented that the 45-kDa protein has a 48-kDa precursor and a 47-kDa putative processing intermediate which have the same carboxy-terminal sequences and undergo the same maturational events as those of the assembly protein. A working model considering the structural relationship of these proteins is presented. Images PMID:1847469

  11. 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

  12. 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

  13. 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

  14. Quality Matters: Extension of Clusters of Residues with Good Hydrophobic Contacts Stabilize (Hyper)Thermophilic Proteins

    PubMed Central

    2015-01-01

    Identifying determinant(s) of protein thermostability is key for rational and data-driven protein engineering. By analyzing more than 130 pairs of mesophilic/(hyper)thermophilic proteins, we identified the quality (residue-wise energy) of hydrophobic interactions as a key factor for protein thermostability. This distinguishes our study from previous ones that investigated predominantly structural determinants. Considering this key factor, we successfully discriminated between pairs of mesophilic/(hyper)thermophilic proteins (discrimination accuracy: ∼80%) and searched for structural weak spots in E. coli dihydrofolate reductase (classification accuracy: 70%). PMID:24437522

  15. Quality matters: extension of clusters of residues with good hydrophobic contacts stabilize (hyper)thermophilic proteins.

    PubMed

    Rathi, Prakash Chandra; Höffken, Hans Wolfgang; Gohlke, Holger

    2014-02-24

    Identifying determinant(s) of protein thermostability is key for rational and data-driven protein engineering. By analyzing more than 130 pairs of mesophilic/(hyper)thermophilic proteins, we identified the quality (residue-wise energy) of hydrophobic interactions as a key factor for protein thermostability. This distinguishes our study from previous ones that investigated predominantly structural determinants. Considering this key factor, we successfully discriminated between pairs of mesophilic/(hyper)thermophilic proteins (discrimination accuracy: ∼80%) and searched for structural weak spots in E. coli dihydrofolate reductase (classification accuracy: 70%).

  16. 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.

  17. 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. PMID:25313039

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

    PubMed

    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

  19. 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

  20. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.

    PubMed

    Fujimoto, Yasuyuki; Itabe, Hiroyuki; Sakai, Jun; Makita, Minoru; Noda, Junich; Mori, Masahiro; Higashi, Yusuke; Kojima, Shinichi; Takano, Tatsuya

    2004-02-01

    Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction. PMID:14741744

  1. 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.

  2. 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. PMID:26970581

  3. 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.

  4. 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. PMID:25832806

  5. Extensive exploration of conformational space improves Rosetta results for short protein domains.

    PubMed

    Li, Yaohang; Bordner, Andrew J; Tian, Yuan; Tao, Xiuping; Gorin, Andrey A

    2008-01-01

    With some simplifications, computational protein folding can be understood as an optimization problem of a potential energy function on a variable space consisting of all conformation for a given protein molecule. It is well known that realistic energy potentials are very "rough" functions, when expressed in the standard variables, and the folding trajectories can be easily trapped in multiple local minima. We have integrated our variation of Parallel Tempering optimization into the protein folding program Rosetta in order to improve its capability to overcome energy barriers and estimate how such improvement will influence the quality of the folded protein domains. Here we report that (1) Parallel Tempering Rosetta (PTR) is significantly better in the exploration of protein structures than previous implementations of the program; (2) systematic improvements are observed across a large benchmark set in the parameters that are normally followed to estimate robustness of the folding; (3) these improvements are most dramatic in the subset of the shortest domains, where high-quality structures have been obtained for >75% of all tested sequences. Further analysis of the results will improve our understanding of protein conformational space and lead to new improvements in the protein folding methodology, while the current PTR implementation should be very efficient for short (up to approximately 80 a.a.) protein domains and therefore may find practical application in system biology studies.

  6. Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension.

    PubMed

    MacDonald, James T; Kabasakal, Burak V; Godding, David; Kraatz, Sebastian; Henderson, Louie; Barber, James; Freemont, Paul S; Murray, James W

    2016-09-13

    The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops.

  7. Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension.

    PubMed

    MacDonald, James T; Kabasakal, Burak V; Godding, David; Kraatz, Sebastian; Henderson, Louie; Barber, James; Freemont, Paul S; Murray, James W

    2016-09-13

    The ability to design and construct structures with atomic level precision is one of the key goals of nanotechnology. Proteins offer an attractive target for atomic design because they can be synthesized chemically or biologically and can self-assemble. However, the generalized protein folding and design problem is unsolved. One approach to simplifying the problem is to use a repetitive protein as a scaffold. Repeat proteins are intrinsically modular, and their folding and structures are better understood than large globular domains. Here, we have developed a class of synthetic repeat proteins based on the pentapeptide repeat family of beta-solenoid proteins. We have constructed length variants of the basic scaffold and computationally designed de novo loops projecting from the scaffold core. The experimentally solved 3.56-Å resolution crystal structure of one designed loop matches closely the designed hairpin structure, showing the computational design of a backbone extension onto a synthetic protein core without the use of backbone fragments from known structures. Two other loop designs were not clearly resolved in the crystal structures, and one loop appeared to be in an incorrect conformation. We have also shown that the repeat unit can accommodate whole-domain insertions by inserting a domain into one of the designed loops. PMID:27573845

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

    DOE PAGESBeta

    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 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

  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. An Extensive Study of Protein Phase Diagram Modification: Increasing Macromolecular Crystallizability by Temperature Screening

    SciTech Connect

    Lin, Yi-Bin; Zhu, Dao-Wei; Wang, Tao; Song, Jian; Zou, Yong-Shui; Zhang, Yong-Lian; Lin, Sheng-Xiang

    2009-02-23

    A new parameter 'relative crystallizability' for protein crystallization has been proposed, and its relationship with protein solubility and crystallization success has been studied (Zhu et al. J. Struct. Biol. 2006, 154, 297). Here we further construct the phase diagrams of a larger number of proteins, study the phase modification as a function of temperature, and establish the relationship between the nucleation zone area (S{sub N}) and crystallization success. The phase diagrams of 10 proteins were constructed and their S{sub N} were compared, demonstrating that temperature modifies the protein nucleation zone. Such modification can significantly enlarge the S{sub N} and increase protein crystallizability. For example, the S{sub N} of ribonuclease S and trypsin increases by 2.4- and 1.6-fold when the temperature moves to 277 K from 295 K, while at the same time the crystallization hits increase from 20.8% to 42.9% and 12.5% to 25%, respectively. S{sub N} of chymotrypsinogen A and concanavalin A increases by 1.6- and 1.7-fold (277 to 295 K), while the hits increase from 37.5% to 54.2% and 43.3% to 73.4%, respectively. Such an excellent agreement strongly supports the validity of protein 'relative crystallizability', and crystallization screening at several temperatures can significantly increase the success for most proteins. A new protein epididymal-specific lipocalin was crystallized by varying temperature, yielding quickly the first crystals, and complete data sets have been collected at 1.97 {angstrom}.

  11. Determination of the unmetabolized 18F-FDG fraction by using an extension of simplified kinetic analysis method: clinical evaluation in paragangliomas

    PubMed Central

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

    2015-01-01

    Summary Tumours with high 18F-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 18F-FDG components in the tissues. We propose a method that enables to calculate different 18F-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 18F-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 (KH). The exact net influx rate constant and vascular volume fraction (Ki and V respectively) were subsequently obtained by the method of least squares. Next, we calculated the mean percentages of metabolised (PM) and unmetabolised (PUM) 18F-FDG components, and the times required to reach 80% of the amount of metabolised 18F-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 standardized uptake value SUVmax (9.7 ± 11, coefficient of variation CV=114%), Ki (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 k3 (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 Ki for in vivo metabolic assessment of PGLs at the molecular level. PMID:26044552

  12. 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. PMID:26044552

  13. 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.

  14. 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

  15. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed Central

    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. Images Fig. 2. Fig. 3. Fig. 4. PMID:7082279

  16. Protein fraction isolated from epididymal fluid re-associates sperm in vitro: possible role of serpins in rat rosettes assembly.

    PubMed

    Monclus, María A; Andreina, Cesari; Cabrillana, María E; Lancellotti, Tania E Saez; Rensetti, Daniel E; Clementi, Marisa A; Boarelli, Paola V; Vincenti, Amanda E; Fornés, Miguel W

    2010-05-01

    In many mammalian species, sperm associate as a consequence of the epididymal transit. From the classic Rouleaux in guinea pig to the most recent work in mouse and echidna, authors have focused mainly on a detailed morphological description of this phenomenon. Some of these articles have also begun to describe the nature of the material present between sperm heads. Here, we try to better understand the factor/s involved in rat sperm association (Rosette). Based on previous work describing the appearance of Rosettes in the distal segments of the rat epididymis, we consider that sperm during their transit must be in contact with factor/s present in the caudal lumen in order to associate with each other. By an in vitro sperm re-associating assay, we try to determine the in vivo phenomenon observed in the lumen. The assay consists of co-incubating non-associated sperm with several protein fractions obtained from epididymal caudal fluid. After establishing the most active fraction, the proteins were characterized by MALDI-TOF mass spectrometry. Among the proteins we found two members of the serine protease inhibitors family; an alpha-1 antitrypsin and a new protein with an alpha-1 antitrypsin like domain which includes a sequence compatible with the serpins' reactive center loop. These serpins may play a role in the assembly/disassembly process of Rosettes by modulating lumenal protease activity. Finally, a biochemical-morphological model which explains the sperm-proteases interaction was proposed. PMID:20143401

  17. A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein.

    PubMed Central

    Roesler, K. R.; Rao, A. G.

    2000-01-01

    The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity. PMID:11045611

  18. Kinetics, aggregation behavior and optimization of the fractionation of whey protein isolate with hydrochloric acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. 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.

  20. A novel protein fraction from Sesbania grandiflora shows potential anticancer and chemopreventive efficacy, in vitro and in vivo.

    PubMed

    Laladhas, Krishna P; Cheriyan, Vino T; Puliappadamba, Vineshkumar T; Bava, Smitha V; Unnithan, Rajesh G; Vijayammal, Parvathy L; Anto, Ruby John

    2010-03-01

    We report mechanism-based evidence for the anticancer efficacy of a protein fraction, SF2 (Sesbania fraction 2) isolated from the flower of the medicinal plant, Sesbania grandiflora (S. grandiflora). The fraction was evaluated in two murine ascites tumour cell lines and human cancer cell lines of different origin for its anticancer effect. SF2 inhibited cell proliferation and induced apoptosis as demonstrated by DNA fragmentation and externalization of phosphatidyl serine in Daltons lymphoma ascites (DLA) and colon cancer cells (SW-480). Sensitivity to SF2 in these cells was associated with activation of caspases 3, 8 and 9, poly (ADP-ribose) polymerase cleavage and cytochrome C release which attests apoptosis induced cell death. Mechanistically, SF2 down-regulated phorbol myristate acetate (PMA) induced NF-kappaB, a transcription factor which controls the expression of genes encoding proteins involved in cell regulation and growth control. Additionally, SF2 also down-regulated anti-apoptotic factors such as Bcl-2, p-Akt and cyclooxygenase-2 induced by the tumour promoter PMA suggestive of a possible explanation for its anticancer effect. In vivo studies using ascites and solid tumour models strongly support in vitro findings as SF2 administration increased the life span and decreased the tumour volume in mice bearing tumour. In vivo toxicological evaluation revealed the pharmacological safety of SF2 and may serve as a potential anticancer drug candidate. PMID:19183244

  1. Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays.

    PubMed

    Salehi-Reyhani, Ali; Gesellchen, Frank; Mampallil, Dileep; Wilson, Rab; Reboud, Julien; Ces, Oscar; Willison, Keith R; Cooper, Jonathan M; Klug, David R

    2015-02-17

    We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.

  2. Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

    PubMed

    Ballot, E; Desbos, A; Auger, C; Monier, J C

    1996-09-01

    Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein. PMID:8811045

  3. 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

  4. Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development

    PubMed Central

    Michaelson, Jennifer S.; Bader, Debra; Kuo, Frank; Kozak, Christine; Leder, Philip

    1999-01-01

    The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx’s physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo. PMID:10444590

  5. Tumor necrosis factor-α suppresses the protein fractional synthesis rate of the small intestine stimulated by glutamine in rats

    PubMed Central

    ZHOU, JIHONG; FAN, SHENGXIAN; CAO, YACHENG; ZHU, MINGFANG; HAN, YONG; CAO, XUEYING; LI, YOUSHENG

    2015-01-01

    The objective of this study was to examine whether and how TNF-α affects glutamine-enhanced protein synthesis and the expression of the amino acid transporter ASCT2 in the small intestine at the mRNA and protein levels. A total of 30 male Sprague-Dawley rats were randomly assigned into three groups, namely the total parenteral nutrition (TPN; control), glutamine-treated (Gln), and glutamine- and tumor necrosis factor-α (TNF-α)-treated (TNF-α) groups. At 30 min prior to examination, all rats were mainlined with [L-15N]leucine. The concentration of TNF-α in plasma and of glutamine in plasma and the small intestine was measured. The fractional synthesis rate (FSR) of protein and the mRNA and protein expression levels of ASCT2 in the small intestine were assessed. The level of TNF-α was highest in the TNF-α group and the glutamine concentration was elevated to a greater extent in the TNF-α group than in the other two groups. However, the FSR of protein in the small intestine was significantly higher in the Gln group compared with that in the TNF-α group. The mRNA and protein expression levels of ASCT2 in the experimental groups were significantly higher that those in the control group, but did not differ significantly between the Gln and TNF-α groups. These results indicate that TNF-α may attenuate glutamine-stimulated protein synthesis in the small intestine in the early stage of sepsis in rats. The mechanism may be that TNF-α inhibits the function of the glutamine transporter in the uptake the glutamine into target cells for protein synthesis. This inhibition may occur at or following protein translation. PMID:25574232

  6. 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

  7. Six subgroups and extensive recent duplications characterize the evolution of the eukaryotic tubulin protein family.

    PubMed

    Findeisen, Peggy; Mühlhausen, Stefanie; Dempewolf, Silke; Hertzog, Jonny; Zietlow, Alexander; Carlomagno, Teresa; Kollmar, Martin

    2014-09-01

    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.

  8. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology.

    PubMed Central

    Gill, D S; Banerjee, A K

    1985-01-01

    The complete nucleotide sequence of the NS mRNA of vesicular stomatitis virus (New Jersey serotype) was established from two cDNA clones spanning the entire coding region of the mRNA. The gene is 856 nucleotides long and can code for a polypeptide of 274 amino acids. Comparison with the nucleotide sequence of the NS gene of the Indiana serotype revealed only 41% sequence homology. The deduced amino acid sequences of the NS proteins were only 32% homologous, with no identical stretches of more than five amino acids. However, at the C-terminal domain there was a conserved region of 21 amino acids with greater than 90% homology. Surprisingly, relative hydropathicity plots also demonstrated the presence of a large number of hydrophilic amino acids sequestered similarly over the N-terminal half of the protein. In addition, the total number of serine and threonine residues, presumptive phosphorylation sites, was similar and included seven serine and three threonine residues located at identical positions. It appears that during divergent evolution of these two vesicular stomatitis virus serotypes from a common ancestor, considerable mutation occurred in the main body of the gene but the overall structure of the protein was retained. The function of the NS protein in relation to the evolution of the two viruses is discussed. Images PMID:2989560

  10. 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.

  11. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

    PubMed

    Staes, An; Van Damme, Petra; Helsens, Kenny; Demol, Hans; Vandekerckhove, Joël; Gevaert, Kris

    2008-04-01

    We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

  12. Preferential binding of radiolabeled zearalenone to a protein fraction of Fusarium roseum graminearum.

    PubMed Central

    Inaba, T; Mirocha, C J

    1979-01-01

    Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)beta-resorcyclic acid lactone] is a hormone produced by Fusarium spp. which regulates the sexual stage in F. roseum. 3H- and 14C-labeled zearalenone were found to bind preferentially to one of two peaks containing uncharacterized proteins obtained from the cytosol of young mycelium and resolved by gel column chromatography. The proteins were partially purified by successive resolution on Sephadex G-100, Sephadex G-200, and BioGel P-300. Free zearalenone (37%) was reisolated from the purified proteins after resolution by thin-layer chromatography or partitioning with ethyl acetate. PMID:760640

  13. 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.

  14. C-terminal extension of calmodulin-like 3 protein from Oryza sativa L.: interaction with a high mobility group target protein.

    PubMed

    Chinpongpanich, Aumnart; Phean-O-Pas, Srivilai; Thongchuang, Mayura; Qu, Li-Jia; Buaboocha, Teerapong

    2015-11-01

    A large number of calmodulin-like (CML) proteins are present in plants, but there is little detailed information on the functions of these proteins in rice (Oryza sativa L.). Here, the CML3 protein from rice (OsCML3) and its truncated form lacking the C-terminal extension (OsCML3m) were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, OsCML3 was mainly localized in the plasma membrane, whereas OsCML3m was found in the nucleus. The rice high mobility group B1 (OsHMGB1) protein was identified as one of the putative OsCML3 target proteins. Bimolecular fluorescence complementation analysis revealed that OsHMGB1 bound OsCML3, OsCML3m or OsCML3s (cysteine to serine mutation at the prenylation site) in the nucleus presumably through the methionine and phenylalanine-rich hydrophobic patches, confirming that OsHMGB1 is a target protein in planta. The effect of OsCML3 or OsCML3m on the DNA-binding ability of OsHMGB1 was measured using an electrophoretic mobility shift assay. OsCML3m decreased the level of OsHMGB1 binding to pUC19 double-stranded DNA whereas OsCML3 did not. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides an intracellular Ca2+ regulatory switch. PMID:26423116

  15. C-terminal extension of calmodulin-like 3 protein from Oryza sativa L.: interaction with a high mobility group target protein.

    PubMed

    Chinpongpanich, Aumnart; Phean-O-Pas, Srivilai; Thongchuang, Mayura; Qu, Li-Jia; Buaboocha, Teerapong

    2015-11-01

    A large number of calmodulin-like (CML) proteins are present in plants, but there is little detailed information on the functions of these proteins in rice (Oryza sativa L.). Here, the CML3 protein from rice (OsCML3) and its truncated form lacking the C-terminal extension (OsCML3m) were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, OsCML3 was mainly localized in the plasma membrane, whereas OsCML3m was found in the nucleus. The rice high mobility group B1 (OsHMGB1) protein was identified as one of the putative OsCML3 target proteins. Bimolecular fluorescence complementation analysis revealed that OsHMGB1 bound OsCML3, OsCML3m or OsCML3s (cysteine to serine mutation at the prenylation site) in the nucleus presumably through the methionine and phenylalanine-rich hydrophobic patches, confirming that OsHMGB1 is a target protein in planta. The effect of OsCML3 or OsCML3m on the DNA-binding ability of OsHMGB1 was measured using an electrophoretic mobility shift assay. OsCML3m decreased the level of OsHMGB1 binding to pUC19 double-stranded DNA whereas OsCML3 did not. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides an intracellular Ca2+ regulatory switch.

  16. 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. PMID:27448590

  17. 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.

  18. 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. PMID:27265169

  19. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.

    PubMed

    Altrock, Wilko D; tom Dieck, Susanne; Sokolov, Maxim; Meyer, Alexander C; Sigler, Albrecht; Brakebusch, Cord; Fässler, Reinhard; Richter, Karin; Boeckers, Tobias M; Potschka, Heidrun; Brandt, Claudia; Löscher, Wolfgang; Grimberg, Dörte; Dresbach, Thomas; Hempelmann, Anne; Hassan, Hadir; Balschun, Detlef; Frey, Julietta U; Brandstätter, Johann H; Garner, Craig C; Rosenmund, Christian; Gundelfinger, Eckart D

    2003-03-01

    Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

  2. The amino acid sequence of protein SCMK-B2C from the high-sulphur fraction of wool keratin.

    PubMed

    Elleman, T C

    1972-08-01

    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.

  3. [Protein fractions and their enzyme activity in rat myocardium after a 22-hour space flight].

    PubMed

    Gaevskaia, M S; Veresotskaia, N A; Kolganova, N S; Kolchina, E V; Kurkina, L M

    1976-01-01

    No significant changes in the content of sarcoplasmatic and myofibrillar proteins were found in the myocardium of rats that made a 22-day flight onboard the biosatellite. On the 2nd and 26th postflight days the activity of aspartate and alanine aminotransferases of sarcoplasmatic proteins was increased and the total activity of lactate dehydrogenase and its isoenzyme spectrum remained unchanged. On the 2nd postflight day the ATPase activity of myosin of the myocardium was substantially lowered and on the 26th postflight day it returned to the normal. This decline in the ATPase activity of myosin can be regarded as an adaptive reaction to weightlessness.

  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 Central

    Vandenplas, Yvan; De Greef, Elisabeth; Hauser, Bruno

    2014-01-01

    Background 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. Methods 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. Results 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. Conclusions 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. Trial registration number ClinicalTrials.gov NCT01998074. PMID:24914098

  5. 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

  6. Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila.

    PubMed

    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.

  7. Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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. PMID:25151510

  9. 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. PMID:22267262

  10. 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

  11. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

  12. Serological serum protein fraction and responses to Brucella melitensis in lambs fed aflatoxins.

    PubMed

    Fernandez, A; Hernandez, M; Sanz, M C; Verde, M T; Ramos, J J

    1997-06-01

    Twenty-four lambs were given 2 ppm aflatoxins (AF) in their diet for 37 d and 12 were kept as a control group. After this time, toxic feed was removed for a further 35 days (clearance period). On day 17 all lambs were vaccinated with B melitensis strain Rev-1, and blood samples were taken regularly to determine the levels of antibodies and serum proteins. Aflatoxins decreased titers of Rose Bengal test and optical densities of ELISA, both in the intoxicated and clearance periods. Complement fixation titers were lower in intoxicated lambs, except on the 23rd day of intoxication, but not statistically different (P > 0.05). No effect of AF was noted on total serum proteins, but albumin and alfa-globulin levels were lower for intoxicated lambs than for the control group. Beta-globulin concentration did not change, and increases in gamma-globulins levels in dosed lambs were observed throughout the experiment. These results suggest that AF causes a failure in the acquired immunity system of lambs by decreasing antibody production and altering serum profile proteins.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

    2016-01-01

    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

  19. 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.

  20. 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.

  1. On the mechanism of the cold ethanol precipitation method of plasma protein fractionation.

    PubMed

    van Oss, C J

    1989-10-01

    Given the negligible difference in the value of the dielectric constant of water at 20 degrees C and that of ethanol solutions at low temperatures, the often advanced explanation for the precipitation of plasma proteins by the cold ethanol process, as being due to a reduction of the dielectric constant and the resulting increase in interprotein charge interactions, is not tenable. It is shown by a surface-thermodynamic approach that, upon dehydration by ethanol, isoelectric serum albumin molecules as well as isoelectric serum gamma globulin molecules will attract each other to a sufficient degree by van der Waals forces to become insoluble in the ethanol-water mixtures used.

  2. Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion.

    PubMed

    Vinokurov, K S; Elpidina, E N; Oppert, B; Prabhakar, S; Zhuzhikov, D P; Dunaevsky, Y E; Belozersky, M A

    2006-10-01

    Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.

  3. Improvement of Surface Functionalities, Including Allergenicity Attenuation, of Whole Buckwheat Protein Fraction by Maillard-Type Glycation with Dextran

    PubMed Central

    Tazawa, Shigeru; Katayama, Shigeru; Hirabayashi, Masahiro; Yamaguchi, Daiki; Nakamura, Soichiro

    2014-01-01

    The purpose of the current study was to determine the effects of the introduction of polysaccharide chains onto the molecular surface of buckwheat proteins on buckwheat protein surface functionality. The whole buckwheat protein fraction (WBP) was prepared using 50 mM phosphate buffer (pH 7.5) containing 0.5 M NaCl and covalently linked with 6 kDa, 17.5 kDa, 40 kDa, 70 kDa, or 200 kDa dextran by Maillard-type glycation through controlled dry-heating at 60°C and 79% relative humidity for two weeks. Conjugation with 40 kDa dextran improved the water solubility and emulsifying properties of WBP without causing a serious loss of available lysine; 84.9% of the free amino groups were conserved. In addition, we found that the introduction of dextran chains onto the molecular surfaces of WBP attenuated the antigenicity of WBP. PMID:25580398

  4. 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

  5. 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

    2016-07-01

    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.

  6. 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

    Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan; Gao, Ning; Woolford, John L

    2016-09-01

    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

  7. 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

    Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan; Gao, Ning; Woolford, John L

    2016-09-01

    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.

  8. 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.

  9. 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.

  10. 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. PMID:26364114

  11. [A study of serum protein fraction binding to indocyanine green (ICG) by combined method of immunoelectrophoresis and ICG fundus videosystem].

    PubMed

    Saito, T; Komatsu, Y; Mori, S; Deguchi, T; Koyama, I; Yoneya, S

    1996-08-01

    Binding characteristics of indocyanine green (ICG) to human serum were investigated, with a combination of immunoelectrophoresis and an ICG fundus video system. Serum samples were obtained from three healthy volunteers, 1 minute after intravenous administration of 50 mg/2 ml ICG, and then fractionated immunoelectrophoretically on agarose plates. Electrophoretic patterns on these plates could be observed with an ICG fundus video system as well as an immunoviewer. Using anti-human sera, only one infrared fluorescent line showing the ICG binding immunoprecipitate was recognized near the area of beta fraction, which was also identified by the use of anti-beta-lipoprotein (Lp) antibody. We also studied the affinity of ICG for apolipoproteins (Apo) AI, B, and CIII, which were the main protein components of serum Lps. Electrophoresis showed that ICG bound only to Apo-B, but not to the others. These results indicated that ICG mainly bound to beta-Lp in the blood, and that ICG angiographic patterns were directly reflecting the dynamics of serum Lps, especially for LDL. The high affinity of ICG for only Apo-B could explain the reason why ICG mainly bound to beta-Lp among several serum Lps, because large amounts of Apo-B are included in beta-Lp but a little in other serum Lps.

  12. Amino acid composition, antinutrients and allergens in the peanut protein fraction obtained by an aqueous enzymatic process.

    PubMed

    Latif, S; Pfannstiel, J; Makkar, H P S; Becker, K

    2013-01-01

    Enzyme-assisted aqueous extraction (EAE) of peanut kernel was used to extract oil and protein. The aqueous fraction (AF) obtained by EAE had a better essential amino acid profile than the residues obtained by solvent extraction (Rs) and cold pressing (Rc). No major difference in the trypsin inhibitor activity among AF, Rs and Rc was observed; however, the trypsin inhibitor activity was drastically reduced in the residue obtained after EAE. AF was subjected to MALDI-TOF/MS, revealing it to be rich in peptides (107) with molecular masses from m/z 700 to 2369Da. AF had an extremely low phytate content and was rich in peptides, which could be used as a food supplement. ESI-MS/MS data were used for the identification of major peanut allergens, viz., Ara h1, h3, h6-8. Their allergenic potential needs to be established. PMID:23017415

  13. Deviation of negatively charged protein fractions in the trochophore and veliger larvae by the larvicidal action of baygon in freshwater pulmonate Gyraulus convexiusculus (Planorbidae).

    PubMed

    Bhide, Mangla; Gupta, Priyamvada

    2006-10-01

    In the present investigation egg capsules of Gyraulus convexiusculus were treated with different concentrations of baygon. A dose and duration dependent deviations in the number of negatively charged protein fractions in the trochophore and veliger larval stages were observed. It resulted into anomalies in the morphogenesis and organogenesis of corresponding larval stages. Most of the protein bands showed the decrease in the protein positive intensities in comparison to control. This suggested that baygon causes larval toxicity in Gyraulus convexiusculus.

  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. 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

  16. Carboxypeptidase I from triticale grains and the hydrolysis of salt-soluble fractions of storage proteins.

    PubMed

    Drzymała, Adam; Prabucka, Beata; Bielawski, Wiesław

    2012-09-01

    Carboxypeptidase I was purified from triticale grains (×Triticosecale Wittm.) by a 5-step purification procedure including gel filtration, cation-exchange chromatography and affinity chromatography. The enzyme was purified 595.9 fold with a 1.58% recovery. Triticale carboxypeptidase I is a homodimer with a molecular weight of 124.2 kDa and a subunit weight of 55.2 kDa. Each subunit is composed of two polypeptide chains (33.4 and 21.3 kDa). Serine was found in the active site of triticale carboxypeptidase I; DFP (diisopropylflourophosphate) and other applied inhibitors of serine proteases inhibited the enzyme activity. Triticale carboxypeptidase I hydrolyzes N-CBZ-dipeptide (N-carbobenzoxy-dipeptide) substrates at low pH. N-CBZ-Phe-Ala, N-CBZ-Phe-Leu and N-CBZ-Ala-Met were hydrolyzed with the highest rates. The lowest K(m) value and the highest k(cat)/K(m) ratio were observed for hydrolysis of N-CBZ-Phe-Ala. Studies on the amino acid sequence revealed that the purified enzyme is homologous to carboxypeptidase I from barley. Analyses of conserved regions in the sequence of triticale carboxypeptidase I revealed the presence of Ser, Asp and His that compose the catalytic triad. Intact storage proteins were poor substrates for carboxypeptidases. Carboxypeptidase I together with carboxypeptidase III effectively degraded albumins proteolytically modified by endopeptidase EP8. Modified globulins were degraded at a slower rate, and all three carboxypeptidases were required for a significantly increased activity. Studies of the expression of the carboxypeptidase I gene revealed that the synthesis of the enzyme occurs mainly in the scutellum of the grain. The enzyme is also expressed in the aleurone layer of the grains, although its function in this tissue is unknown.

  17. The particularities of protein fraction in the apoptosis of lymphocytes of patients with asthma.

    PubMed

    Vodounon, C A; Abramova, Z I; Aikou, N; Sezan, A; Kotchoni, S O; Akpona, S A

    2013-12-15

    The emergence of various diseases specifically severe diseases such as bronchial asthma is associated with apoptosis of lymphocytes. One of the major biochemical features of apoptosis is chromosome DNA fragmentation implemented by apoptotic nucleases. The inactivation of these apoptotic nucleases produces undigested DNA and is linked to a number of autoimmune disorders. Instead of this we have studied the enzymatic activities of the cytoplasmic and nucleic proteins of lymphocytes from healthy donors and patients with bronchial asthma. The study of enzymatic activities of the nuclease of lymphocytes was assessed by flow cytometry, spectrofluorimetry and electrophoresis method in agarose gel. In the peripheral blood cells of healthy donors undergoing apoptosis, we found a DNAse activated by Ca2+ and Mg2+ ions. Lymphocytes of patients with bronchial asthma contain DNAses, the activity of which depends on the seriousness of the desease. In patients cells, the activity of the Mn2+-dependent DNAse increases, whereas the activity of the Ca2+, Mg(2+)-dependent DNase decreases. Taking into consideration the role of the Ca2+, Mg(2+)-dependent DNAse in apoptosis, we can propose that there is a link between the reduction of the rate of apoptosis of lymphocytes in patients with bronchial asthma and the dysfunction of the induction of "apoptotical" Ca2+, Mg(2+)-dependent nuclease. According to the results obtained, we can assume that why apoptosis of lymphocytes resist in patients with bronchial asthma is a reduction in concentration of endocellular calcium and an increase of manganese ions content, which results in the triggering of activation mechanism for Mn(2+)-dependent endonuclease activity. This leads to the change of DNA fragmentation nature in lymphocytes and as a consequence, to disorders in process of apoptotic bodies' formation, thus, hindering apoptosis of lymphocytes in patients with bronchial asthma. PMID:24517002

  18. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits.

    PubMed

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-09-19

    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

  19. 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

  20. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits.

    PubMed

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-09-19

    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.

  1. 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.

  2. Subcellular Fractionation and Localization Studies Reveal a Direct Interaction of the Fragile X Mental Retardation Protein (FMRP) with Nucleolin

    PubMed Central

    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. PMID:24658146

  3. 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

  4. 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.

  5. 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

  6. Recombinational DNA repair: the RecF and RecR proteins limit the extension of RecA filaments beyond single-strand DNA gaps.

    PubMed

    Webb, B L; Cox, M M; Inman, R B

    1997-10-31

    In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair. PMID:9363943

  7. Fractionation of whey protein isolate with supercritical carbon dioxide to produce enriched α-lactalbumin and β-lactoglobulin food ingredients.

    PubMed

    Bonnaillie, Laetitia M; Tomasula, Peggy M

    2012-05-23

    An environmentally friendly protein fractionation process using supercritical carbon dioxide (SCO(2)) as an acid was developed to produce enriched α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) fractions from whey protein isolate solutions containing from 2 to 10% WPI. This study investigated the effects of pH, temperature, WPI concentration, and residence time on the precipitation kinetics and recovery yields of individual whey proteins and the relative enrichment and composition of both protein fractions. At 5.5-34 MPa and 60-65 °C, solubilized SCO(2) decreased solution pH and induced the formation and precipitation of α-LA aggregates. Gel electrophoresis and HPLC of the enriched fractions demonstrated the production of ≥ 60% pure α-LA, and ≥ 70% pure β-LG, under various operating conditions, from WPI containing ∼57% β-LG and 21% α-LA. The enriched fractions are ready-to-use food ingredients with neutral pH, untainted by acids and contaminants.

  8. 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.

  9. Storage stability of keratinocyte growth factor-2 in lyophilized formulations: effects of formulation physical properties and protein fraction at the solid-air interface.

    PubMed

    Devineni, Dilip; Gonschorek, Christoph; Cicerone, Marcus T; Xu, Yemin; Carpenter, John F; Randolph, Theodore W

    2014-10-01

    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 〈u(2)〉(-1) for hydrogen atoms (fast β relaxation), and the relaxation time τ(β), which correlates with relaxation due to fast Johari-Goldstein motions in the glass (Xu et al., 2013) [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

  10. Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration.

    PubMed Central

    Dunkley, P R; Holmes, H; Rodnight, R

    1977-01-01

    Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories. PMID:869930

  11. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  12. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. PMID:26299399

  13. Vaccination of mice with a soluble protein fraction of Mycobacterium leprae provides consistent and long-term protection against M. leprae infection.

    PubMed Central

    Gelber, R H; Murray, L; Siu, P; Tsang, M

    1992-01-01

    Groups of BALB/c mice were vaccinated intradermally with either Freund's incomplete adjuvant (FIA) alone, 10(7) heat-killed Mycobacterium leprae organisms in FIA, or a number of fractions of M. leprae containing soluble and/or cell wall components. At 1, 3, 6, 9, and 12 months later, vaccinated mice were challenged in the right hind footpad with 5,000 live M. leprae organisms, and vaccine protection was assessed 6 to 8 months later, at the peak of M. leprae multiplication in the negative control (FIA alone), by the two-sample rank-sum test. In these studies, a cell wall fraction rich in peptidoglycan was consistently ineffective. Both heat-killed M. leprae and a fraction containing cell wall and fixed proteins generally protected when the interval between vaccination and challenge was 1 or 3 months but not subsequently. On the other hand, soluble proteins of M. leprae alone or in combination (with cell wall fractions) consistently (14 of 14 instances) afforded highly significant protection (P less than or equal to 0.01) at all challenge intervals up to 1 year after vaccination. These results suggest that the soluble protein fraction of M. leprae offers promise for a vaccine against leprosy. PMID:1563772

  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. 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.

  16. Decolorization of direct dyes by salt fractionated turnip proteins enhanced in the presence of hydrogen peroxide and redox mediators.

    PubMed

    Matto, Mahreen; Husain, Qayyum

    2007-09-01

    The present paper demonstrates the effect of salt fractionated turnip (Brassica rapa) proteins on the decolorization of direct dyes, used in textile industry, in the presence of various redox mediators. The rate and extent of decolorization of dyes was significantly enhanced by the presence of different types of redox mediators. Six out of 10 investigated compounds have shown their potential in enhancing the decolorization of direct dyes. The performance was evaluated at different concentrations of mediator and enzyme. The efficiency of each natural mediator depends on the type of dye treated. The decolorization of all tested direct dyes was maximum in the presence of 0.6mM redox mediator at pH 5.5 and 30 degrees C. Complex mixtures of dyes were also maximally decolorized in the presence of 0.6mM redox mediator (1-hydroxybenzotriazole/violuric acid). In order to examine the operational stability of the enzyme preparation, the enzyme was exploited for the decolorization of mixtures of dyes for different times in a stirred batch process. There was no further change in decolorization of an individual dye or their mixtures after 60 min; the enzyme caused more than 80% decolorization of all dyes in the presence of 1-hydroxybenzotriazole/violuric acid. However, there was no desirable increase in dye decolorization of the mixtures on overnight stay. Total organic carbon analysis of treated dyes or their mixtures showed that these results were quite comparable to the loss of color from solutions. However, the treatment of such polluted water in the presence of redox mediators caused the formation of insoluble precipitate, which could be removed by the process of centrifugation. The results suggested that catalyzed oxidative coupling reactions might be important for natural transformation pathways for dyes and indicate their potential use as an efficient means for removal of dyes color from waters and wastewaters.

  17. 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

  18. 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.

  19. A modified rinsing method for the determination of the S, W-S and D + U fraction of protein and starch in feedstuff within the in situ technique.

    PubMed

    de Jonge, L H; van Laar, H; Hendriks, W H; Dijkstra, J

    2013-08-01

    A modified rinsing method for the in situ technique was developed to separate, isolate and characterise the soluble (S), the insoluble washout (W-S) and the non-washout fractions (D + U) within one procedure. For non-incubated bags (t = 0 h), this method was compared with the conventional, Combined Fractionation (CF) method that measures the D + U and S fractions in separate steps and subsequently calculates the W-S fraction. The modified method was based on rinsing of nylon bags in a closed vessel containing a buffer solution (pH 6.2) during 1 h, where shaking speeds of 40, 100, and 160 strokes per minutes (spm) were evaluated, and tested for six feed ingredients (faba beans, maize, oats, peas, soya beans and wheat) and four forages (two ryegrass silages and two maize silages). The average recoveries as the sum of all fractions were 0.972 ± 0.041 for N and 0.990 ± 0.050 for starch (mean ± s.d.). The mean W-S fraction increased with increasing shaking speed and varied between 0.017 (N) and 0.083 (starch) at 40 spm and 0.078 (N) and 0.303 (starch) at 160 spm, respectively. For ryegrass silages, the W-S fraction was absent at all shaking speeds, but was present in the CF method. The modified method, in particular at 40 and 100 spm, reduced the loss of small particles during rinsing, resulting in lower W-S and higher D + U fractions for N and starch compared with the CF method. For soya beans and ryegrass silage, the modified method reduced the S fraction of N compared with the CF method. The results obtained at 160 spm showed the best comparison with those from the CF method. The W-S fraction of the feedstuff obtained at 160 spm contained mainly particles smaller than 40 μm (0.908 ± 0.086). In most feedstuff, starch was the most abundant chemical component in the W-S fraction and its content (726 ± 75 g/kg DM) was higher than in the D + U fraction (405 ± 177 g/kg DM). Alkaline-soluble proteins were the dominant N-containing components in the W-S fraction of

  20. 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.

  1. 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. PMID:25981407

  2. A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms.

    PubMed

    West, Rolieria; Whitmon, Jennifer; Williamson, Yulanda M; Moura, Hercules; Nelson, Marguerite; Melnick, Nikkol; Tondella, Maria Lucia C; Schieltz, David; Rees, Jon; Woolfitt, Adrian R; Barr, John R; Ades, Edwin W; Carlone, George M; Sampson, Jacquelyn S

    2012-03-16

    Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

  3. Preferential DNA-protein cross-linking by NiCl2 in magnesium-insoluble regions of fractionated Chinese hamster ovary cell chromatin.

    PubMed

    Patierno, S R; Sugiyama, M; Basilion, J P; Costa, M

    1985-11-01

    Intracellular nickel ions (Ni2+) have been shown to cause single-strand breaks in DNA, that were rapidly repaired, and DNA-protein cross-links, that persisted for at least 24 h following removal of extracellular ionic nickel. In this study, we have used the techniques of alkaline elution, chromatin fractionation, and sodium dodecyl sulfate:polyacrylamide gel electrophoresis to examine the DNA-protein cross-linking induced by NiCl2 in Chinese hamster ovary cells. Continuous treatment of logarithmically growing Chinese hamster ovary cells with 2.5 mM NiCl2 in complete medium resulted in DNA single-strand breaks within 1 h, followed by a time-dependent increase in the induction of DNA-protein cross-links at 2, 3, and 6 h. Since the entry of nickel into cells was maximal within 2 h of exposure, the time delay for the formation of DNA-protein cross-links was not limited by metal uptake. The nickel-induced DNA-protein cross-linking appeared to require active cell cycling, since single-strand breaks but no cross-linking could be detected in confluent cells treated with 1, 2.5, or 5 mM NiCl2 for 3 h. DNA-protein cross-linking induced by nickel occurred in late S phase of the cell cycle. High-molecular-weight nonhistone chromatin proteins and possibly histone H1 migrating at the Mr 30,000 range became cross-linked to DNA after treatment of cells with NiCl2. All nickel-cross-linked proteins were concentrated in the magnesium-insoluble regions of fractionated chromatin and were stable to urea, 2-mercaptoethanol, and Nonidet P-40. Some proteins (Mr 48,000, 52,000, 55,000, 70,000, and 95,000), the association of which with DNA was also stable to Sarkosyl, salt, and EDTA, were detectable in DNA rigorously fractionated from untreated cells. Nickel therefore appeared to cause the cross-linking of proteins that normally reside in close association with DNA. Alterations of the normal association of these proteins with DNA by nickel may be an early event in the nickel transformation

  4. Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension.

    PubMed Central

    Geisler, N; Fischer, S; Vandekerckhove, J; Plessmann, U; Weber, K

    1984-01-01

    The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6439558

  5. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

    SciTech Connect

    Bartles, J.R.; Feracci, H.M.; Stieger, B.; Hubbard, A.L.

    1987-09-01

    We have used pulse-chase metabolic radiolabeling with L-(/sup 35/S)methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor (ASGP-R)) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates.

  6. p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.

    PubMed Central

    Benton, B M; Berrios, S; Fisher, P A

    1988-01-01

    A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed. Images PMID:3133549

  7. Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery.

    PubMed

    Pirmoradian, Mohammad; Astorga-Wells, Juan; Zubarev, Roman A

    2015-12-01

    Prefractionation of proteins is often employed to improve analysis specificity in proteomics. Prefractionation based on the isoelectric point (pI) is particularly attractive because pI is a well-defined parameter and it is orthogonal to hydrophobicity on which reversed-phase chromatography is based. However, direct capillary electrophoresis of blood proteins is challenging due to its high content of salts and charged small molecules. Here, we couple an online desalinator device to our multijunction capillary isoelectric focusing (MJ-CIEF) instrument and perform direct isoelectric separation of human blood plasma. In a proof-of-principle experiment, pooled samples of patients with progressive mild cognitive impairment and corresponding healthy controls were investigated. Injection of 3 μL of plasma containing over 100 μg of proteins into the desalinator was followed by pI fractionation with MJ-CIEF in less than 1 h. Shotgun proteomics of 12 collected fractions from each of the 5 replicates of pooled samples resulted in the identification and accurate quantification (median CV between the replicates is <4%) of nearly 365 protein groups from 4030 unique peptides (with <1% FDR for both peptides and proteins). The obtained results include several proteins previously reported as AD markers. The isoelectric point of each quantified protein was calculated using a set of 7 synthetic peptides spiked into the samples. Several proteins with a significant pI shift between their isoforms in the patient and control samples were identified. The presented method is straightforward, robust, and scalable; therefore, it can be used in both biological and clinical applications.

  8. Novel inhibitory action of tunicamycin homologues suggests a role for dynamic protein fatty acylation in growth cone-mediated neurite extension

    PubMed Central

    1994-01-01

    dynamic protein acylation in growth cone- mediated extension of neuronal processes. PMID:8106550

  9. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    SciTech Connect

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E. . E-mail: glenn.morris@rjah.nhs.uk

    2005-09-10

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with {beta}-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.

  10. Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension

    PubMed Central

    Geisler, N.; Fischer, S.; Vandekerckhove, J.; Damme, J. Van; Plessmann, U.; Weber, K.

    1985-01-01

    NF-H has the highest mol. wt. of the three mammalian neurofilament components (NF-L, NF-M, NF-H). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis, NF-H contains sequences which identify it as an integral intermediate filament (IF) protein in its amino-terminal region. We have isolated and partially characterized a basic, non-α-helical segment located at the amino-terminal end with properties similar to headpieces of other non-epithelial IF proteins. The highly α-helical 40-K fragment excised by chymotrypsin is now identified by the amino acid sequence of a 17-K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled-coil arrays. NF-H and NF-M show 45% sequence identity in this region. The extra mass of NF-H in comparison with most other IF proteins arises from a carboxy-terminal extension thought to be responsible for inter-neurofilament cross-bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The NF-H tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino-terminal part. Having now delineated the intermediate filament-type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled-coil interactions. They all carry unique carboxy-terminal extensions which increase in length from NF-L to NF-H and seem to extend from the filament wall. ImagesFig. 1. PMID:16453600

  11. A Profile of an Endosymbiont-enriched Fraction of the Coral Stylophora pistillata Reveals Proteins Relevant to Microbial-Host Interactions*

    PubMed Central

    Weston, Andrew J.; Dunlap, Walter C.; Shick, J. Malcolm; Klueter, Anke; Iglic, Katrina; Vukelic, Ana; Starcevic, Antonio; Ward, Malcolm; Wells, Mark L.; Trick, Charles G.; Long, Paul F.

    2012-01-01

    This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal “bleaching” stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure. PMID:22351649

  12. Even with nonnative interactions, the updated folding transition states of the homologs Proteins G & L are extensive and similar

    PubMed Central

    Baxa, Michael C.; Yu, Wookyung; Adhikari, Aashish N.; Ge, Liang; Xia, Zhen; Zhou, Ruhong; Freed, Karl F.; Sosnick, Tobin R.

    2015-01-01

    Experimental and computational folding studies of Proteins L & G and NuG2 typically find that sequence differences determine which of the two hairpins is formed in the transition state ensemble (TSE). However, our recent work on Protein L finds that its TSE contains both hairpins, compelling a reassessment of the influence of sequence on the folding behavior of the other two homologs. We characterize the TSEs for Protein G and NuG2b, a triple mutant of NuG2, using ψ analysis, a method for identifying contacts in the TSE. All three homologs are found to share a common and near-native TSE topology with interactions between all four strands. However, the helical content varies in the TSE, being largely absent in Proteins G & L but partially present in NuG2b. The variability likely arises from competing propensities for the formation of nonnative β turns in the naturally occurring proteins, as observed in our TerItFix folding algorithm. All-atom folding simulations of NuG2b recapitulate the observed TSEs with four strands for 5 of 27 transition paths [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517–520]. Our data support the view that homologous proteins have similar folding mechanisms, even when nonnative interactions are present in the transition state. These findings emphasize the ongoing challenge of accurately characterizing and predicting TSEs, even for relatively simple proteins. PMID:26100906

  13. Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast

    PubMed Central

    2004-01-01

    Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40–220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3′-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA–protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program. PMID:15024427

  14. DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis.

    PubMed

    Zaremba, Mindaugas; Toliusis, Paulius; Grigaitis, Rokas; Manakova, Elena; Silanskas, Arunas; Tamulaitiene, Giedre; Szczelkun, Mark D; Siksnys, Virginijus

    2014-12-16

    The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6-7 nucleotides) downstream of the asymmetric recognition sequence 5'-GCCGC-3'. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage. PMID:25429977

  15. Analyzing the Effects of Climate Factors on Soybean Protein, Oil Contents, and Composition by Extensive and High-Density Sampling in China.

    PubMed

    Song, Wenwen; Yang, Ruping; Wu, Tingting; Wu, Cunxiang; Sun, Shi; Zhang, Shouwei; Jiang, Bingjun; Tian, Shiyan; Liu, Xiaobing; Han, Tianfu

    2016-05-25

    From 2010 to 2013, 763 soybean samples were collected from an extensive area of China. The correlations between seed compositions and climate data were analyzed. The contents of crude protein and water-soluble protein, total amount of protein plus oil, and most of the amino acids were positively correlated with an accumulated temperature ≥15 °C (AT15) and the mean daily temperature (MDT) but were negatively correlated with hours of sunshine (HS) and diurnal temperature range (DTR). The correlations of crude oil and most fatty acids with climate factors were opposite to those of crude protein. Crude oil content had a quadratic regression relationship with MDT, and a positive correlation between oil content and MDT was found when the daily temperature was <19.7 °C. A path analysis indicated that DTR was the main factor that directly affected soybean protein and oil contents. The study illustrated the effects of climate factors on soybean protein and oil contents and proposed agronomic practices for improving soybean quality in different regions of China. The results provide a foundation for the regionalization of high-quality soybean production in China and similar regions in the world.

  16. Analyzing the Effects of Climate Factors on Soybean Protein, Oil Contents, and Composition by Extensive and High-Density Sampling in China.

    PubMed

    Song, Wenwen; Yang, Ruping; Wu, Tingting; Wu, Cunxiang; Sun, Shi; Zhang, Shouwei; Jiang, Bingjun; Tian, Shiyan; Liu, Xiaobing; Han, Tianfu

    2016-05-25

    From 2010 to 2013, 763 soybean samples were collected from an extensive area of China. The correlations between seed compositions and climate data were analyzed. The contents of crude protein and water-soluble protein, total amount of protein plus oil, and most of the amino acids were positively correlated with an accumulated temperature ≥15 °C (AT15) and the mean daily temperature (MDT) but were negatively correlated with hours of sunshine (HS) and diurnal temperature range (DTR). The correlations of crude oil and most fatty acids with climate factors were opposite to those of crude protein. Crude oil content had a quadratic regression relationship with MDT, and a positive correlation between oil content and MDT was found when the daily temperature was <19.7 °C. A path analysis indicated that DTR was the main factor that directly affected soybean protein and oil contents. The study illustrated the effects of climate factors on soybean protein and oil contents and proposed agronomic practices for improving soybean quality in different regions of China. The results provide a foundation for the regionalization of high-quality soybean production in China and similar regions in the world. PMID:27022763

  17. Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens.

    PubMed

    Hennebert, Elise; Wattiez, Ruddy; Waite, J Herbert; Flammang, Patrick

    2012-01-01

    Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named 'Sea star footprint proteins' (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes.

  18. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed

    Murray, P J; Spithill, T W; Handman, E

    1989-07-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens.

  19. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed Central

    Murray, P J; Spithill, T W; Handman, E

    1989-01-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens. Images PMID:2731987

  20. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products.

    PubMed Central

    Harlow, E; Franza, B R; Schley, C

    1985-01-01

    Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins. Images PMID:3894685

  1. Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

    PubMed

    Kähne, Thilo; Richter, Sandra; Kolodziej, Angela; Smalla, Karl-Heinz; Pielot, Rainer; Engler, Alexander; Ohl, Frank W; Dieterich, Daniela C; Seidenbecher, Constanze; Tischmeyer, Wolfgang; Naumann, Michael; Gundelfinger, Eckart D

    2016-07-01

    Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down-regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label-free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin-remodeling proteins, and RNA-binding proteins, were regulated in at least three brain regions pointing to common, cross-regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up-regulated only in the hippocampus, while Septin-9 was down-regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area-specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing. PMID

  2. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  3. Fractional enrichment of proteins using [2-(13)C]-glycerol as the carbon source facilitates measurement of excited state 13Cα chemical shifts with improved sensitivity.

    PubMed

    Ahlner, Alexandra; Andresen, Cecilia; Khan, Shahid N; Kay, Lewis E; Lundström, Patrik

    2015-07-01

    A selective isotope labeling scheme based on the utilization of [2-(13)C]-glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state (13)Cα chemical shifts using Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold relative to labeling schemes based on [2-(13)C]-glucose, effectively doubling the sensitivity of NMR experiments. Applications to a binding reaction involving an SH3 domain from the protein Abp1p and a peptide from the protein Ark1p establish that accurate excited state (13)Cα chemical shifts can be obtained from RD experiments, with errors on the order of 0.06 ppm for exchange rates ranging from 100 to 1000 s(-1), despite the small fraction of (13)Cα-(13)Cβ spin-pairs that are present for many residue types. The labeling approach described here should thus be attractive for studies of exchanging systems using (13)Cα spin probes.

  4. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples

    PubMed Central

    Belov, Larissa; Matic, Kieran J.; Hallal, Susannah; Best, O. Giles; Mulligan, Stephen P.; Christopherson, Richard I.

    2016-01-01

    Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19+ EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals. PMID:27086589

  5. Organellar RNA editing and plant-specific extensions of pentatricopeptide repeat proteins in jungermanniid but not in marchantiid liverworts.

    PubMed

    Rüdinger, Mareike; Polsakiewicz, Monika; Knoop, Volker

    2008-07-01

    The pyrimidine exchange type of RNA editing in land plant (embryophyte) organelles has largely remained an enigma with respect to its biochemical mechanisms, the underlying specificities, and its raison d'être. Apparently arising with the earliest embryophytes, RNA editing is conspicuously absent in one clade of liverworts, the complex thalloid Marchantiidae. Several lines of evidence suggest that the large gene family of organelle-targeted RNA-binding pentatricopeptide repeat (PPR) proteins plays a fundamental role in the sequence-specific editing of organelle transcripts. We here describe the identification of PPR protein genes with plant-specific carboxyterminal (C-terminal) sequence signatures (E, E+, and DYW domains) in ferns, lycopodiophytes, mosses, hornworts, and jungermanniid liverworts, one subclass of the basal most clade of embryophytes, on DNA and cDNA level. In contrast, we were unable to identify these genes in a wide sampling of marchantiid liverworts (including the phylogenetic basal genus Blasia)--taxa for which no RNA editing is observed in the organelle transcripts. On the other hand, we found significant diversity of this type of PPR proteins also in Haplomitrium, a genus with an extremely high rate of RNA editing and a phylogenetic placement basal to all other liverworts. Although the presence of modularly extended PPR proteins correlates well with organelle RNA editing, the now apparent complete loss of an entire gene family from one clade of embryophytes, the marchantiid liverworts, remains puzzling.

  6. Mutation of FdC2 gene encoding a ferredoxin-like protein with C-terminal extension causes yellow-green leaf phenotype in rice.

    PubMed

    Li, Chunmei; Hu, Yan; Huang, Rui; Ma, Xiaozhi; Wang, Yang; Liao, Tingting; Zhong, Ping; Xiao, Fuliang; Sun, Changhui; Xu, Zhengjun; Deng, Xiaojian; Wang, Pingrong

    2015-09-01

    Ferredoxins (Fds) are small iron-sulfur proteins that mediate electron transfer in a wide range of metabolic reactions. Besides Fds, there is a type of Fd-like proteins designated as FdC, which have conserved elements of Fds, but contain a significant C-terminal extension. So far, only two FdC genes of Arabidopsis (Arabidopsis thaliana) have been identified in higher plants and thus the functions of FdC proteins remain largely unknown. In this study, we isolated a yellow-green leaf mutant, 501ys, in rice (Oryza sativa). The mutant exhibited yellow-green leaf phenotype and reduced chlorophyll level. The phenotype of 501ys was caused by mutation of a gene on rice chromosome 3. Map-based cloning of this mutant resulted in identification of OsFdC2 gene (LOC_Os03g48040) showing high identity with Arabidopsis FdC2 gene (AT1G32550). OsFdC2 was expressed most abundantly in leaves and its encoded protein was targeted to the chloroplast. In 501ys mutant, a missense mutation was detected in DNA sequence of the gene, resulting in an amino acid change in the encoded protein. The mutant phenotype was rescued by introduction of the wild-type gene. Therefore, we successfully identified FdC2 gene via map-based cloning approach, and demonstrated that mutation of this gene caused yellow-green leaf phenotype in rice.

  7. Mutation of FdC2 gene encoding a ferredoxin-like protein with C-terminal extension causes yellow-green leaf phenotype in rice.

    PubMed

    Li, Chunmei; Hu, Yan; Huang, Rui; Ma, Xiaozhi; Wang, Yang; Liao, Tingting; Zhong, Ping; Xiao, Fuliang; Sun, Changhui; Xu, Zhengjun; Deng, Xiaojian; Wang, Pingrong

    2015-09-01

    Ferredoxins (Fds) are small iron-sulfur proteins that mediate electron transfer in a wide range of metabolic reactions. Besides Fds, there is a type of Fd-like proteins designated as FdC, which have conserved elements of Fds, but contain a significant C-terminal extension. So far, only two FdC genes of Arabidopsis (Arabidopsis thaliana) have been identified in higher plants and thus the functions of FdC proteins remain largely unknown. In this study, we isolated a yellow-green leaf mutant, 501ys, in rice (Oryza sativa). The mutant exhibited yellow-green leaf phenotype and reduced chlorophyll level. The phenotype of 501ys was caused by mutation of a gene on rice chromosome 3. Map-based cloning of this mutant resulted in identification of OsFdC2 gene (LOC_Os03g48040) showing high identity with Arabidopsis FdC2 gene (AT1G32550). OsFdC2 was expressed most abundantly in leaves and its encoded protein was targeted to the chloroplast. In 501ys mutant, a missense mutation was detected in DNA sequence of the gene, resulting in an amino acid change in the encoded protein. The mutant phenotype was rescued by introduction of the wild-type gene. Therefore, we successfully identified FdC2 gene via map-based cloning approach, and demonstrated that mutation of this gene caused yellow-green leaf phenotype in rice. PMID:26259181

  8. A protein extract and a cysteine protease inhibitor enriched fraction from Jatropha curcas seed cake have in vitro anti-Toxoplasma gondii activity.

    PubMed

    Soares, A M S; Carvalho, L P; Melo, E J T; Costa, H P S; Vasconcelos, I M; Oliveira, J T A

    2015-06-01

    Toxoplasma gondii is a parasite of great medical and veterinary importance that has worldwide distribution and causes toxoplasmosis. There are few treatments available for toxoplasmosis and the search for plant extracts and compounds with anti-Toxoplasma activity is of utmost importance for the discovery of new active drugs. The objective of this study was to investigate the action of a protein extract and a protease inhibitor enriched fraction from J. curcas seed cake on developing tachyzoites of T. gondii-infected Vero cells. The protein extract (JcCE) was obtained after solubilization of the J. curcas seed cake with 100 mM sodium borate buffer, pH 10, centrifugation and dialysis of the resulting supernatant with the extracting buffer. JcCE was used for the in vitro assays of anti-Toxoplasma activity at 0.01, 0.1, 0.5, 1.5, 3.0 and 5.0 mg/ml concentration for 24 h. The results showed that JcCE reduced the percentage of infection and the number of intracellular parasites, but had no effect on the morphology of Vero cells up to 3.0 mg/mL. The cysteine protease inhibitor enriched fraction, which was obtained after chromatography of JcCE on Sephadex G-75 and presented a unique protein band following SDS-PAGE, reduced both the number of T. gondii infected cells and intracellular parasites. These results suggest that both JcCE and the cysteine protease inhibitor enriched fraction interfere with the intracellular growth of T. gondii. PMID:25816973

  9. Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

    PubMed

    Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J

    2016-03-15

    Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM. PMID:26714847

  10. Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.).

    PubMed

    McCarthy, Ian D; Nicholls, Ruth; Malham, Shelagh K; Whiteley, Nia M

    2016-01-01

    For the first time, use of the flooding dose technique using (3)H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of (3)H-phenylalanine into the posterior adductor muscle. Incorporation of (3)H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1-6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d(-1) for the gill, 2.5±0.3%d(-1) for the mantle and 2.6±0.3%d(-1) for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day(-1). The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

  11. Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

    PubMed

    Shankaran, Mahalakshmi; Shearer, Todd W; Stimpson, Stephen A; Turner, Scott M; King, Chelsea; Wong, Po-Yin Anne; Shen, Ying; Turnbull, Philip S; Kramer, Fritz; Clifton, Lisa; Russell, Alan; Hellerstein, Marc K; Evans, William J

    2016-03-15

    Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

  12. Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System

    PubMed Central

    Galgano, Alessia; Forrer, Michael; Jaskiewicz, Lukasz; Kanitz, Alexander; Zavolan, Mihaela; Gerber, André P.

    2008-01-01

    Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs. PMID:18776931

  13. Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

    PubMed Central

    2011-01-01

    Background Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues. Results We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. Conclusion For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Reviewers This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian. PMID:22024092

  14. Fractional randomness

    NASA Astrophysics Data System (ADS)

    Tapiero, Charles S.; Vallois, Pierre

    2016-11-01

    The premise of this paper is that a fractional probability distribution is based on fractional operators and the fractional (Hurst) index used that alters the classical setting of random variables. For example, a random variable defined by its density function might not have a fractional density function defined in its conventional sense. Practically, it implies that a distribution's granularity defined by a fractional kernel may have properties that differ due to the fractional index used and the fractional calculus applied to define it. The purpose of this paper is to consider an application of fractional calculus to define the fractional density function of a random variable. In addition, we provide and prove a number of results, defining the functional forms of these distributions as well as their existence. In particular, we define fractional probability distributions for increasing and decreasing functions that are right continuous. Examples are used to motivate the usefulness of a statistical approach to fractional calculus and its application to economic and financial problems. In conclusion, this paper is a preliminary attempt to construct statistical fractional models. Due to the breadth and the extent of such problems, this paper may be considered as an initial attempt to do so.

  15. A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism.

    PubMed

    Chronis, Demosthenis; Chen, Shiyan; Lu, Shunwen; Hewezi, Tarek; Carpenter, Sara C D; Loria, Rosemary; Baum, Thomas J; Wang, Xiaohong

    2013-04-01

    Nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide for secretion, a mono-ubiquitin domain, and a 12 amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell, and was up-regulated in the parasitic second-stage juvenile, correlating with the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success, and that over-expression of the secreted Gr(Δ) (SP) UBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we found that Gr(Δ) (SP) UBCEP12 is processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta, and that GrCEP12 suppresses resistance gene-mediated cell death. A target search showed that expression of RPN2a, a gene encoding a subunit of the 26S proteasome, was dramatically suppressed in Gr(Δ) (SP) UBCEP12 but not GrCEP12 over-expression plants when compared with control plants. Together, these results suggest that, when delivered into host plant cells, Gr(Δ) (SP) UBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation.

  16. The effect of dietary intake of the acidic protein fraction of bovine colostrum on influenza A (H1N1) virus infection.

    PubMed

    Xu, Mei Ling; Kim, Hyoung Jin; Chang, Don Yong; Kim, Hong-Jin

    2013-06-01

    Acidic protein levels in the milk decrease markedly as lactation progresses, suggesting that it is an important part of the colostrum. However, little attention has been paid to their biological function. In this study, we isolated the acidic protein fraction of bovine colostrum (AFC, isoelectric point <5) by anion-exchange chromatography, and investigated the effect of its dietary intake on influenza A (H1N1) virus infection. 100% of mice infected with 1 LD50 of the virus survived when administered AFC for 14 days prior to infection, compared with 33% survival when administered phosphate buffered saline (PBS). Moreover, consumption of AFC reduced the weight loss associated with infection. We propose that dietary intake of AFC has a prophylactic effect on influenza A virus infection. PMID:23620352

  17. Hepatoprotective effect of a protein-enriched fraction from the maggots (Musca domestica) against CCl4-induced hepatic damage in rats.

    PubMed

    Wang, Fu-Rong; Ai, Hui; Chen, Xiao-Min; Lei, Chao-Liang

    2007-06-01

    The hepatoprotective potential of a protein-enriched fraction (PEF) isolated from the maggots of housefly (Musca domestica) was evaluated in rats against carbon tetrachloride (CCl4)-induced acute hepatic damage. Activities of serum aspartate aminotransferase and alanine aminotransferase increased by 4- and 13-fold induced by CCl4, were significantly inhibited by pretreatment with 50, 100 and 200 mg PEF/kg. The formation of malondialdehyde was also significantly decreased in PEF-treated group compared with CCl4-treated group. The treatments with PEF also elevated total protein levels significantly. These results were further supplemented by histopathological examination of liver sections. Hyperplasia of kupffer cells was observed after treatment with PEF (100 and 200 mg/kg). We conclude that PEF is effective in this model of liver damage.

  18. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

    PubMed Central

    Wu, An-Guo; Kam-Wai Wong, Vincent; Zeng, Wu; Liu, Liang; Yuen-Kwan Law, Betty

    2015-01-01

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future. PMID:26598009

  19. Influence of black gram (Vigna mungo) trypsin inhibitory fraction on the hepatic protein catabolism in male albino mice.

    PubMed

    Kamalakannan, V; Sathyamoorthy, A V; Motlag, D B

    1984-01-01

    The effect of black gram and black gram trypsin inhibitor on the protein catabolism of male albino mice has been investigated. Group 1 was given autoclaved black gram (control), Group II raw black gram and Group III the autoclaved black gram incorporated with 1% black gram trypsin inhibitor. Blood as well as urinary urea and creatine were found to be elevated in Groups II and III. Increased levels of arginase, ornithine transcarbamylase and transaminases were noted in Groups II and III. The results suggested an enhanced catabolism of proteins evoked by the native black gram trypsin inhibitor.

  20. Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster.

    PubMed Central

    Verrelli, B C; Eanes, W F

    2000-01-01

    PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution. PMID:11102370

  1. Utility of a Protein Fraction with Cathepsin L-Like Activity Purified from Cysticercus Fluid of Taenia solium in the Diagnosis of Human Cysticercosis

    PubMed Central

    Zimic, Mirko; Pajuelo, Mónica; Rueda, Daniel; López, César; Arana, Yanina; Castillo, Yesenia; Calderón, Maritza; Rodriguez, Silvia; Sheen, Patricia; Vinetz, Joseph M.; Gonzales, Armando; García, Héctor H.; Gilman, Robert H.

    2009-01-01

    Neurocysticercosis, an endemic parasitic disease in most developing countries, is caused by Taenia solium and compromises the human central nervous system. Cathepsin L-like proteases are secreted by several parasites including T. solium and constitute important antigens for immunodiagnostics. A protein fraction with cathepsin L-like activity was purified from the cysticercus fluid by size exclusion and ion exchange chromatography. Cathepsin L-like activity was measured fluorometrically by detecting the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC. The purified protein fraction included antigens of 53 and 25 kD that were tested in a Western immunoblot and in an enzyme-linked immunosorbent assay (ELISA) for detection of human cysticercosis. The sensitivity of the Western immunoblot was 96% for patients infected with multiple cysts and 78% for patients with a single cyst. Specificity was 98%. The sensitivity of the ELISA was 98% in patients with multiple cysts and 84% in patients with a single cyst. Specificity was 92.7%. PMID:19478259

  2. Induction of Defense Reactions in Sugar Beet and Wheat by Treatment with Cell Wall Protein Fractions from the Mycoparasite Pythium oligandrum.

    PubMed

    Takenaka, Shigehito; Nishio, Zenta; Nakamura, Yumi

    2003-10-01

    ABSTRACT To detect molecules with elicitor properties from Pythium oligandrum, cell wall protein fractions (CWPs) were extracted from 10 P. oligandrum isolates and examined for elicitor activity in sugar beet and wheat. P. oligandrum isolates were divided into two groups based on the number of major proteins in CWP: isolates with two major proteins (D-type) and isolates with one major protein (S-type). Sugar beet seedlings treated with both types of CWP through their roots showed enhanced activities of phenylalanine ammonia lyase and chitinase, and D-type-treated seedlings also showed significantly higher cell wall-bound phenolic compounds, mainly ferulic acid, compared with the distilled-water-treatment control. Damping-off severity was significantly reduced on seedlings treated with both types of CWP compared with the control, following challenge with Rhizoctonia solani AG2-2. Both types of CWP significantly reduced the number of infected spikelets developed from the injected spikelet compared with the control, following challenge with Fusarium graminearum. Neither type of CWP resulted in any reduction in pathogen growth rate in plate tests. These results demonstrate that CWPs of P. oligandrum have elicitor properties in sugar beet and wheat.

  3. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    PubMed

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects. PMID:27435508

  4. Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells.

    PubMed

    Volstatova, Tereza; Havlik, Jaroslav; Potuckova, Miroslava; Geigerova, Martina

    2016-08-10

    Adhesion to the intestinal epithelium is considered an important feature of probiotic bacteria, which may increase their persistence in the intestine, allowing them to exert their beneficial health effect or promote the colonisation process. However, this feature might be largely dependent on the host specificity or diet. In the present study, we investigated the effect of selected milks and milk protein fractions on the ability of selected lactobacilli to adhere to the cells of an intestinal model based on co-culture Caco-2/HT29-MTX cell lines. Most milk digesta did not significantly affect bacterial adhesion except for UHT-treated milk and sheep milk. The presence of UHT-treated milk digesta reduced the adhesion of Lactobacillus gasseri R by 61% but not that of Lactobacillus casei FMP. However, sheep milk significantly increased the adherence of L. casei FMP (P < 0.05) but not of L. gasseri R. Among the protein fractions, rennet casein (RCN) and bovine serum albumin (BSA) showed reproducible patterns and strain-specific effects on bacterial adherence. While RCN reduced the adherence of L. gasseri R to <50% compared to the control, it did not have a significant effect on L. casei FMP. In contrast, BSA reduced L. casei FMP adherence to a higher extent than that of L. gasseri R. Whey protein (WH) tended to increase the adherence of both strains by 130%-180%. Recently, interactions between the host diet and its microbiota have attracted considerable interest. Our results may explain one of the aspects of the role of milk in the development of microbiota or support of probiotic supplements. Based on our data, we conclude that the persistence of probiotic strains supplemented as part of dairy food or constitutional microbiota in the gut might be affected negatively or positively by the food matrix through complex strain or concentration dependent effects.

  5. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion.

    PubMed

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-01-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P<0.05) among the feeds. The spectral bands features were significantly different (P<0.05) among the feeds. Spectral intensities of A_Cell, H_1415 and H_1370 were weakly positively correlated with in situ rumen digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region.

  6. Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower ( Helianthus annuus L.).

    PubMed

    Queiroz, M A A; Fukushima, R S; Gomide, C A; Braga, M R

    2008-07-01

    Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper, these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B2 and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B1 and C when CW replaced NDF; however, there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF. Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B1 (starch + pectin) values were lower than pectin determined through wet chemistry. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B1 but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B3) be adopted for the digestible cell wall carbohydrates.

  7. ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

    EPA Science Inventory

    Introduction

    Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

  8. Antihepatitis B virus activity of a protein-enriched fraction from housefly (Musca domestica) in a stable HBV-producing cell line.

    PubMed

    Lu, Xuemei; Jin, Xiaobao; Wang, Jie; Chu, Fujiang; Zhu, Jiayong

    2014-01-01

    Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

  9. Chromatographic methods of fractionation.

    PubMed

    Friesen, A D

    1987-01-01

    Chromatography's functional versatility, separation efficiency, gentle non-denaturing separating process and ease of automation and scale-up make it attractive for industrial scale protein purification. The Winnipeg Rh Institute's new Plasma Fractionation facility is an example of the use of chromatography for the large scale purification of plasma protein fractions. The fractionation facility has a capacity to process 800 litres of plasma per batch into blood clotting factor VIII and IX, albumin and intravenous immune serum globulin (i.v. ISG). Albumin and i.v. ISG are purified using ion exchange columns of DEAE-Sepharose (230 litre size), DEAE-Biogel (150 litre size) and CM-Sepharose (150 litre size). The chromatographic process is automated using a Modicon 584 Programmable Logic Controller to regulate valves, pumps and sensors which control plasma flow during fractionation. The stainless steel tanks and piping are automatically cleaned-in-place. The high degree of automation and cleaning provides efficient operation and sanitary processing. Chromatographic methods (DEAE-Sepharose and metal chelation) are also being used at the pilot scale to purify the human blood products superoxide dismutase and hemoglobin from outdated red blood cells. Characterization of the protein fractions produced by chromatography has shown them to be of equal or higher quality than fractions produced by other techniques.

  10. The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

    PubMed

    Lesot, H; Fausser, J L; Akiyama, S K; Staub, A; Black, D; Kubler, M D; Ruch, J V

    1992-03-01

    Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation. PMID:1597256

  11. Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

    SciTech Connect

    Tan, Wen Siang; McNae, Iain W.; Ho, Kok Lian; Walkinshaw, Malcolm D.

    2007-08-01

    Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was found to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.

  12. Extensive protein hydrolysate formula effectively reduces regurgitation in infants with positive and negative challenge tests for cow’s milk allergy

    PubMed Central

    Vandenplas, Y; De Greef, E

    2014-01-01

    Aim 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. Methods 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. Results 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). Conclusion 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. PMID:24575806

  13. Endothelial Cell Sensitization by Death Receptor Fractions of an Anti-Dengue Nonstructural Protein 1 Antibody Induced Plasma Leakage, Coagulopathy, and Mortality in Mice.

    PubMed

    Sun, Der-Shan; Chang, Ying-Chen; Lien, Te-Sheng; King, Chwan-Chuen; Shih, Yung-Luen; Huang, Hsuan-Shun; Wang, Teng-Yi; Li, Chen-Ru; Lee, Chin-Cheng; Hsu, Ping-Ning; Chang, Hsin-Hou

    2015-09-15

    The mechanisms leading to the life-threatening dengue hemorrhagic fever (DHF) remain elusive. DHF preferentially occurs during secondary dengue infections, suggesting that aberrant immune responses are involved in its development. We previously demonstrated that the autoantibodies elicited by dengue virus (DENV) nonstructural protein 1 (NS1; anti-NS1 Igs) induce plasma leakage and mortality in mice with warfarinized anticoagulant suppression. However, the involved pathogenic Ig fractions of anti-NS1 Igs remain unclear. In this study, the autoreactive Igs in patients with DHF and in NS1-immunized rabbits crossreacted with TNF-related apoptosis-inducing ligand receptor 1 (death receptor [DR]4). Challenges with the DENV in a subcytotoxic dose sensitized endothelial cells to apoptosis. Treatments with the autoantibodies induced proapoptotic activities and suppressed the surface expression of endothelial anticoagulant thrombomodulin. Combined treatments comprising the DENV and DR4 affinity-purified fractions of anti-NS1 IgGs (anti-NS1-DR4 Ig), but not preimmune control IgGs, in subcytotoxic doses led to apoptosis in endothelial cells. Treatments with the anti-NS1-DR4 Ig led to plasma leakage, coagulopathy, and morality in mice with warfarinized anticoagulant suppression. These results suggest that DR4-induced endothelial cell sensitization through NS1-elicited autoantibodies exacerbates anticoagulant suppression, vascular injury, and plasma leakage. Detecting and blocking anti-DR Igs in patients may be novel strategies for managing severe DENV infection. PMID:26259584

  14. Endothelial Cell Sensitization by Death Receptor Fractions of an Anti-Dengue Nonstructural Protein 1 Antibody Induced Plasma Leakage, Coagulopathy, and Mortality in Mice.

    PubMed

    Sun, Der-Shan; Chang, Ying-Chen; Lien, Te-Sheng; King, Chwan-Chuen; Shih, Yung-Luen; Huang, Hsuan-Shun; Wang, Teng-Yi; Li, Chen-Ru; Lee, Chin-Cheng; Hsu, Ping-Ning; Chang, Hsin-Hou

    2015-09-15

    The mechanisms leading to the life-threatening dengue hemorrhagic fever (DHF) remain elusive. DHF preferentially occurs during secondary dengue infections, suggesting that aberrant immune responses are involved in its development. We previously demonstrated that the autoantibodies elicited by dengue virus (DENV) nonstructural protein 1 (NS1; anti-NS1 Igs) induce plasma leakage and mortality in mice with warfarinized anticoagulant suppression. However, the involved pathogenic Ig fractions of anti-NS1 Igs remain unclear. In this study, the autoreactive Igs in patients with DHF and in NS1-immunized rabbits crossreacted with TNF-related apoptosis-inducing ligand receptor 1 (death receptor [DR]4). Challenges with the DENV in a subcytotoxic dose sensitized endothelial cells to apoptosis. Treatments with the autoantibodies induced proapoptotic activities and suppressed the surface expression of endothelial anticoagulant thrombomodulin. Combined treatments comprising the DENV and DR4 affinity-purified fractions of anti-NS1 IgGs (anti-NS1-DR4 Ig), but not preimmune control IgGs, in subcytotoxic doses led to apoptosis in endothelial cells. Treatments with the anti-NS1-DR4 Ig led to plasma leakage, coagulopathy, and morality in mice with warfarinized anticoagulant suppression. These results suggest that DR4-induced endothelial cell sensitization through NS1-elicited autoantibodies exacerbates anticoagulant suppression, vascular injury, and plasma leakage. Detecting and blocking anti-DR Igs in patients may be novel strategies for managing severe DENV infection.

  15. Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

    PubMed Central

    Shcherbakova, Larisa A.; Odintsova, Tatyana I.; Stakheev, Alexander A.; Fravel, Deborah R.; Zavriev, Sergey K.

    2016-01-01

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed. PMID:26779237

  16. Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis.

    PubMed

    Belanche, A; de la Fuente, G; Pinloche, E; Newbold, C J; Balcells, J

    2012-11-01

    Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

  17. Safety evaluation of a whey protein fraction containing a concentrated amount of naturally occurring TGF-β2.

    PubMed

    Forster, Roy; Bourtourault, Michel; Chung, Yong Joo; Silvano, Jérémy; Sire, Guillaume; Spezia, François; Puel, Caroline; Descotes, Jacques; Mikogami, Takashi

    2014-08-01

    TM0601p is a whey protein isolate derived from cow milk, containing a concentrated amount of transforming growth factor β2 (TGF-β2), and is intended for nutritional use in infants and adults. In vivo and in vitro studies have been performed to evaluate the safety of this product. In a 13-week toxicity study, treatment of adult Sprague-Dawley rats by gavage at up to 2000mg/kg/day did not result in any significant findings other than minor non-adverse changes in urinary parameters in females. The no-observed-adverse-effect level (NOAEL) was established as 2000mg/kg/day. In a juvenile toxicity study, rat pups received 600mg/kg/day by gavage from postnatal day (PND) 7 to PND 49. Transient lower bodyweight gain in the pre-weaning period was attributed to gastrointestinal effects of the viscous test material; following weaning, bodyweight gain was comparable to the vehicle controls. Reduced eosinophil counts and changes in urinary parameters (females) were recorded in treated pups at PND 49, and higher thymus weights were recorded in males only at the end of the recovery period (Day 77). None of the findings were considered adverse. There were no other significant findings and the NOAEL was established as 600mg/kg/day. No evidence of genotoxicity was seen in the bacterial reverse mutation test or the in vitro micronucleus test. Overall the results obtained present a reassuring safety profile for TM0601p. PMID:24842704

  18. Herbal adaptogens combined with protein fractions from bovine colostrum and hen egg yolk reduce liver TNF-α expression and protein carbonylation in Western diet feeding in rats

    PubMed Central

    2014-01-01

    Background We examined if a purported anti-inflammatory supplement (AF) abrogated Western-diet (WD)-induced liver pathology in rats. AF contained: 1) protein concentrates from bovine colostrum and avian egg yolk; 2) herbal adaptogens and antioxidants; and 3) acetyl-L-carnitine. Methods Nine month-old male Brown Norway rats were allowed ad libitum access to WD for 41–43 days and randomly assigned to WD + AF feeding twice daily for the last 31–33 days (n = 8), or WD and water-placebo feeding twice daily for the last 31–33 days (n = 8). Rats fed a low-fat/low-sucrose diet (CTL, n = 6) for 41–43 days and administered a water-placebo twice daily for the last 31–33 days were also studied. Twenty-four hours following the last gavage-feed, liver samples were analyzed for: a) select mRNAs (via RT-PCR) as well as genome-wide mRNA expression patterns (via RNA-seq); b) lipid deposition; and, c) protein carbonyl and total antioxidant capacity (TAC). Serum was also examined for TAC, 8-isoprostane and clinical chemistry markers. Results WD + AF rats experienced a reduction in liver Tnf-α mRNA (-2.8-fold, p < 0.01). Serum and liver TAC was lower in WD + AF versus WD and CTL rats (p < 0.05), likely due to exogenous antioxidant ingredients provided through AF as evidenced by a tendency for mitochondrial SOD2 mRNA to increase in WD + AF versus CTL rats (p = 0.07). Liver fat deposition nor liver protein carbonyl content differed between WD + AF versus WD rats, although liver protein carbonyls tended to be lower in WD + AF versus CTL rats (p = 0.08). RNA-seq revealed that 19 liver mRNAs differed between WD + AF versus WD when both groups were compared with CTL rats (+/- 1.5-fold, p < 0.01). Bioinformatics suggest that AF prevented WD-induced alterations in select genes related to the transport and metabolism of carbohydrates in favor of select genes related to lipid transport and metabolism. Finally, serum clinical

  19. Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension.

    PubMed

    Simon, Manuel; Frey, Raphael; Zangemeister-Wittke, Uwe; Plückthun, Andreas

    2013-11-20

    The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

  20. The C-terminal extension peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs) affects their solubility and stability: comparative analyses of the biochemical and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs with or without their C-terminal extension peptides.

    PubMed

    Takahashi, Shigekazu; Uchida, Akira; Nakayama, Katsumi; Satoh, Hiroyuki

    2014-02-01

    Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.

  1. Fractionation of thylakoid membranes from Porphyridium purpureum using the detergent N-lauryl-β-iminodipropionate : A study on the chlorophyll-protein and pigment composition of the membrane-intrinsic antenna complexes of a red alga.

    PubMed

    Marquardt, J; Ried, A

    1992-06-01

    Two green fractions, thought to represent the chlorophyll-antennae of photosystems I (PSI) and II (PSII), were isolated from the red alga Porphyridium purpureum by solubilisation of the thylakoid membranes using the detergent N-lauryl-ß-iminodipropionate and subsequent sucrose-density-gradient centrifugation. No release of pigments from the pigment-protein complexes was detected during isolation. The fractions were analyzed with respect to their chlorophyll-protein pattern, spectral properties and pigment composition. The supposed PSII antenna fraction contained both the major carotenoids of P. purpureum, β-carotene and zeaxanthin, and showed a long-wavelength absorption maximum at 672 nm and a low-temperature fluorescence maximum at 692-694 nm. Polypeptides of this fraction cross-reacted with antibodies raised against the PSII polypeptides D1, CP43 and CP47 from higher plants. The PSI fraction could perform P-700 photooxidation and showed a long-wavelength absorbance maximum at 679 nm and a low-temperature fluorescence maximum at 718 nm. It contained β-carotene as the only carotenoid. The fluorescence excitation spectrum of the fraction and measurements of the photochemical activity of a thylakoid preparation excited with light that is preferentially absorbed either by chlorophyll (433 nm) or by carotenoids (495 nm) indicate that β-carotene serves as a very efficient antenna-pigment in PSI. In contrast, only a small amount of energy transfer from the carotenoids to chlorophyll could be observed with the supposed PSII fraction.

  2. AGRICULTURAL EXTENSION.

    ERIC Educational Resources Information Center

    FARQUHAR, R.N.

    AUSTRALIAN AGRICULTURAL EXTENSION HAS LONG EMPHASIZED TECHNICAL ADVISORY SERVICE AT THE EXPENSE OF THE SOCIOECONOMIC ASPECTS OF FARM PRODUCTION AND FARM LIFE. ONLY IN TASMANIA HAS FARM MANAGEMENT BEEN STRESSED. DEMANDS FOR THE WHOLE-FARM APPROACH HAVE PRODUCED A TREND TOWARD GENERALISM FOR DISTRICT OFFICERS IN MOST STATES. THE FEDERAL GOVERNMENT,…

  3. Effects of spontaneous heating on forage protein fractions and in situ disappearance kinetics of crude protein for alfalfa-orchardgrass hays packaged in large round bales.

    PubMed

    Coblentz, W K; Hoffman, P C; Martin, N P

    2010-03-01

    During 2006 and 2007, forages from 3 individual hay harvests were used to assess the effects of spontaneous heating on concentrations of crude protein (CP), neutral detergent insoluble CP (NDICP), acid detergent insoluble CP (ADICP), and in situ disappearance kinetics of CP and NDICP for large round bales of mixed alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.). Over the 3 harvests, 96 large round bales were made at preset bale diameters of 0.9, 1.2, or 1.5m and at moisture concentrations ranging from 9.3 to 46.6%. Internal bale temperatures were monitored daily during an outdoor storage period. The change in concentrations of NDICP (poststorage - prestorage) increased with heating degree days (HDD) >30 degrees C in a relationship best explained with a nonlinear model {Y=24.9 - [22.7 x (e(-0.000010 x x x x))]; R(2)=0.892} that became asymptotic at +24.9 percentage units of CP, thereby indicating that NDICP increases rapidly within bales that heat spontaneously. When maximum internal bale temperature (MAX) was used as the independent variable, the best regression model was quadratic and the coefficient of determination was still relatively high (R(2)=0.716). The change in concentrations of ADICP (poststorage - prestorage; DeltaADICP) also increased with HDD and was best fitted to a nonlinear model {Y=14.9 - [15.7 x (e(-0.0000019 x x x x))]} with a very high coefficient of determination (R(2)=0.934). A similar quartic response was observed for the regression of DeltaADICP on MAX (R(2)=0.975). Increases in DeltaADICP as a result of heating (HDD or MAX) were paralleled by concurrent increases in hemicellulose at relatively low increments of heating, but the inverse relationship was observed as hemicelluloses likely became reactive and concentrations decreased in more severely heated hays. Changes in ruminal disappearance rate of CP were best fitted to cubic models for regressions on both HDD (R(2)=0.939) and MAX (R(2)=0.876); these changes

  4. Radiating subdispersive fractional optical solitons

    SciTech Connect

    Fujioka, J. Espinosa, A.; Rodríguez, R. F.; Malomed, B. A.

    2014-09-01

    It was recently found [Fujioka et al., Phys. Lett. A 374, 1126 (2010)] that the propagation of solitary waves can be described by a fractional extension of the nonlinear Schrödinger (NLS) equation which involves a temporal fractional derivative (TFD) of order α > 2. In the present paper, we show that there is also another fractional extension of the NLS equation which contains a TFD with α < 2, and in this case, the new equation describes the propagation of radiating solitons. We show that the emission of the radiation (when α < 2) is explained by resonances at various frequencies between the pulses and the linear modes of the system. It is found that the new fractional NLS equation can be derived from a suitable Lagrangian density, and a fractional Noether's theorem can be applied to it, thus predicting the conservation of the Hamiltonian, momentum and energy.

  5. Beef Species Symposium: an assessment of the 1996 Beef NRC: metabolizable protein supply and demand and effectiveness of model performance prediction of beef females within extensive grazing systems.

    PubMed

    Waterman, R C; Caton, J S; Löest, C A; Petersen, M K; Roberts, A J

    2014-07-01

    Interannual variation of forage quantity and quality driven by precipitation events influence beef livestock production systems within the Southern and Northern Plains and Pacific West, which combined represent 60% (approximately 17.5 million) of the total beef cows in the United States. The beef cattle requirements published by the NRC are an important tool and excellent resource for both professionals and producers to use when implementing feeding practices and nutritional programs within the various production systems. The objectives of this paper include evaluation of the 1996 Beef NRC model in terms of effectiveness in predicting extensive range beef cow performance within arid and semiarid environments using available data sets, identifying model inefficiencies that could be refined to improve the precision of predicting protein supply and demand for range beef cows, and last, providing recommendations for future areas of research. An important addition to the current Beef NRC model would be to allow users to provide region-specific forage characteristics and the ability to describe supplement composition, amount, and delivery frequency. Beef NRC models would then need to be modified to account for the N recycling that occurs throughout a supplementation interval and the impact that this would have on microbial efficiency and microbial protein supply. The Beef NRC should also consider the role of ruminal and postruminal supply and demand of specific limiting AA. Additional considerations should include the partitioning effects of nitrogenous compounds under different physiological production stages (e.g., lactation, pregnancy, and periods of BW loss). The intent of information provided is to aid revision of the Beef NRC by providing supporting material for changes and identifying gaps in existing scientific literature where future research is needed to enhance the predictive precision and application of the Beef NRC models. PMID:24398839

  6. Xenopus laevis nucleotide binding protein 1 (xNubp1) is important for convergent extension movements and controls ciliogenesis via regulation of the actin cytoskeleton.

    PubMed

    Ioannou, Andriani; Santama, Niovi; Skourides, Paris A

    2013-08-15

    Nucleotide binding protein 1 (Nubp1) is a highly conserved phosphate loop (P-loop) ATPase involved in diverse processes including iron-sulfur protein assembly, centrosome duplication and lung development. Here, we report the cloning, expression and functional characterization of Xenopus laevis Nubp1. We show that xNubp1 is expressed maternally, displays elevated expression in neural tissues and is required for convergent extension movements and neural tube closure. In addition, xNubp1knockdown leads to defective ciliogenesis of the multi-ciliated cells of the epidermis as well as the monociliated cells of the gastrocoel roof plate. Specifically, xNubp1 is required for basal body migration, spacing and docking in multi-ciliated cells and basal body positioning and axoneme elongation in monociliated gastrocoel roof plate cells. Live imaging of the different pools of actin and basal body migration during the process of ciliated cell intercalation revealed that two independent pools of actin are present from the onset of cell intercalation; an internal network surrounding the basal bodies, anchoring them to the cell cortex and an apical pool of punctate actin which eventually matures into the characteristic apical actin network. We show that xNubp1 colocalizes with the apical actin network of multiciliated cells and that problems in basal body transport in xNubp1 morphants are associated with defects of the internal network of actin, while spacing and polarity issues are due to a failure of the apical and sub-apical actin pools to mature into a network. Effects of xNubp1 knockdown on the actin cytoskeleton are independent of RhoA localization and activation, suggesting that xNubp1 may have a direct role in the regulation of the actin cytoskeleton.

  7. Structural and Temporal Requirements of Wnt/PCP Protein Vangl2 Function for Convergence and Extension Movements and Facial Branchiomotor Neuron Migration in Zebrafish

    PubMed Central

    Pan, Xiufang; Sittaramane, Vinoth; Gurung, Suman; Chandrasekhar, Anand

    2014-01-01

    Van gogh-like 2 (Vangl2), a core component of the Wnt/Planar Cell Polarity (PCP) signaling pathway, is a four-pass transmembrane protein with N-terminal and C-terminal domains located in the cytosol, and is structurally conserved from flies to mammals. In vertebrates, Vangl2 plays an essential role in convergence and extension (CE) movements during gastrulation and in facial branchiomotor (FBM) neuron migration in the hindbrain. However, the roles of specific Vangl2 domains, of membrane association, and of specific extracellular and intracellular motifs have not been examined, especially in the context of FBM neuron migration. Through heat shock-inducible expression of various Vangl2 transgenes, we found that membrane associated functions of the N-terminal and C-terminal domains of Vangl2 are involved in regulating FBM neuron migration. Importantly, through temperature shift experiments, we found that the critical period for Vangl2 function coincides with the initial stages of FBM neuron migration out of rhombomere 4. Intriguingly, we have also uncovered a putative nuclear localization motif in the C-terminal domain that may play a role in regulating CE movements. PMID:24333599

  8. The effect of high and low levels of supplementation on milk production, nitrogen utilization efficiency, and milk protein fractions in late-lactation dairy cows.

    PubMed

    Reid, M; O'Donovan, M; Murphy, J P; Fleming, C; Kennedy, E; Lewis, E

    2015-08-01

    To fill the feed deficit in the autumn/late lactation period in a seasonal grazing system, supplementation is required. This study aimed to investigate the use of baled grass silage or concentrate as supplementation to grazing dairy cows in late lactation. Eighty-four grass-based spring-calving dairy cows, averaging 212d in milk, were allocated to 1 of 6 treatments [high grass allowance (HG), low grass allowance (LG), grass with a low concentrate allocation (GCL), grass with a low grass silage allocation (GSL), grass with a high concentrate allocation (GCH), and grass with a high grass silage allocation (GSH)] to measure the effects of using baled grass silage or concentrate as supplements to grazed grass. Effects on intake, milk yield, milk composition and N fractions, and N utilization efficiency were measured. Treatments HG and LG received 17 and 14kg of dry matter (DM) grass/cow per d, respectively. Treatments GCL and GSL were offered 14kg of DM grass/cow per d and 3kg of DM of supplementation/cow per d. Treatments GCH and GSH were offered 11kg of DM grass/cow per d and 6kg of DM of supplementation/cow per d. Milk yield was greatest in the GCH treatment and milk solids yield was greatest in both concentrate-supplemented treatments. The HG and LG treatments excreted a greater quantity of N as a proportion of N intake than the supplemented treatments. The HG treatment also excreted the greatest total quantity of N. This indicates an improvement in N utilization efficiency when supplementation is offered compared with grazing only. Offering 6kg of DM of either grass silage or concentrate as supplementation decreased milk true protein concentration compared with offering a grass-only diet. This suggests that increasing the proportion of supplementation relative to grass may negatively affect milk processability, which is associated with milk true protein concentration.

  9. Insoluble fraction of buckwheat (Fagopyrum esculentum Moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by Caco-2 cells.

    PubMed

    Metzger, Brandon T; Barnes, David M; Reed, Jess D

    2007-07-25

    Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.

  10. Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica.

    PubMed

    Ai, Hui; Wang, Furong; Zhang, Na; Zhang, Lingyao; Lei, Chaoliang

    2013-01-01

    In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents.

  11. Lyapunov functions for fractional order systems

    NASA Astrophysics Data System (ADS)

    Aguila-Camacho, Norelys; Duarte-Mermoud, Manuel A.; Gallegos, Javier A.

    2014-09-01

    A new lemma for the Caputo fractional derivatives, when 0<α<1, is proposed in this paper. This result has proved to be useful in order to apply the fractional-order extension of Lyapunov direct method, to demonstrate the stability of many fractional order systems, which can be nonlinear and time varying.

  12. Fraction Reduction through Continued Fractions

    ERIC Educational Resources Information Center

    Carley, Holly

    2011-01-01

    This article presents a method of reducing fractions without factoring. The ideas presented may be useful as a project for motivated students in an undergraduate number theory course. The discussion is related to the Euclidean Algorithm and its variations may lead to projects or early examples involving efficiency of an algorithm.

  13. Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Ikeda, Nobuhito; Nakayama, Yuji; Nakazawa, Natsumi; Yoshida, Akio; Ninomiya, Haruaki; Shirayoshi, Yasuaki

    2016-01-01

    Background The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP+ cells include undifferentiated cells with a capacity to develop into tumors. Methods PrP+ cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT–PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5GFP/+ (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. Results PrP+ cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP+ cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP+ cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP+ cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP+/SSEA1– cells did not proliferate and expressed cardiac cell markers, while PrP+/SSEA1+ did proliferate. Conclusion PrP+ cells isolated from EB included undifferentiated cells in day 21. PrP+/SSEA1– cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes. PMID:27493483

  14. Biorefining of wheat straw using an acetic and formic acid based organosolv fractionation process.

    PubMed

    Snelders, Jeroen; Dornez, Emmie; Benjelloun-Mlayah, Bouchra; Huijgen, Wouter J J; de Wild, Paul J; Gosselink, Richard J A; Gerritsma, Jort; Courtin, Christophe M

    2014-03-01

    To assess the potential of acetic and formic acid organosolv fractionation of wheat straw as basis of an integral biorefinery concept, detailed knowledge on yield, composition and purity of the obtained streams is needed. Therefore, the process was performed, all fractions extensively characterized and the mass balance studied. Cellulose pulp yield was 48% of straw dry matter, while it was 21% and 27% for the lignin and hemicellulose-rich fractions. Composition analysis showed that 67% of wheat straw xylan and 96% of lignin were solubilized during the process, resulting in cellulose pulp of 63% purity, containing 93% of wheat straw cellulose. The isolated lignin fraction contained 84% of initial lignin and had a purity of 78%. A good part of wheat straw xylan (58%) ended up in the hemicellulose-rich fraction, half of it as monomeric xylose, together with proteins (44%), minerals (69%) and noticeable amounts of acids used during processing.

  15. Fractional factorial approach combining 4 Escherichia coli strains, 3 culture media, 3 expression temperatures and 5 N-terminal fusion tags for screening the soluble expression of recombinant proteins.

    PubMed

    Noguère, Christophe; Larsson, Anna M; Guyot, Jean-Christophe; Bignon, Christophe

    2012-08-01

    Producing recombinant proteins in Escherichia coli (E. coli) is generally performed using a trial and error approach with the different expression variables being tested independently from each other. As a consequence, variable interactions are lost which makes the trial and error approach quite time-consuming. In this paper, we report how switching from a trial and error to a fractional factorial approach allows testing in less than 2 weeks four expression variables (E. coli strains, culture media, expression temperatures and N-terminal fusion tags) in a single experiment. The method, called "Fusion-InFFact", was validated using four test proteins. In all cases, Fusion-InFFact allowed finding conditions for expressing high yields of soluble proteins. The method was originally set-up for high throughput structural genomics programs, but can be used in any recombinant protein expression project. PMID:22705765

  16. Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

    PubMed

    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

    2015-09-01

    Despite significant efforts in the past decade toward 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 nonsmall 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 prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was 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 using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

  17. Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence

    PubMed Central

    Mosnier, Amandine; Hologne, Maggy; Martin, Amandine; Lindgren, Lena; Punginelli, Claire; Lays, Claire; Walker, Olivier; Charbit, Alain; Telouk, Philippe; Conlan, Wayne; Terradot, Laurent; Sjöstedt, Anders; Henry, Thomas

    2016-01-01

    The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF. PMID:27602570

  18. Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence.

    PubMed

    Rigard, Mélanie; Bröms, Jeanette E; Mosnier, Amandine; Hologne, Maggy; Martin, Amandine; Lindgren, Lena; Punginelli, Claire; Lays, Claire; Walker, Olivier; Charbit, Alain; Telouk, Philippe; Conlan, Wayne; Terradot, Laurent; Sjöstedt, Anders; Henry, Thomas

    2016-09-01

    The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF. PMID:27602570

  19. Extensive nonadditivity of privacy.

    PubMed

    Smith, Graeme; Smolin, John A

    2009-09-18

    Quantum information theory establishes the ultimate limits on communication and cryptography in terms of channel capacities for various types of information. The private capacity is particularly important because it quantifies achievable rates of quantum key distribution. We study the power of quantum channels with limited private capacity, focusing on channels that dephase in random bases. These display extensive nonadditivity of private capacity: a channel with 2logd input qubits that has a private capacity less than 2, but when used together with a second channel with zero private capacity, the joint capacity jumps to (1/2)logd. In contrast to earlier work which found nonadditivity vanishing as a fraction of input size or conditional on unproven mathematical assumptions, this provides a natural setting manifesting nonadditivity of privacy of the strongest possible sort. PMID:19792417

  20. Non-extensive radiobiology

    SciTech Connect

    Sotolongo-Grau, O.; Rodriguez-Perez, D.; Antoranz, J. C.; Sotolongo-Costa, O.

    2011-03-14

    The expression of survival factors for radiation damaged cells is based on probabilistic assumptions and experimentally fitted for each tumor, radiation and conditions. Here we show how the simplest of these radiobiological models can be derived from the maximum entropy principle of the classical Boltzmann-Gibbs expression. We extend this derivation using the Tsallis entropy and a cutoff hypothesis, motivated by clinical observations. A generalization of the exponential, the logarithm and the product to a non-extensive framework, provides a simple formula for the survival fraction corresponding to the application of several radiation doses on a living tissue. The obtained expression shows a remarkable agreement with the experimental data found in the literature, also providing a new interpretation of some of the parameters introduced anew. It is also shown how the presented formalism may have direct application in radiotherapy treatment optimization through the definition of the potential effect difference, simply calculated between the tumour and the surrounding tissue.

  1. Non-extensive radiobiology

    NASA Astrophysics Data System (ADS)

    Sotolongo-Grau, O.; Rodriguez-Perez, D.; Antoranz, J. C.; Sotolongo-Costa, O.

    2011-03-01

    The expression of survival factors for radiation damaged cells is based on probabilistic assumptions and experimentally fitted for each tumor, radiation and conditions. Here we show how the simplest of these radiobiological models can be derived from the maximum entropy principle of the classical Boltzmann-Gibbs expression. We extend this derivation using the Tsallis entropy and a cutoff hypothesis, motivated by clinical observations. A generalization of the exponential, the logarithm and the product to a non-extensive framework, provides a simple formula for the survival fraction corresponding to the application of several radiation doses on a living tissue. The obtained expression shows a remarkable agreement with the experimental data found in the literature, also providing a new interpretation of some of the parameters introduced anew. It is also shown how the presented formalism may have direct application in radiotherapy treatment optimization through the definition of the potential effect difference, simply calculated between the tumour and the surrounding tissue.

  2. Wheat streak mosaic virus infects systemically despite extensive coat protein deletions: identification of virion assembly and cell-to-cell movement determinants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral coat proteins function in virion assembly and virus biology in a tightly coordinated manner with a role for virtually every amino acid. In this study, we demonstrated that the coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus; family Potyviridae) is unusually tolerant o...

  3. The use of 99mTc-HYNIC-TOC and 18F-FDG PET/CT in the evaluation of duodenal neuroendocrine tumor with atypical and extensive metastasis responding dramatically to a single fraction of PRRT with 177Lu-DOTATATE.

    PubMed

    Basu, Sandip; Abhyankar, Amit

    2014-12-01

    This report describes a case of extensive diffuse bone marrow involvement with bilateral breast metastases from duodenal neuroendocrine tumor giving rise to a superscan-like appearance on somatostatin receptor-targeted (99m)Tc-hydrazinonicotinamide-TOC scintigraphy. The metastatic lesions demonstrated partial concordance with (18)F-FDG PET/CT findings, signifying varying tumor biology and heterogeneity among metastatic lesions in the same individual, as illustrated with a dual-tracer approach. There was a dramatic symptomatic and biochemical response and better health-related quality of life with a single fraction of peptide receptor radionuclide therapy with (177)Lu-DOTATATE, and radiologically there was stable disease at that point.

  4. Adaptive fractionation therapy: II. Biological effective dose

    NASA Astrophysics Data System (ADS)

    Chen, Mingli; Lu, Weiguo; Chen, Quan; Ruchala, Kenneth; Olivera, Gustavo

    2008-10-01

    Radiation therapy is fractionized to differentiate the cell killing between the tumor and organ at risk (OAR). Conventionally, fractionation is done by dividing the total dose into equal fraction sizes. However, as the relative positions (configurations) between OAR and the tumor vary from fractions to fractions, intuitively, we want to use a larger fraction size when OAR and the tumor are far apart and a smaller fraction size when OAR and the tumor are close to each other. Adaptive fractionation accounts for variations of configurations between OAR and the tumor. In part I of this series, the adaptation minimizes the OAR (physical) dose and maintains the total tumor (physical) dose. In this work, instead, the adaptation is based on the biological effective dose (BED). Unlike the linear programming approach in part I, we build a fraction size lookup table using mathematical induction. The lookup table essentially describes the fraction size as a function of the remaining tumor BED, the OAR/tumor dose ratio and the remaining number of fractions. The lookup table is calculated by maximizing the expected survival of OAR and preserving the tumor cell kill. Immediately before the treatment of each fraction, the OAR-tumor configuration and thus the dose ratio can be obtained from the daily setup image, and then the fraction size can be determined by the lookup table. Extensive simulations demonstrate the effectiveness of our method compared with the conventional fractionation method.

  5. Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis.

    PubMed

    de Jesus Pereira, Nathália Cristina; Régis, Wiliam César Bento; Costa, Lourena Emanuele; de Oliveira, Jamil Silvano; da Silva, Alanna Gomes; Martins, Vivian Tamietti; Duarte, Mariana Costa; de Souza, José Roberto Rodrigues; Lage, Paula Sousa; Schneider, Mônica Santos; Melo, Maria Norma; Soto, Manuel; Soares, Sandra Aguiar; Tavares, Carlos Alberto Pereira; Chávez-Fumagalli, Miguel Angel; Coelho, Eduardo Antonio Ferraz

    2015-06-01

    The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the

  6. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

    PubMed Central

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H.; Gutiérrez, Andrés H.; Rueda, Luis D.; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H.; Sheen, Patricia

    2011-01-01

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection. PMID:22119017

  7. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs.

    PubMed

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H; Gutiérrez, Andrés H; Rueda, Luis D; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H; Sheen, Patricia

    2012-01-15

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.

  8. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    PubMed

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P<0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition.

  9. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    PubMed

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P<0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  10. Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation1

    PubMed Central

    Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-01-01

    Germination and thermoinhibition in lettuce (Lactuca sativa ‘Jianyexianfeng No. 1’) seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P < 0.05) in abundance in at least one germination treatment. Nineteen proteins increasing and one protein decreasing in abundance during germination had higher abundance in germinated 15°C, 15°C after imbibition at 25°C for 48 h, and 25°C in KNO3 seeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  11. Assessment of the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of hydrolysates of bovine brisket sarcoplasmic proteins produced by papain and characterisation of associated bioactive peptidic fractions.

    PubMed

    Di Bernardini, Roberta; Mullen, Anne Maria; Bolton, Declan; Kerry, Joseph; O'Neill, Eileen; Hayes, Maria

    2012-01-01

    The main objective was to investigate the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of sarcoplasmic proteins isolated from the brisket muscle (Pectoralis profundus) of 3 (Bos taurus) cattle and hydrolysed with papain for 24 h at 37°C. Sarcoplasmic protein hydrolysates were ultra-filtered using molecular weight cut off (MWCO) membranes and 10-kDa and 3-kDa filtrates were obtained. The total sarcoplasmic protein extracts and the 3-kDa filtrates were tested for angiotensin I-converting enzyme inhibitory (ACE-I) activities. The total hydrolysates, 10-kDa and 3-kDa filtrates were also tested for their associated antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe(2+) metal chelating ability assay. The peptidic content of the total hydrolysates, the 10-kDa and the 3-kDa filtrates were analysed using an ORBITRAP mass spectrometer, and mass spectral data obtained were analysed using TurboSEQUEST. Eleven peptides were characterised from the total hydrolysates, fifteen from the 10-kDa filtrate fractions, whilst nine peptides were characterised from the 3-kDa filtrate fractions. Similarities between the amino acid sequences of the peptides identified in this study and previously identified antioxidant and ACE-I inhibitory peptides detailed in the BIOPEP database were outlined. PMID:21880436

  12. The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress

    PubMed Central

    Johnson, Kristen A.; Taghon, Geoffrey J. F.; Scott, Jordan L.; Stahelin, Robert V.

    2016-01-01

    VP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly at the plasma membrane and subsequent virus like particle formation. Additionally, PI(4,5)P2 was required for formation of extensive oligomers of VP40, suggesting PS and PI(4,5)P2 have different roles in VP40 assembly where PS regulates formation of hexamers from VP40 dimers and PI(4,5)P2 stabilizes and/or induces extensive VP40 oligomerization at the plasma membrane. PMID:26753796

  13. Interaction of modified tail-anchored proteins with liposomes: effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b₅.

    PubMed

    Sakamoto, Yoichi; Miura, Masahiro; Takeuchi, Fusako; Park, Sam-Yong; Tsubaki, Motonari

    2012-03-01

    A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b(5) (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH(2)-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial liposomes.

  14. Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins.

    PubMed

    Rahman, Safikur; Ali, Syed Ausaf; Islam, Asimul; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2016-02-01

    Here we have generated two different denatured states using heat- and guanidinium chloride (GdmCl)-induced denaturations of three disulfide bond free proteins (barstar, cytochrome-c and myoglobin). We have observed that these two denatured states of barstar and myoglobin are structurally and energetically different, for, heat-induced denatured state contains many un-melted residual structure that has a significant amount of secondary and tertiary interactions. We show that structural properties of the denatured state determine the magnitude of the protein stabilization in terms of Gibbs free energy change (ΔGD°) induced by an osmolyte, i.e., the greater the exposed surface area, the greater is the stabilization. Furthermore, we predicted the m-values (ability of osmolyte to fold or unfold proteins) using Tanford's transfer-free energy model for the transfer of proteins to osmolyte solutions. We observed that, for each protein, m-value is comparable with our experimental data in cases of TMAO (trimethylamine-N-oxide) and sarcosine. However, a significant discrepancy between predicted and experimental m-values were observed in the case of glycine-betaine.

  15. Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes.

    PubMed

    Hresko, Richard C; Murata, Haruhiko; Mueckler, Mike

    2003-06-13

    By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt. PMID:12682057

  16. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  17. Matrix fractional systems

    NASA Astrophysics Data System (ADS)

    Tenreiro Machado, J. A.

    2015-08-01

    This paper addresses the matrix representation of dynamical systems in the perspective of fractional calculus. Fractional elements and fractional systems are interpreted under the light of the classical Cole-Cole, Davidson-Cole, and Havriliak-Negami heuristic models. Numerical simulations for an electrical circuit enlighten the results for matrix based models and high fractional orders. The conclusions clarify the distinction between fractional elements and fractional systems.

  18. Pasta supplemented with isolated lupin protein fractions reduces body weight gain and food intake of rats and decreases plasma glucose concentration upon glucose overload trial.

    PubMed

    Capraro, Jessica; Magni, Chiara; Scarafoni, Alessio; Caramanico, Rosita; Rossi, Filippo; Morlacchini, Mauro; Duranti, Marcello

    2014-02-01

    The supplementation of foods with biologically active compounds can be a powerful approach for improving diet and well being. In this study we separately included in pasta matrices a concentrate of γ-conglutin, a glucose-lowering protein from Lupinus albus seeds, an isolate of the other main lupin storage proteins and ovalbumin, at a ratio corresponding to 125 mg of pure protein in 100 g of pasta. With these products we fed rats made hyperglycaemic, for 3 weeks. Among the most relevant changes measured in body and blood parameters were: (i) a significant reduction in food intake of rats fed γ-conglutin concentrate supplemented pasta and a significant limitation in the body weight increase in rats fed α, β and δ-conglutin isolate supplemented pasta, while the food conversion indices were unchanged; (ii) a reduction in glycaemia upon glucose overload trial, especially in the γ-conglutin concentrate supplemented pasta fed animals, at a dose of 45 mg per kg body weight. The correlations among the measured parameters are discussed. Overall, the results evidence the potentiality of supplementing traditional foods with exogenous nutraceutical seed proteins to control body weight gain and glycaemia.

  19. X-ray crystal structures of Escherichia coli RNA polymerase with switch region binding inhibitors enable rational design of squaramides with an improved fraction unbound to human plasma protein

    PubMed Central

    Molodtsov, Vadim; Fleming, Paul R.; Eyermann, Charles J.; Ferguson, Andrew D.; Foulk, Melinda A.; McKinney, David C.; Masse, Craig E.; Buurman, Ed T.; Murakami, Katsuhiko S.

    2015-01-01

    Squaramides constitute a novel class of RNA polymerase inhibitors of which genetic evidence and computational modeling previously have suggested an inhibitory mechanism mediated by binding to the RNA polymerase switch region. An iterative chemistry program increased the fraction unbound to human plasma protein from below minimum detection levels, i.e. <1%, to 4~6%, while retaining biochemical potency. Since in vitro antimicrobial activity against an efflux-negative strain of Haemophilus influenzae was 4~8-fold higher, the combined improvement was at least 20~60-fold. Co-crystal structures of Escherichia coli RNA polymerase with two key squaramides showed displacement of the switch 2, predicted to interfere with the conformational change of clamp domain and/or with binding of non-template DNA, a mechanism akin to that of natural product myxopyronin. Furthermore, the structures confirmed the chemical features required for biochemical potency. The terminal isoxazole and benzyl rings bind into distinct relatively narrow, hydrophobic pockets and both are required for biochemical potency. In contrast, the linker composed of squarate and piperidine accesses different conformations in their respective co-crystal structures with RNA polymerase, reflecting its main role of proper orientation of the aforementioned terminal rings. These observations further explain the tolerance of hydrophilic substitutions in the linker region that was exploited to improve the fraction unbound to human plasma protein while retaining biochemical potency. PMID:25798859

  20. Quantitative Proteomic Analysis of the Orbital Frontal Cortex in Rats Following Extended Exposure to Caffeine Reveals Extensive Changes to Protein Expression: Implications for Neurological Disease.

    PubMed

    Franklin, Jane L; Mirzaei, Mehdi; Wearne, Travis A; Homewood, Judi; Goodchild, Ann K; Haynes, Paul A; Cornish, Jennifer L

    2016-05-01

    Caffeine is a plant-derived psychostimulant and a common additive found in a wide range of foods and pharmaceuticals. The orbitofrontal cortex (OFC) is rapidly activated by flavours, integrates gustatory and olfactory information, and plays a critical role in decision-making, with dysfunction contributing to psychopathologies and neurodegenerative conditions. This study investigated whether long-term consumption of caffeine causes changes to behavior and protein expression in the OFC. Male adult Sprague-Dawley rats (n = 8 per group) were treated for 26 days with either water or a 0.6 g/L caffeine solution. Locomotor behavior was measured on the first and last day of treatment, then again after 9 days treatment free following exposure to a mild stressor. When tested drug free, caffeine-treated animals were hyperactive compared to controls. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free shotgun proteomics found 157 proteins differentially expressed in the caffeine-drinking rats compared to control. Major proteomic effects were seen for cell-to-cell communication, cytoskeletal regulation, and mitochondrial function. Similar changes have been observed in neurological disorders including Alzheimer's disease, Parkinson's disease, and schizophrenia.

  1. Losses of nitrogen fractions from herring to brine during marinating.

    PubMed

    Szymczak, Mariusz; Kołakowski, Edward

    2012-05-01

    Fish meat is characterized by a high content of valuable nutrients. During the marinating process, however, the process of proteins diffusion and other nitrogen fractions from fish to the surrounding brine is commonly observed. It was determined that the total nitrogen loss from herring meat of industrial maturity (4-5day) amounted to 6-19% of raw material nitrogen. Extension of the marinating time to 16-18days increases nitrogen losses even to 18-27%. Less loss was observed during marinating of fresh than of frozen herring and during marinating of carcasses, as opposite to fillets. Higher nitrogen content in fish was not proved to influence higher nitrogen losses in a brine. The majority of loss consisted of nitrogen fractions soluble in TCA, of which one third was formed by α-amine nitrogen. Nitrogen contained in brine suspension accounted for only 1.5-4% of total nitrogen losses. With increasing salt or acid concentration the amount of total nitrogen loss was lower from fresh herring and higher from frozen one. Higher salt concentration significantly reduced the amount of non-protein nitrogen and all its fractions during marinating of fresh and frozen herring. In case of acetic acid, the influence of its concentration was diverse and depended on type of herring and its dressing.

  2. Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration.

    PubMed

    Fullerton, Gary D; Kanal, Kalpana M; Cameron, Ivan L

    2006-01-01

    How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.

  3. pH Shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions.

    PubMed

    Jiang, Jiang; Xiong, Youling L; Chen, Jie

    2010-07-14

    Soy protein isolate (SPI), beta-conglycinin (7S), and glycinin (11S) were subjected to pH-shifting treatments, that is, unfolding at pH 1.5 or 12.0 followed by refolding at pH 7.0, to induce molten globule structures. Treated samples were analyzed for protein solubility, thermal stability, and aggregation in 0, 0.1, and 0.6 M NaCl solutions at pH 2.0-8.0. The pH(12) shifting resulted in drastic increases (up to 2.5-fold) in SPI solubility in the pH 6.0-7.0 range, especially at 0 M NaCl. The pH(1.5) shifting had a generally lesser effect on solubility. 11S exhibited a solubility pattern similar to that of SPI, but the solubility of 7S was unaffected by pH shifting except at 0.6 M NaCl. The pH shifting, notably at pH 12.0, produced soluble, disulfide-linked polymers from 11S and reduced (P < 0.05) its enthalpy but not its temperature of denaturation. Soy proteins structurally altered by pH shifting had a reduced sensitivity to thermal aggregation.

  4. Impact of total solid content and extraction pH on enzyme-aided recovery of protein from defatted rapeseed (Brassica rapa L.) press cake and physicochemical properties of the protein fractions.

    PubMed

    Rommi, Katariina; Ercili-Cura, Dilek; Hakala, Terhi K; Nordlund, Emilia; Poutanen, Kaisa; Lantto, Raija

    2015-03-25

    Pectinase treatment was used to facilitate protein recovery from defatted rapeseed (Brassica rapa) cold-pressing residue in water-lean conditions and without pH adjustment. Effect of extraction pH on protein yield and physiochemical properties of the protein concentrates was assessed. Enzymatic hydrolysis of carbohydrates was feasible at high (40%) solid content and improved protein recovery at pH 6. Comparable protein yields (40-41% of total protein) from enzyme-aided water extraction (pH 6) and nonenzymatic alkaline extraction (pH10) at 10% solid content suggested that after enzymatic treatment, rapeseed protein could be extracted without exposure to alkali. However, water extraction required dilute conditions, whereas alkaline extraction was feasible also at 20% solid content. The water extracts possessed better protein solubility, higher ζ-potential, and smaller particle size than isoelectric precipitates from alkaline extraction, indicating higher dispersion stability. This is suggested to be mediated by electrostatic interactions between proteins and pectic carbohydrates in the water extracts.

  5. Initialized Fractional Calculus

    NASA Technical Reports Server (NTRS)

    Lorenzo, Carl F.; Hartley, Tom T.

    2000-01-01

    This paper demonstrates the need for a nonconstant initialization for the fractional calculus and establishes a basic definition set for the initialized fractional differintegral. This definition set allows the formalization of an initialized fractional calculus. Two basis calculi are considered; the Riemann-Liouville and the Grunwald fractional calculi. Two forms of initialization, terminal and side are developed.

  6. Tempered fractional calculus

    NASA Astrophysics Data System (ADS)

    Sabzikar, Farzad; Meerschaert, Mark M.; Chen, Jinghua

    2015-07-01

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered fractional difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series.

  7. Tempered fractional calculus

    SciTech Connect

    Sabzikar, Farzad; Meerschaert, Mark M.; Chen, Jinghua

    2015-07-15

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered fractional difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series.

  8. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation.

    PubMed

    Liu, Fahui; Teodorowicz, Małgorzata; Wichers, Harry J; van Boekel, Martinus A J S; Hettinga, Kasper A

    2016-08-24

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE receptor (sRAGE). Samples consisting of mixtures of whey protein and lactose were heated at 130 °C. An in vitro infant digestion model was used to study the influence of heat treatment on the digestibility of whey proteins. The amount of sRAGE-binding ligands before and after digestion was measured by an ELISA-based sRAGE-binding assay. Water activity did not significantly affect the extent of digestibility of whey proteins dry heated at pH 5 (ranging from 3.3 ± 0.2 to 3.6 ± 0.1% for gastric digestion and from 53.5 ± 1.5 to 64.7 ± 1.1% for duodenal digestion), but there were differences in cleavage patterns of peptides among the samples heated at different pH values. Formation of sRAGE-binding ligands depended on the formation of aggregates and was limited in the samples heated at pH 5. Moreover, the sRAGE-binding activity of digested sample was changed by protease degradation and correlated with the digestibility of samples. In conclusion, generation of sRAGE-binding ligands during extensive heat treatment of whey protein/lactose mixtures is limited in acidic heating condition and dependent on glycation and aggregation. PMID:27460534

  9. Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes.

    PubMed

    Dam, Svend; Thaysen-Andersen, Morten; Stenkjær, Eva; Lorentzen, Andrea; Roepstorff, Peter; Packer, Nicolle H; Stougaard, Jens

    2013-07-01

    Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.

  10. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  11. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  12. The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH.

    PubMed Central

    Proft, T; Hilbert, H; Layh-Schmitt, G; Herrmann, R

    1995-01-01

    Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta- and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated beta-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain. PMID:7768845

  13. Podophyllum hexandrum fraction (REC-2006) shows higher radioprotective efficacy in the p53-carrying hepatoma cell line: a role of cell cycle regulatory proteins.

    PubMed

    Singh, Pankaj Kumar; Kumar, Raj; Sharma, Ashok; Arora, Rajesh; Chawla, Raman; Jain, Swatantra Kumar; Sharma, Rakesh Kumar

    2009-09-01

    The present study was carried out to evaluate the radioprotective efficacy of Podophyllum hexandrum fraction (REC-2006) in hepatoma cell lines having different p53 statuses. Higher radioresistance was observed in the HepG2 (p53(++)) cell line in comparison to the Hep3B (p53(-)) cell line, indicating a plausible role of p53 in radioresistance. REC-2006 exhibited nearly twice the survival in p53-expressing HepG2 cells compared with p53-negative Hep3B cells. REC-2006 treatment alone induced p53 expression as compared with untreated controls. However, REC-2006 reduced p53 expression when treated 2 hours before irradiation as compared with the irradiated HepG2 controls, indicating that REC-2006 modulates the expression of p53 to mitigate its apoptotic effect. Induction of p21 in the REC-2006 + radiation treatment group downregulated the expression of cyclin E and CDK2, leading to a delay in the G1 phase of HepG2 cells, which provided time for DNA repair or related processes. However, no significant difference in CDC2 expression in both cell lines suggested that G2 phase arrest might not be the only responsible factor for REC-2006-mediated radioprotection. Significant induction of PCNA and GADD45 expression in HepG2 cells suggested that REC-2006 increased the percentage survival of HepG2 cells by increasing the span of time as well as efficacy for repair processes. In conclusion, REC-2006 modulated the expression of p53 and thereby promoted cell cycle arrest in the G1 phase, encouraging cell proliferation and DNA repair and thus providing significantly higher protection against acute gamma-radiation in the HepG2 cell line.

  14. How Many proteins are Missed in Quantitative proteomics Based on Ms/Ms sequencing Methods?

    PubMed Central

    Mulvey, Claire; Thur, Bettina; Crawford, Mark; Godovac-Zimmermann, Jasminka

    2014-01-01

    Current bottom-up quantitative proteomics methods based on MS/MS sequencing of peptides are shown to be strongly dependent on sample preparation. Using cytosolic proteins from MCF-7 breast cancer cells, it is shown that protein pre-fractionation based on pI and MW is more effective than pre-fractionation using only MW in increasing the number of observed proteins (947 vs. 704 proteins) and the number of spectral counts per protein. Combination of MS data from the different pre-fractionation methods results in further improvements (1238 proteins). We discuss that at present the main limitation on quantitation by MS/MS sequencing is not MS sensitivity and protein abundance, but rather extensive peptide overlap and limited MS/MS sequencing throughput, and that this favors internally calibrated methods such as SILAC, ICAT or ITRAQ over spectral counting methods in attempts to drastically improve proteome coverage of biological samples. PMID:25729266

  15. TEMPERED FRACTIONAL CALCULUS

    PubMed Central

    MEERSCHAERT, MARK M.; SABZIKAR, FARZAD; CHEN, JINGHUA

    2014-01-01

    Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series. PMID:26085690

  16. Instrument and method to determine the electrophoretic mobility of nanoparticles and proteins by combining electrical and flow field-flow fractionation.

    PubMed

    Johann, Christoph; Elsenberg, Stephan; Schuch, Horst; Rösch, Ulrich

    2015-04-21

    A new FFF method is presented which combines asymmetrical flow-FFF (AF4) and electrical FFF (ElFFF) in one channel to electrical asymmetrical flow-FFF (EAF4) to overcome the restrictions of pure ElFFF. It allows for measuring electrophoretic mobility (μ) as a function of size. The method provides an absolute value and does not require calibration. Results of μ for two particle standards are in good agreement with values determined by phase analysis light scattering (PALS). There is no requirement for low ionic strength carriers with EAF4. This overcomes one of the main limitations of ElFFF, making it feasible to measure proteins under physiological conditions. EAF4 has the capability to determine μ for individual populations which are resolved into separate peaks. This is demonstrated for a mixture of three polystyrene latex particles with different sizes as well as for the monomer and dimer of BSA and an antibody. The experimental setup consists of an AF4 channel with added electrodes; one is placed beneath the frit at the bottom wall and the other covers the inside of the upper channel plate. This design minimizes contamination from the electrolysis reactions by keeping the particles distant from the electrodes. In addition the applied voltage range is low (1.5-5 V), which reduces the quantity of gaseous electrolysis products below a threshold that interferes with the laminar flow profile or detector signals. Besides measuring μ, the method can be useful to improve the separation between sample components compared to pure flow-FFF. For two proteins (BSA and a monoclonal antibody), enhanced resolution of the monomer and dimer is achieved by applying an electric field.

  17. University of Wisconsin - Extension

    MedlinePlus

    ... Academic Affairs From the Provost Scholarship Governance Advancing Innovation Reimagining UW-Extension Awards Provost Staff Jobs Available ... extension , business and entrepreneurship , and broadcast and media innovations . Content on this page requires a newer version ...

  18. A dot-ELISA using a partially purified cathepsin-L-like protein fraction from Taenia solium cysticerci, for the diagnosis of human neurocysticercosis

    PubMed Central

    Piña, R; Gutiérrez, A H; Gilman, R H; Rueda, D; Sifuentes, C; Flores, M; Sheen, P; Rodriguez, S; GarcÍa, H H; Zimic, M

    2011-01-01

    Human neurocysticercosis (NCC), caused by the cestode Taenia solium, is responsible for a significant amount of neurological morbidity and epilepsy in developing countries. The disease remains highly endemic in many areas, despite several efforts and interventions to control it. A simple, cheap and fast diagnostic assay that is suitable for use in field conditions is highly desired. In immunodiagnostics based on western immunoblots or standard ELISA, a cathepsin-L-like protein purified from the cysticercus fluid has previously performed well as an antigen. In a recent study in Peru, the same 53/25-kDa antigen was therefore used in the development of a dot-ELISA that could be employed for mass screenings under field conditions. The assay was standardized and tested not only against sera from a large group of NCC cases but also against sera from patients with other common parasitic infections, so that sensitivity and specificity could be assessed. For NCC, the assay gave better sensitivity in the detection of individuals with extraparenchymal cysts (94·4%–100%) or multiple parenchymal cysts (74·6%–80·0%) than in the detection of individuals with single parenchymal cysts (29·4%–45·1%). The assay also showed a high specificity for NCC (99·0%–100%), with a very low level of cross-reactivity with other parasitic infections. The dot-ELISA developed in this study is a highly specific, simple, cheap and rapid test for NCC that could be used under field conditions, even in the low-resource settings that are common in developing countries. PMID:21871167

  19. Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

    PubMed Central

    Teigelkamp, S; Newman, A J; Beggs, J D

    1995-01-01

    Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP. Images PMID:7781612

  20. Cooled artery extension

    NASA Technical Reports Server (NTRS)

    Gernert, Nelson J. (Inventor)

    1990-01-01

    An artery vapor trap. A heat pipe artery is constructed with an extension protruding from the evaporator end of the heat pipe beyond the active area of the evaporator. The vapor migrates into the artery extension because of gravity or liquid displacement, and cooling the extension condenses the vapor to liquid, thus preventing vapor lock in the working portion of the artery by removing vapor from within the active artery. The condensed liquid is then transported back to the evaporator by the capillary action of the artery extension itself or by wick located within the extension.

  1. Pseudo Algebraically Closed Extensions

    NASA Astrophysics Data System (ADS)

    Bary-Soroker, Lior

    2009-07-01

    This PhD deals with the notion of pseudo algebraically closed (PAC) extensions of fields. It develops a group-theoretic machinery, based on a generalization of embedding problems, to study these extensions. Perhaps the main result is that although there are many PAC extensions, the Galois closure of a proper PAC extension is separably closed. The dissertation also contains the following subjects. The group theoretical counterpart of pseudo algebraically closed extensions, the so-called projective pairs. Applications to seemingly unrelated subjects, e.g., an analog of Dirichlet's theorem about primes in arithmetic progression for polynomial rings in one variable over infinite fields.

  2. Fractional Calculus in Hydrologic Modeling: A Numerical Perspective

    SciTech Connect

    David A. Benson; Mark M. Meerschaert; Jordan Revielle

    2012-01-01

    Fractional derivatives can be viewed either as a handy extension of classical calculus or, more deeply, as mathematical operators defined by natural phenomena. This follows the view that the diffusion equation is defined as the governing equation of a Brownian motion. In this paper, we emphasize that fractional derivatives come from the governing equations of stable Levy motion, and that fractional integration is the corresponding inverse operator. Fractional integration, and its multi-dimensional extensions derived in this way, are intimately tied to fractional Brownian (and Levy) motions and noises. By following these general principles, we discuss the Eulerian and Lagrangian numerical solutions to fractional partial differential equations, and Eulerian methods for stochastic integrals. These numerical approximations illuminate the essential nature of the fractional calculus.

  3. Extensions developed for NOVA/Husky cloud point equation

    SciTech Connect

    Seglin, L. )

    1988-10-24

    Extensions have been developed for the NOVA/Husky cloud point equation that improve the determination of constants for the equation, and optimize the use of available distillate fractions for producing diesel fuels with acceptable cloud points.

  4. Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

    PubMed Central

    Gong, Ting; Liao, Yuan; He, Fei; Yang, Yang; Yang, Dan-Dan; Chen, Xiang-Dong

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4. PMID:23264647

  5. Type Safe Extensible Programming

    NASA Astrophysics Data System (ADS)

    Chae, Wonseok

    2009-10-01

    Software products evolve over time. Sometimes they evolve by adding new features, and sometimes by either fixing bugs or replacing outdated implementations with new ones. When software engineers fail to anticipate such evolution during development, they will eventually be forced to re-architect or re-build from scratch. Therefore, it has been common practice to prepare for changes so that software products are extensible over their lifetimes. However, making software extensible is challenging because it is difficult to anticipate successive changes and to provide adequate abstraction mechanisms over potential changes. Such extensibility mechanisms, furthermore, should not compromise any existing functionality during extension. Software engineers would benefit from a tool that provides a way to add extensions in a reliable way. It is natural to expect programming languages to serve this role. Extensible programming is one effort to address these issues. In this thesis, we present type safe extensible programming using the MLPolyR language. MLPolyR is an ML-like functional language whose type system provides type-safe extensibility mechanisms at several levels. After presenting the language, we will show how these extensibility mechanisms can be put to good use in the context of product line engineering. Product line engineering is an emerging software engineering paradigm that aims to manage variations, which originate from successive changes in software.

  6. Dividing Fractions: A Pedagogical Technique

    ERIC Educational Resources Information Center

    Lewis, Robert

    2016-01-01

    When dividing one fraction by a second fraction, invert, that is, flip the second fraction, then multiply it by the first fraction. To multiply fractions, simply multiply across the denominators, and multiply across the numerators to get the resultant fraction. So by inverting the division of fractions it is turned into an easy multiplication of…

  7. Differential Gene Expression Profiles of Radioresistant Non-Small-Cell Lung Cancer Cell Lines Established by Fractionated Irradiation: Tumor Protein p53-Inducible Protein 3 Confers Sensitivity to Ionizing Radiation

    SciTech Connect

    Lee, Young Sook; Oh, Jung-Hwa; Yoon, Seokjoo; Kwon, Myung-Sang

    2010-07-01

    Purpose: Despite the widespread use of radiotherapy as a local and regional modality for the treatment of cancer, some non-small-cell lung cancers commonly develop resistance to radiation. We thus sought to clarify the molecular mechanisms underlying resistance to radiation. Methods and Materials: We established the radioresistant cell line H460R from radiosensitive parental H460 cells. To identify the radioresistance-related genes, we performed microarray analysis and selected several candidate genes. Results: Clonogenic and MTT assays showed that H460R was 10-fold more resistant to radiation than H460. Microarray analysis indicated that the expression levels of 1,463 genes were altered more than 1.5-fold in H460R compared with parental H460. To evaluate the putative functional role, we selected one interesting gene tumor protein p53-inducible protein 3 (TP53I3), because that this gene was significantly downregulated in radioresistant H460R cells and that it was predicted to link p53-dependent cell death signaling. Interestingly, messenger ribonucleic acid expression of TP53I3 differed in X-ray-irradiated H460 and H460R cells, and overexpression of TP53I3 significantly affected the cellular radiosensitivity of H460R cells. Conclusions: These results show that H460R may be useful in searching for candidate genes that are responsible for radioresistance and elucidating the molecular mechanism of radioresistance.

  8. Transcriptional response of four C1q domain containing protein (C1qDC) genes from Venerupis philippinarum exposed to the water soluble fraction of No.0 diesel oil.

    PubMed

    Zhang, Linbao; Sun, Wei; Cai, Wengui; Zhang, Zhe; Chen, Haigang; Ma, Shengwei; Jia, Xiaoping

    2016-10-01

    As pattern recognitionreceptors, the C1q-domain-containing (C1qDC) proteins play an important role in the pathogen recognition and complement pathway activation. In the present study, four novel C1q domain containing proteins (designated as VpC1qDC1, VpC1qDC2, VpC1qDC3 and VpC1qDC4) were cloned and characterized from clam Venerupis philippinarum. The four VpC1qDCs all possessed the conserved features critical for the fundamental structure and function of the C1q family. The four VpC1qDCs genes showed differential response profiles after exposure to the water soluble fraction of No.0 diesel oil (WSFD). More notably, VpC1qDC1 and VpC1qDC3 were more sensitive to low concentration of WSFD, as their mRNA level changed by higher magnitudes. In addition, VpC1qDC2 and VpC1qDC4 displayed notable increases with larger amplitude to high concentration of WSFD. All these results suggested that the transcriptional response of VpC1qDCs genes were probably a protective mechanism of the cell to oils pollution. The diverse expression patterns of VpC1qDCs demonstrated that VpC1qDC1 and VpC1qDC3 were sensitive responders to environmental stress in V. philippinarum. PMID:27261881

  9. Transcriptional response of four C1q domain containing protein (C1qDC) genes from Venerupis philippinarum exposed to the water soluble fraction of No.0 diesel oil.

    PubMed

    Zhang, Linbao; Sun, Wei; Cai, Wengui; Zhang, Zhe; Chen, Haigang; Ma, Shengwei; Jia, Xiaoping

    2016-10-01

    As pattern recognitionreceptors, the C1q-domain-containing (C1qDC) proteins play an important role in the pathogen recognition and complement pathway activation. In the present study, four novel C1q domain containing proteins (designated as VpC1qDC1, VpC1qDC2, VpC1qDC3 and VpC1qDC4) were cloned and characterized from clam Venerupis philippinarum. The four VpC1qDCs all possessed the conserved features critical for the fundamental structure and function of the C1q family. The four VpC1qDCs genes showed differential response profiles after exposure to the water soluble fraction of No.0 diesel oil (WSFD). More notably, VpC1qDC1 and VpC1qDC3 were more sensitive to low concentration of WSFD, as their mRNA level changed by higher magnitudes. In addition, VpC1qDC2 and VpC1qDC4 displayed notable increases with larger amplitude to high concentration of WSFD. All these results suggested that the transcriptional response of VpC1qDCs genes were probably a protective mechanism of the cell to oils pollution. The diverse expression patterns of VpC1qDCs demonstrated that VpC1qDC1 and VpC1qDC3 were sensitive responders to environmental stress in V. philippinarum.

  10. Exact solution to fractional logistic equation

    NASA Astrophysics Data System (ADS)

    West, Bruce J.

    2015-07-01

    The logistic equation is one of the most familiar nonlinear differential equations in the biological and social sciences. Herein we provide an exact solution to an extension of this equation to incorporate memory through the use of fractional derivatives in time. The solution to the fractional logistic equation (FLE) is obtained using the Carleman embedding technique that allows the nonlinear equation to be replaced by an infinite-order set of linear equations, which we then solve exactly. The formal series expansion for the initial value solution of the FLE is shown to be expressed in terms of a series of weighted Mittag-Leffler functions that reduces to the well known analytic solution in the limit where the fractional index for the derivative approaches unity. The numerical integration to the FLE provides an excellent fit to the analytic solution. We propose this approach as a general technique for solving a class of nonlinear fractional differential equations.

  11. Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona.

    PubMed

    Mudalige, Thilak K; Qu, Haiou; Linder, Sean W

    2015-07-21

    Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

  12. The Hexane Fraction of cyperus rotundus Prevents Non-Alcoholic Fatty Liver Disease Through the Inhibition of Liver X Receptor α-Mediated Activation of Sterol Regulatory Element Binding Protein-1c.

    PubMed

    Oh, Gyun-Sik; Yoon, Jin; Lee, Gang Gu; Kwak, Jong Hwan; Kim, Seung-Whan

    2015-01-01

    The goals of this study were (1) to examine the effects of Cyperus rotundus (CR) rhizome on cellular lipogenesis and non-alcoholic/diet-induced fatty liver disease, and (2) to elucidate the molecular mechanism behind its actions. The present investigation showed that the hexane fraction of CR rhizome (CRHF) reduced the elevated transcription levels of sterol regulatory element binding protein-1c (SREBP-1c) in primary hepatocytes following exposure to the liver X receptor α (LXRα) agonist. The SREBP-1c gene is a master regulator of lipogenesis and a key target of LXRα. CRHF inhibited not only the LXRα-dependent activation of the synthetic LXR response element (LXRE) promoter, but also the activation of the natural SREBP-1c promoter. Moreover, CRHF decreased (a) the recruitment of RNA polymerase II to the LXRE of the SREBP-1c gene; (b) the LXRα-dependent up-regulation of various lipogenic genes; and (c) the LXRα-mediated accumulation of triglycerides in primary hepatocytes. Furthermore, CRHF ameliorated fatty liver disease and reduced the expression levels of hepatic lipogenic genes in high sucrose diet (HSD)-fed mice. Interestingly, CRHF did not affect the expression of ATP-binding cassette transporter A1, another important LXR target gene that is required for reverse cholesterol transport (RCT) and protects against atherosclerosis. Taken together, these results suggest that CRHF might be a novel therapeutic remedy for fatty liver disease through the selective inhibition of the lipogenic pathway. PMID:25967664

  13. Transformations between Extensive and Intensive Versions of Thermodynamic Relationships

    ERIC Educational Resources Information Center

    Eberhart, James G.

    2010-01-01

    Most thermodynamic properties are either extensive (e.g., volume, energy, entropy, amount, etc.) or intensive (e.g., temperature, pressure, chemical potential, mole fraction, etc.). By the same token most of the mathematical relationships in thermodynamics can be written in extensive or intensive form. The basic laws of thermodynamics are usually…

  14. An Appetite for Fractions

    ERIC Educational Resources Information Center

    Wilkerson, Trena L.; Bryan, Tommy; Curry, Jane

    2012-01-01

    This article describes how using candy bars as models gives sixth-grade students a taste for learning to represent fractions whose denominators are factors of twelve. Using paper models of the candy bars, students explored and compared fractions. They noticed fewer different representations for one-third than for one-half. The authors conclude…

  15. (Carbon isotope fractionation inplants)

    SciTech Connect

    O'Leary, M.H.

    1990-01-01

    The objectives of this research are: To develop a theoretical and experimental framework for understanding isotope fractionations in plants; and to develop methods for using this isotope fractionation for understanding the dynamics of CO{sub 2} fixation in plants. Progress is described.

  16. The Future of Fractions

    ERIC Educational Resources Information Center

    Usiskin, Zalman P.

    2007-01-01

    In the 1970s, the movement to the metric system (which has still not completely occurred in the United States) and the advent of hand-held calculators led some to speculate that decimal representation of numbers would render fractions obsolete. This provocative proposition stimulated Zalman Usiskin to write "The Future of Fractions" in 1979. He…

  17. Acetylation in vitro of constituent polypeptides by smooth endoplasmic reticulum (SER) and Golgi membrane fractions

    SciTech Connect

    Sambasivam, H.; Murray, R.K.

    1986-05-01

    Many polypeptides of the membranes of the ER are phosphorylated. To determine if any such polypeptides are acetylated, microsomal and other classical subcellular fractions were incubated with (/sup 3/H) acetyl-CoA; the specific activity of the microsomal fraction (MF) was the greatest. SDS-PAGE revealed that some 20 polypeptides of the MF were acetylated; 2-D electrophoretograms extended this number to approximately 60. Separation of the MF into smooth (S) and rough (R) fractions showed that the great majority of the labelled polypeptides belonged to the former. Isolation of a Golgi fraction revealed that its acetylation activity was approximately 3-fold greater than the SER fraction. Extensive proteolytic digestion of the MF followed by radiochromatography disclosed some 9 components whose precise nature (acetylated amino acids and/or sialic acids, etc.) is under study. Assuming that the majority of the radioactivity is in the former components and that a similar process occurs in vivo, the authors suggest that the Golgi apparatus may be a major site of acetylation of membrane and possibly other proteins.

  18. Fractional calculus in bioengineering.

    PubMed

    Magin, Richard L

    2004-01-01

    Fractional calculus (integral and differential operations of noninteger order) is not often used to model biological systems. Although the basic mathematical ideas were developed long ago by the mathematicians Leibniz (1695), Liouville (1834), Riemann (1892), and others and brought to the attention of the engineering world by Oliver Heaviside in the 1890s, it was not until 1974 that the first book on the topic was published by Oldham and Spanier. Recent monographs and symposia proceedings have highlighted the application of fractional calculus in physics, continuum mechanics, signal processing, and electromagnetics, but with few examples of applications in bioengineering. This is surprising because the methods of fractional calculus, when defined as a Laplace or Fourier convolution product, are suitable for solving many problems in biomedical research. For example, early studies by Cole (1933) and Hodgkin (1946) of the electrical properties of nerve cell membranes and the propagation of electrical signals are well characterized by differential equations of fractional order. The solution involves a generalization of the exponential function to the Mittag-Leffler function, which provides a better fit to the observed cell membrane data. A parallel application of fractional derivatives to viscoelastic materials establishes, in a natural way, hereditary integrals and the power law (Nutting/Scott Blair) stress-strain relationship for modeling biomaterials. In this review, I will introduce the idea of fractional operations by following the original approach of Heaviside, demonstrate the basic operations of fractional calculus on well-behaved functions (step, ramp, pulse, sinusoid) of engineering interest, and give specific examples from electrochemistry, physics, bioengineering, and biophysics. The fractional derivative accurately describes natural phenomena that occur in such common engineering problems as heat transfer, electrode/electrolyte behavior, and sub

  19. Fractional calculus in bioengineering.

    PubMed

    Magin, Richard L

    2004-01-01

    Fractional calculus (integral and differential operations of noninteger order) is not often used to model biological systems. Although the basic mathematical ideas were developed long ago by the mathematicians Leibniz (1695), Liouville (1834), Riemann (1892), and others and brought to the attention of the engineering world by Oliver Heaviside in the 1890s, it was not until 1974 that the first book on the topic was published by Oldham and Spanier. Recent monographs and symposia proceedings have highlighted the application of fractional calculus in physics, continuum mechanics, signal processing, and electromagnetics, but with few examples of applications in bioengineering. This is surprising because the methods of fractional calculus, when defined as a Laplace or Fourier convolution product, are suitable for solving many problems in biomedical research. For example, early studies by Cole (1933) and Hodgkin (1946) of the electrical properties of nerve cell membranes and the propagation of electrical signals are well characterized by differential equations of fractional order. The solution involves a generalization of the exponential function to the Mittag-Leffler function, which provides a better fit to the observed cell membrane data. A parallel application of fractional derivatives to viscoelastic materials establishes, in a natural way, hereditary integrals and the power law (Nutting/Scott Blair) stress-strain relationship for modeling biomaterials. In this review, I will introduce the idea of fractional operations by following the original approach of Heaviside, demonstrate the basic operations of fractional calculus on well-behaved functions (step, ramp, pulse, sinusoid) of engineering interest, and give specific examples from electrochemistry, physics, bioengineering, and biophysics. The fractional derivative accurately describes natural phenomena that occur in such common engineering problems as heat transfer, electrode/electrolyte behavior, and sub

  20. Stable Chlorine Isotope Fractionation

    NASA Astrophysics Data System (ADS)

    Sharp, Z.

    2006-12-01

    Chlorine isotope partitioning between different phases is not well understood. Pore fluids can have δ37Cl values as low as -8‰, with neoform sediments having strongly positive values. Most strikingly, volcanic gases have δ37Cl values that cover a range in excess of 14‰ (Barnes et al., this meeting). The large range is difficult to explain in terms of equilibrium fractionation, which, although calculated to be very large for Cl in different oxidation states, should be less than 2‰ between chloride species (Schauble et al., 2003, GCA). To address the discrepancy between Nature and theory, we have measured Cl isotope fractionation for selected equilibrium and disequilibrium experiments in order to identify mechanisms that might lead to large fractionations. 1) NaCl (s,l) NaCl (v): NaCl was sealed in an evacuated silica tube and heated at one end, causing vaporization and reprecipitation of NaCl (v) at the cool end of the tube. The fractionation is 0.2‰ at 700°C (halite-vapor) and 0.7‰ at 800°C (liquid-vapor), respectively. The larger fractionation at higher temperature may be related to equilibrium fractionation between liquid and gas vs. `stripping' of the solid in the lower T experiments. 2) Sodalite NaCl(l): Nepheline and excess NaCl were sealed in a Pt crucible at 825°C for 48 hrs producing sodalite. The measured newly-formed sodalite-NaCl fractionation is -0.2‰. 3) Volatilization of HCl: Dry inert gas was bubbled through HCl solutions and the vapor was collected in a downstream water trap. There was no fractionation for 12.4M HCl (HCl fuming) vapor at 25°C. For a 1 M boiling HCl solution, the HCl-vapor fractionation was ~9‰. The difference is probably related to the degree of dissociation in the acid, with HCl dissolved in water for the highly acidic solutions, and dissociated H3O+ and Cl- for lower concentrations. The HCl volatilization experiments are in contrast to earlier vapor-liquid experiments in NaCl-H2O system, where fractionation was

  1. Intracellular Cadmium Isotope Fractionation

    NASA Astrophysics Data System (ADS)

    Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.

    2011-12-01

    Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.

  2. Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft-and the Non-Raft Brush Border Membrane Fractions of NHE3

    PubMed Central

    Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E.; Donowitz, Mark; Yun, C. Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

    2014-01-01

    Background/Aims Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3− mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. PMID:24297041

  3. An Analysis of the Extension Worker's Knowledge of Extension Programs.

    ERIC Educational Resources Information Center

    Stonecipher, Charles Leroy

    Forty-four employees of the General Extension Division of the Nebraska Agricultural Extension Service and 119 Agricultural Extension employees of the University of Nebraska comprised the population of this study comparing personnel's knowledge of Agricultural Extension and General Extension programs and differences in knowledge of programs…

  4. soil organic matter fractionation

    NASA Astrophysics Data System (ADS)

    Osat, Maryam; Heidari, Ahmad

    2010-05-01

    Carbon is essential for plant growth, due to its effects on other soil properties like aggregation. Knowledge of dynamics of organic matter in different locations in the soil matrix can provide valuable information which affects carbon sequestration and soil the other soil properties. Extraction of soil organic matter (SOM) fractions has been a long standing approach to elucidating the roles of soil organic matter in soil processes. Several kind fractionation methods are used and all provide information on soil organic matter function. Physical fractionation capture the effects on SOM dynamics of the spatial arrangement of primary and secondary organomineral particles in soil while chemical fractionation can not consider the spatial arrangement but their organic fractions are suitable for advanced chemical characterization. Three method of physical separation of soil have been used, sieving, sedimentation and densitometry. The distribution of organic matter within physical fractions of the soil can be assessed by sieving. Sieving separates soil particles based strictly on size. The study area is located on north central Iran, between 35° 41'- 36° 01' N and 50° 42'- 51° 14' E. Mean annual precipitation about 243.8 mm and mean annual air temperature is about 14.95 °C. The soil moisture and temperature regime vary between aridic-thermic in lower altitudes to xeric-mesic in upper altitudes. More than 36 surface soil samples (0-20 cm) were collected according to land-use map units. After preliminary analyzing of samples 10 samples were selected for further analyses in five size fractions and three different time intervals in September, January and April 2008. Fractionation carried out by dry sieving in five classes, 1-2 mm, 0.5-1 mm, 270 μm-0.5mm, 53-270 μm and <53 μm. Organic matter and C/N ratio were determined for all fractions at different time intervals. Chemical fractionation of organic matter also carried out according to Tan (2003), also Mineralogical

  5. Fractional Noether Theorem Based on Extended Exponentially Fractional Integral

    NASA Astrophysics Data System (ADS)

    Long, Zi-Xuan; Zhang, Yi

    2013-10-01

    Based on the new type of fractional integral definition, namely extended exponentially fractional integral introduced by EI-Nabulsi, we study the fractional Noether symmetries and conserved quantities for both holonomic system and nonholonomic system. First, the fractional variational problem under the sense of extended exponentially fractional integral is established, the fractional d'Alembert-Lagrange principle is deduced, then the fractional Euler-Lagrange equations of holonomic system and the fractional Routh equations of nonholonomic system are given; secondly, the invariance of fractional Hamilton action under infinitesimal transformations of group is also discussed, the corresponding definitions and criteria of fractional Noether symmetric transformations and quasi-symmetric transformations are established; finally, the fractional Noether theorems for both holonomic system and nonholonomic system are explored. What's more, the relationship between the fractional Noether symmetry and conserved quantity are revealed.

  6. Mobile Applications for Extension

    ERIC Educational Resources Information Center

    Drill, Sabrina L.

    2012-01-01

    Mobile computing devices (smart phones, tablets, etc.) are rapidly becoming the dominant means of communication worldwide and are increasingly being used for scientific investigation. This technology can further our Extension mission by increasing our power for data collection, information dissemination, and informed decision-making. Mobile…

  7. Today's Youth: Extension's Challenge

    ERIC Educational Resources Information Center

    Robinson, Russell D.

    1970-01-01

    The challenge to Extension youth work today is programing to deal with political and value issues in a way appealing to the small group who want to make things happen" without losing the majority, who let things happen" or wonder what happened. (EB)

  8. Vocabulary Extension through Poetry.

    ERIC Educational Resources Information Center

    Surajlal, K. C.

    1986-01-01

    Based on the notion that teaching vocabulary extension in isolation makes little impact on students, a three-part exercise, designed to develop students' vocabulary through poetry while providing meaningful enjoyment, uses the poem "The Hawk" by A. C. Benson. In the first class period, students are introduced to both the exercise and the poem and…

  9. Volatile fractionation and tektite source material

    NASA Technical Reports Server (NTRS)

    Walter, Louis S.

    1989-01-01

    The arguments used by Love and Woronow (1988) to assess the role played in the origin of bediasites by extensive volatile fractionation are critically examined. Using the ratios of 'refractory' oxides, CaO, Al2O3, and MgO, to the 'volatile' oxides, Na2O and K2O, these authors concluded that vapor fractionation did not play a significant role. In this paper, experimental evidence is presented that shows that the assumption of volatility for the alkali elements (as least with respect to silica) to be not valid under the conditions under which tektites formed. It is shown that the results of vapor fractionation in experiments on glasses of tektite composition are approximately parallel the trends seen in bediasite analysis.

  10. Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts.

    PubMed

    Vasconcelos, A C; Mendiola-Morgenthaler, L R; Floyd, G L; Salisbury, J L

    1976-07-01

    Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.The envelope membranes were separated into two fractions in the gradients according to their banding densities. Electron micrographs showed that the light envelope fraction consisted mostly of single-membrane vesicles, whereas the heavy envelope fraction consisted of multiple layers of folded membranes. Both envelope fractions were ultrastructurally distinct from the thylakoid membranes. PMID:16659627

  11. On the Singular Perturbations for Fractional Differential Equation

    PubMed Central

    Atangana, Abdon

    2014-01-01

    The goal of this paper is to examine the possible extension of the singular perturbation differential equation to the concept of fractional order derivative. To achieve this, we presented a review of the concept of fractional calculus. We make use of the Laplace transform operator to derive exact solution of singular perturbation fractional linear differential equations. We make use of the methodology of three analytical methods to present exact and approximate solution of the singular perturbation fractional, nonlinear, nonhomogeneous differential equation. These methods are including the regular perturbation method, the new development of the variational iteration method, and the homotopy decomposition method. PMID:24683357

  12. FRACTIONAL CRYSTALLIZATION FEED ENVELOPE

    SciTech Connect

    HERTING DL

    2008-03-19

    Laboratory work was completed on a set of evaporation tests designed to establish a feed envelope for the fractional crystallization process. The feed envelope defines chemical concentration limits within which the process can be operated successfully. All 38 runs in the half-factorial design matrix were completed successfully, based on the qualitative definition of success. There is no feed composition likely to be derived from saltcake dissolution that would cause the fractional crystallization process to not meet acceptable performance requirements. However, some compositions clearly would provide more successful operation than other compositions.

  13. Release Fraction Evaluation

    SciTech Connect

    Bamberger, Judith A.; Glissmeyer, John A.

    2004-01-01

    This document presents results of experiments conducted to measure release fractions during certain tank retrieval processes. The tests were performed in a 1/4 scale model of a waste storage tank. The retrieval processes simulated were: (1) Discharging liquid or slurry from the mouth of a vertically oriented two-in. Schedule 40 pipe. The discharging material was in free-fall from the mouth of the pipe near the top of the tank into a liquid or slurry pool at the bottom of the tank. (2) The jet from a 9/16-in.-diameter nozzle transferring liquid or slurry waste from one side of the tank to the other. The discharging liquid was aimed at the opposite side of the tank from the nozzle and either impacted the tank wall or fell into a liquid or slurry pool in the bottom of the tank. (3) A high pressure fan jet of liquid striking a steel plate or simulated waste from a stand-off distance of a few inches. For each process, a water-soluble fluorescent dye was added to the liquid fraction as a tracer. Kaolin clay was used to represent the solids. The tank was covered and there was no forced ventilation in the tank during the tests. Six air samples were collected during each test. The air samples were collected at fixed positions in the tank. The air sample filters were dried and weighed to determine the solids collection. The fluorescent dye was then leached from each filter and quantified with a fluorometer to determine the collection of liquid. Samples of the slurry and liquid simulants were also collected to determine the quantities of simulant used in each test. To calculate the release fraction, the quantity collected on each air sample was adjusted for the fraction of the tank volume sampled and divided by the quantity of material exposed in the simulation. The method was not as sensitive for the solids content as it was for the liquid content, but in those instances where a solids release fraction was determined, it was in relatively good agreement with that of the

  14. Momentum fractionation on superstrata

    DOE PAGESBeta

    Bena, Iosif; Martinec, Emil; Turton, David; Warner, Nicholas P.

    2016-05-11

    Superstrata are bound states in string theory that carry D1, D5, and momentum charges, and whose supergravity descriptions are parameterized by arbitrary functions of (at least) two variables. In the D1-D5 CFT, typical three-charge states reside in highdegree twisted sectors, and their momentum charge is carried by modes that individually have fractional momentum. Understanding this momentum fractionation holographically is crucial for understanding typical black-hole microstates in this system. We use solution-generating techniques to add momentum to a multi-wound supertube and thereby construct the first examples of asymptotically-flat superstrata. The resulting supergravity solutions are horizonless and smooth up to well-understood orbifoldmore » singularities. Upon taking the AdS3 decoupling limit, our solutions are dual to CFT states with momentum fractionation. We give a precise proposal for these dual CFT states. Lastly, our construction establishes the very nontrivial fact that large classes of CFT states with momentum fractionation can be realized in the bulk as smooth horizonless supergravity solutions.« less

  15. Understanding Fraction Multiplication.

    ERIC Educational Resources Information Center

    Bezuk, Nadine S.; Armstrong, Barbara E.

    1992-01-01

    Presents five activities to help students construct meaning for multiplying fractions through real-world problem contexts, physical or pictorial models, the recognition of patterns, and the use of calculators. In the context of a garden plot, worksheets examine various aspects of parts of plots, patterns in plots, and a maximization problem.…

  16. Fractions through Fruit Salad.

    ERIC Educational Resources Information Center

    Lincoln, Lisa

    1987-01-01

    The mathematics concept of fractions was taught to a group of learning disabled, dyslexic, and multiply handicapped students (15-20 years old) by preparing a fruit salad. Enthusiastic student participation and enhanced knowledge illustrated the effectiveness of employing several sensory modes in learning activities. (CB)

  17. Field-Flow Fractionation.

    ERIC Educational Resources Information Center

    Caldwell, Karin D.

    1988-01-01

    Describes a technique for separating samples that range over 15 orders of magnitude in molecular weight. Discusses theory, apparatus, and sample preparation techniques. Lists several types of field-flow fractionation (FFF) and their uses: sedimentation FFF, thermal FFF, flow FFF, electrical FFF, and steric FFF. (ML)

  18. Avoidance of Fractions.

    ERIC Educational Resources Information Center

    Hart, Kathleen; Kerslake, Daphne

    The Concepts in Secondary Mathematics and Science (CSMS) and Strategies and Errors in Secondary Mathematics (SESM) research projects based at Chelsa College, England, have shown the marked reluctance of secondary school students to use fractions when solving mathematical problems, even though they have been taught the topic for a number of years.…

  19. Sweet Work with Fractions

    ERIC Educational Resources Information Center

    Vinogradova, Natalya; Blaine, Larry

    2013-01-01

    Almost everyone loves chocolate. However, the same cannot be said about fractions, which are loved by markedly fewer. Middle school students tend to view them with wary respect, but little affection. The authors attempt to sweeten the subject by describing a type of game involving division of chocolate bars. The activity they describe provides a…

  20. Videodisc Instruction in Fractions.

    ERIC Educational Resources Information Center

    Carnine, Douglas; And Others

    1987-01-01

    How laser videodisc technology can be used to improve mathematics instruction is described, with note of the development of a videodisc curriculum on mastering fractions. Relevant research is reviewed, as well as how teachers can use the technology. The instructional design is described, and field-testing and revision reported. (MNS)

  1. Momentum fractionation on superstrata

    NASA Astrophysics Data System (ADS)

    Bena, Iosif; Martinec, Emil; Turton, David; Warner, Nicholas P.

    2016-05-01

    Superstrata are bound states in string theory that carry D1, D5, and momentum charges, and whose supergravity descriptions are parameterized by arbitrary functions of (at least) two variables. In the D1-D5 CFT, typical three-charge states reside in high-degree twisted sectors, and their momentum charge is carried by modes that individually have fractional momentum. Understanding this momentum fractionation holographically is crucial for understanding typical black-hole microstates in this system. We use solution-generating techniques to add momentum to a multi-wound supertube and thereby construct the first examples of asymptotically-flat superstrata. The resulting supergravity solutions are horizonless and smooth up to well-understood orbifold singularities. Upon taking the AdS3 decoupling limit, our solutions are dual to CFT states with momentum fractionation. We give a precise proposal for these dual CFT states. Our construction establishes the very nontrivial fact that large classes of CFT states with momentum fractionation can be realized in the bulk as smooth horizonless supergravity solutions.

  2. Weighted average finite difference methods for fractional diffusion equations

    NASA Astrophysics Data System (ADS)

    Yuste, S. B.

    2006-07-01

    A class of finite difference methods for solving fractional diffusion equations is considered. These methods are an extension of the weighted average methods for ordinary (non-fractional) diffusion equations. Their accuracy is of order (Δ x) 2 and Δ t, except for the fractional version of the Crank-Nicholson method, where the accuracy with respect to the timestep is of order (Δ t) 2 if a second-order approximation to the fractional time-derivative is used. Their stability is analyzed by means of a recently proposed procedure akin to the standard von Neumann stability analysis. A simple and accurate stability criterion valid for different discretization schemes of the fractional derivative, arbitrary weight factor, and arbitrary order of the fractional derivative, is found and checked numerically. Some examples are provided in which the new methods' numerical solutions are obtained and compared against exact solutions.

  3. An Introduction to Continued Fractions.

    ERIC Educational Resources Information Center

    Moore, Charles G.

    Provided is an introduction to the properties of continued fractions for the intellectually curious high school student. Among the topics included are (1) Expansion of Rational Numbers into Simple Continued Fractions, (2) Convergents, (3) Continued Fractions and Linear Diophantine Equations of the Type am + bn = c, (4) Continued Fractions and…

  4. Agricultural Extension: Who Uses It?

    ERIC Educational Resources Information Center

    Nolan, Michael; Lasley, Paul

    1979-01-01

    A Missouri study conducted to determine agricultural extension usage patterns found that heavy users of extension publications tended to be younger farmers, those with a relatively large amount of land, and pork producers. Extension meetings were a less frequent source of information than either publications or county extension office visits. (LRA)

  5. LIMB demonstration project extension

    SciTech Connect

    Not Available

    1990-09-21

    The purpose of the DOE limestone injection multistage burner (LIMB) Demonstration Project Extension is to extend the data base on LIMB technology and to expand DOE's list of Clean Coal Technologies by demonstrating the Coolside process as part of the project. The main objectives of this project are: to demonstrate the general applicability of LIMB technology by testing 3 coals and 4 sorbents (total of 12 coal/sorbent combinations) at the Ohio Edison Edgewater plant; and to demonstrate that Coolside is a viable technology for improving precipitator performance and reducing sulfur dioxide emissions while acceptable operability is maintained. Progress is reported. 3 figs.

  6. Testing fractional action cosmology

    NASA Astrophysics Data System (ADS)

    Shchigolev, V. K.

    2016-08-01

    The present work deals with a combined test of the so-called Fractional Action Cosmology (FAC) on the example of a specific model obtained by the author earlier. In this model, the effective cosmological term is proportional to the Hubble parameter squared through the so-called kinematic induction. The reason of studying this cosmological model could be explained by its ability to describe two periods of accelerated expansion, that is in agreement with the recent observations and the cosmological inflation paradigm. First of all, we put our model through the theoretical tests, which gives a general conception of the influence of the model parameters on its behavior. Then, we obtain some restrictions on the principal parameters of the model, including the fractional index, by means of the observational data. Finally, the cosmography parameters and the observational data compared to the theoretical predictions are presented both analytically and graphically.

  7. [Fractionated P-bilirubins].

    PubMed

    Schou, C S; Mortensen, H

    1989-08-14

    A diazo-based dry film technique for the estimation of different bilirubins in plasma is now available. This procedure separates bilirubins from icteric sera into three separate fractions: bilirubin (unconjugated), bilirubin-glucuronides (mono + diglucuronide) and bilirubin-albumin. In newborns with prolonged jaundice classification of hyperbilirubinemia is of importance for choice of treatment. While binding of bilirubin and bilirubin-glucuronides to albumin is non covalent, reversible, bilirubin-albumin appears to be firmly associated with albumin by covalent bonds. This causes delayed clearance of this bilirubin fraction from plasma as the half-life of albumin is approximately 18 days. Hence the substance concentration of bilirubin-albumin will decrease at a slower rate than will bilirubin and bilirubin-glucuronide, despite hepatobiliary recovery. Bilirubin-albumin may therefore prove of value in the differentiation between different clinical entities with hyperbilirubinemia. PMID:2773134

  8. New Dry Fractionation Methods

    NASA Technical Reports Server (NTRS)

    McKay, David S.; Cooper, Bonnie L.

    2010-01-01

    This slide presentation describes new fractionation methods that are used to create dust that is respirable for testing the effects of inhalation of lunar dust in preparation for future manned lunar exploration. Because lunar dust is a very limited commodity, a method that does not result in loss of the material had to be developed. The dust separation system that is described incorporates some traditional methods, while preventing the dust from being contaminated or changed in reactivity properties while also limiting losses.

  9. Fractional Galilean symmetries

    NASA Astrophysics Data System (ADS)

    Hosseiny, Ali; Rouhani, Shahin

    2016-09-01

    We generalize the differential representation of the operators of the Galilean algebras to include fractional derivatives. As a result a whole new class of scale invariant Galilean algebras are obtained. The first member of this class has dynamical index z = 2 similar to the Schrödinger algebra. The second member of the class has dynamical index z = 3 / 2, which happens to be the dynamical index Kardar-Parisi-Zhang equation.

  10. Model Fractional Chern Insulators.

    PubMed

    Behrmann, Jörg; Liu, Zhao; Bergholtz, Emil J

    2016-05-27

    We devise local lattice models whose ground states are model fractional Chern insulators-Abelian and non-Abelian topologically ordered states characterized by exact ground state degeneracies at any finite size and infinite entanglement gaps. Most saliently, we construct exact parent Hamiltonians for two distinct families of bosonic lattice generalizations of the Z_{k} parafermion quantum Hall states: (i) color-entangled fractional Chern insulators at band filling fractions ν=k/(C+1) and (ii) nematic states at ν=k/2, where C is the Chern number of the lowest band. In spite of a fluctuating Berry curvature, our construction is partially frustration free: the ground states reside entirely within the lowest band and exactly minimize a local (k+1) body repulsion term by term. In addition to providing the first known models hosting intriguing states such as higher Chern number generalizations of the Fibonacci anyon quantum Hall states, the remarkable stability and finite-size properties make our models particularly well suited for the study of novel phenomena involving, e.g., twist defects and proximity induced superconductivity, as well as being a guide for designing experiments.

  11. Model Fractional Chern Insulators

    NASA Astrophysics Data System (ADS)

    Behrmann, Jörg; Liu, Zhao; Bergholtz, Emil J.

    2016-05-01

    We devise local lattice models whose ground states are model fractional Chern insulators—Abelian and non-Abelian topologically ordered states characterized by exact ground state degeneracies at any finite size and infinite entanglement gaps. Most saliently, we construct exact parent Hamiltonians for two distinct families of bosonic lattice generalizations of the Zk parafermion quantum Hall states: (i) color-entangled fractional Chern insulators at band filling fractions ν =k /(C +1 ) and (ii) nematic states at ν =k /2 , where C is the Chern number of the lowest band. In spite of a fluctuating Berry curvature, our construction is partially frustration free: the ground states reside entirely within the lowest band and exactly minimize a local (k +1 ) body repulsion term by term. In addition to providing the first known models hosting intriguing states such as higher Chern number generalizations of the Fibonacci anyon quantum Hall states, the remarkable stability and finite-size properties make our models particularly well suited for the study of novel phenomena involving, e.g., twist defects and proximity induced superconductivity, as well as being a guide for designing experiments.

  12. Model Fractional Chern Insulators.

    PubMed

    Behrmann, Jörg; Liu, Zhao; Bergholtz, Emil J

    2016-05-27

    We devise local lattice models whose ground states are model fractional Chern insulators-Abelian and non-Abelian topologically ordered states characterized by exact ground state degeneracies at any finite size and infinite entanglement gaps. Most saliently, we construct exact parent Hamiltonians for two distinct families of bosonic lattice generalizations of the Z_{k} parafermion quantum Hall states: (i) color-entangled fractional Chern insulators at band filling fractions ν=k/(C+1) and (ii) nematic states at ν=k/2, where C is the Chern number of the lowest band. In spite of a fluctuating Berry curvature, our construction is partially frustration free: the ground states reside entirely within the lowest band and exactly minimize a local (k+1) body repulsion term by term. In addition to providing the first known models hosting intriguing states such as higher Chern number generalizations of the Fibonacci anyon quantum Hall states, the remarkable stability and finite-size properties make our models particularly well suited for the study of novel phenomena involving, e.g., twist defects and proximity induced superconductivity, as well as being a guide for designing experiments. PMID:27284668

  13. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  14. LIMB Demonstration Project Extension

    SciTech Connect

    Not Available

    1989-11-15

    The basic goal of the Limestone Injection Mitigation Burner (LIMB) demonstration is to extend LIMB technology development to a full- scale application on a representative wall-fired utility boiler. The successful retrofit of LIMB to an existing boiler is expected to demonstrate that (a) reductions of 50 percent or greater in SO{sub x} and NO{sub x} emissions can be achieved at a fraction of the cost of add-on FGD systems, (b) boiler reliability, operability, and steam production can be maintained at levels existing prior to LIMB retrofit, and (c) technical difficulties attributable to LIMB operation, such as additional slagging and fouling, changes in ash disposal requirements, and an increased particulate load, can be resolved in a cost-effective manner. The primary fuel to be used will be an Ohio bituminous coal having a nominal sulfur content of 3 percent or greater.

  15. Pepsin degradation of Cry1A(b) protein purified from genetically modified maize (Zea mays).

    PubMed

    de Luis, Ruth; Lavilla, María; Sánchez, Lourdes; Calvo, Miguel; Pérez, María D

    2010-02-24

    The aim of this work was to study the in vitro digestion of Cry1A(b) protein by pepsin. To perform this work, a protein fraction purified from transgenic maize by immunoadsorption was employed. The undigested fraction showed several bands of molecular weight ranging between 14 and 70 kDa when assayed by SDS-PAGE. These bands were identified as corresponding to Cry1A(b) protein by immunochemical techniques and mass spectrometry. The rate of degradation of the purified fraction by pepsin estimated by ELISA was found to be about 75% within 30 min, and the protein concentration remained constant up to 4 h. In all treated samples, the full-length protein and fragments present in Cry1A(b) fraction were absent and peptides of less than 8.5 kDa were mainly found by SDS-PAGE and mass spectrometry. These peptides did not react with antiserum against Cry1A(b) protein by Western blotting. These results suggest that Cry1A(b) fraction purified from transgenic maize is rapidly and extensively degraded by pepsin, giving peptides of low molecular mass.

  16. Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

    NASA Technical Reports Server (NTRS)

    Melan, M. A.; Cosgrove, D. J.

    1988-01-01

    Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

  17. Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114.

    PubMed

    González de la Vara, Luis E; Lino Alfaro, Bárbara

    2009-04-15

    The detergent Triton X-114, because of its convenient cloud point temperature (22 degrees C), has been used extensively to extract membrane proteins and to separate them in two phases according to their hydropathy. The upper detergent-poor phase contains mostly hydrophilic proteins, whereas hydrophobic ones are found mainly in the lower detergent-rich phase. In this work, we developed a method to fractionate membrane proteins and estimate their hydropathy based on a series of cloud point partitions with Triton X-114. With this method, beetroot plasma membrane proteins were separated in different fractions according to their hydropathy, following the binomial distribution law as expected. This method revealed the presence of both hydrophilic and hydrophobic Ca(2+)-dependent protein kinases in those membranes. At least five distinct Ca(2+)-dependent kinases were observed in in-gel kinase activity assays. This separation procedure was also used as the first step in the purification of a hydrophobic 60-kDa kinase.

  18. [Ablative and fractional lasers].

    PubMed

    Beylot, C; Grognard, C; Michaud, T

    2009-10-01

    The use of pulsed or scanning Carbon Dioxide, and pulsed Erbium-YAG lasers allows the programmable and reproducible photocoagulation of thin layers of the epidermis and superficial dermis. Thermal damage depends on the type of laser and is greater with CO(2) lasers. The degree of neocollagenesis is proportional to the thermal damage and is better with CO(2) lasers. Their main indication is the correction of photoaged facial skin but they can also be used for corrective dermatology, e.g. for scars and genodermatosis. Results are highly satisfactory but the technique is invasive and the patient experiences a social hindrance of around two weeks. Fractionated techniques treat 25% of the defective skin area at each session in noncontiguous microzones; four sessions are therefore necessary to treat the entire cutaneous surface. The treatment is given under topical anesthesia and is much less invasive, particularly with nonablative fractional laser treatment in which photothermolysis does not penetrate below the epidermis and/or the effects are slight, with no or very little social isolation. However, the results are much less satisfactory than the results of ablative laser and there is no firming effect. Other zones than the face can be treated. With the fractional CO(2) and Erbium ablative lasers, which have multiplied over the past 2 years, the much wider impacts cause perforation of the epidermis and there is a zone of ablation by laser photovaporization, with a zone of thermal damage below. The results are better in correcting photoaging of the face, without, however, achieving the efficacy of ablative lasers, which remain the reference technique. However, the effects are not insignificant, requiring at least 5 days of social isolation.

  19. Membrane and membrane-associated proteins in Triton X-114 extracts of Mycobacterium bovis BCG identified using a combination of gel-based and gel-free fractionation strategies.

    PubMed

    Målen, Hiwa; Berven, Frode S; Søfteland, Tina; Arntzen, Magnus Øverlie; D'Santos, Clive S; De Souza, Gustavo Antonio; Wiker, Harald G

    2008-05-01

    Tuberculosis is an ancient disease that remains a significant global health problem. Because many membrane and membrane-associated proteins of this pathogen represent potential targets for drugs, diagnostic probes or vaccine components, we have analysed Mycobacterium bovis, bacillus Calmette-Guérin (BCG) substrain Moreau, using Triton X-114 for extraction of lipophilic proteins, followed by identification with LC coupled MS/MS. We identified 351 different proteins in total, and 103 (29%) were predicted as integral membrane proteins with at least one predicted transmembrane region and another 84 (23.9%) proteins had a positive grand average of hydropathicity (GRAVY) value, indicating increased probability for membrane association. Altogether 43 predicted lipoproteins (Lpps) were identified which is close to 50% of the total number of Lpps in the genome. Fifty-four proteins, including twenty-four predicted integral membrane proteins and seven predicted Lpps are described for the first time. The proportion of hydrophobic membrane and membrane-associated proteins shows that Triton X-114 is a highly efficient method for extraction of membrane proteins from bacteria, without the need for preisolation of membranes. ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase appear to represent major enzyme complexes in the membrane of Mycobacterium tuberculosis complex organisms.

  20. Protein Bodies of the Soybean

    PubMed Central

    Tombs, M. P.

    1967-01-01

    Some microscope observations of the protein bodies of the cotyledon cells of the soybean (Glycine max) are described, together with changes in their appearance which occur on germination. Density gradient centrifugation permits the isolation of protein bodies from soymeal. They contain about 70% of the protein of the bean. Only 1 protein could be detected in them: glycinin, the major soybean protein. The protein bodies were fractionated to light and heavy fractions. The former contained 97.5% protein, the latter 78.5%. RNA, phytic acid and lipids were also present. The 2 fractions probably differ only in the extent of contamination by other cell fragments. Images PMID:16656574

  1. Fractional channel multichannel analyzer

    DOEpatents

    Brackenbush, Larry W.; Anderson, Gordon A.

    1994-01-01

    A multichannel analyzer incorporating the features of the present invention obtains the effect of fractional channels thus greatly reducing the number of actual channels necessary to record complex line spectra. This is accomplished by using an analog-to-digital converter in the asynscronous mode, i.e., the gate pulse from the pulse height-to-pulse width converter is not synchronized with the signal from a clock oscillator. This saves power and reduces the number of components required on the board to achieve the effect of radically expanding the number of channels without changing the circuit board.

  2. Turning an Extension Aide into an Extension Agent

    ERIC Educational Resources Information Center

    Seevers, Brenda; Dormody, Thomas J.

    2010-01-01

    For any organization to remain sustainable, a renewable source of faculty and staff needs to be available. The Extension Internship Program for Juniors and Seniors in High School is a new tool for recruiting and developing new Extension agents. Students get "hands on" experience working in an Extension office and earn college credit while in high…

  3. Repair of hair bundles in sea anemones by secreted proteins.

    PubMed

    Watson, G M; Mire, P; Hudson, R R

    1998-01-01

    Sea anemones are sessile invertebrates that detect movements of prey using numerous hair bundles located on tentacles surrounding their mouth. Previously we found that hair bundles of anemones are structurally and functionally similar to those of vertebrates. After 10-15 min exposure to calcium depleted buffers, hair bundles in chickens suffer moderate damage from which they recover in 12 h without requiring new protein synthesis [Zhao, Yamoah and Gillespie, Proc. Natl. Acad. Sci. USA 94 (1996) 15469-15474]. We find that after 1 h exposure to calcium free seawater, hair bundles of anemones suffer extensive damage from which they recover in 4 h, apparently because of newly synthesized, secretory proteins called 'repair proteins'. Recovery is delayed in a dose dependent fashion by cycloheximide. In the presence of exogenously added repair proteins, recovery occurs within 8 min and is cycloheximide insensitive. Recovery is ascertained by a bioassay performed on intact specimens, by electrophysiology, and by timelapse video microscopy. Fraction beta, a chromatographic fraction with bioactivity comparable to the complete mixture of repair proteins, consists of complexes having an estimated mass of 2000 kDa. Avidin based cytochemistry suggests that biotinylated fraction beta binds to damaged hair bundles. SDS-PAGE gel electrophoresis demonstrates that fraction beta contains 8-10 polypeptides of 90 kDa or smaller. At least four of these polypeptides apparently are consumed during the repair process. Negatively stained samples of fraction beta are shown by transmission electron microscopy to include filamentous structures similar in length (150 nm) and width (6 nm) to linkages between stereocilia. The filamentous structures can be associated with globular structures (20 nm in diameter). A model is presented wherein repair proteins comprise replacement linkages and enzymes that attach linkages to appropriate membrane proteins. PMID:9472741

  4. Field-flow fractionation of chromosomes

    SciTech Connect

    Giddings, J.C.

    1993-04-01

    The first topic of this project involved the preparation, fractionation by sedimentation/steric Field Flow Fractionation (FFF), and modeling of metaphase chromosomes. After numerous unsuccessful attempts to prepare chromosomes, we have implemented a procedure (in collaboration with Los Alamos National Laboratory) to prepare metaphase chromosomes from Chinese hamster cells. Extensive experimentation was necessary to identify a suitable FFF channel surface to minimize chromosome adsorption and a carrier liquid to stabilize and disperse the chromosomes. Under suitable operating conditions, the Chinese hamster chromosomes were purified from cell debris and partially fractionated. The purified, preenriched chromosomes that can be prepared by sedimentation/steric FFF or produced continuously by continuous SPLITT fractionation provide an enriched feed material for subsequent flow cytometry. In the second project component, flow FFF permitted successful separations of single- from double-stranded circular DNA, double-stranded circular DNAs of various sizes, and linear double-stranded DNA fragments of various lengths. Diffusion coefficients extracted from retention data agreed well with literature data as well as predictions of major polymer theories. The capacity of FFF separations was evaluated to examine potential applications to long DNA chains.

  5. Introducing the fractional order robotic Darwinian PSO

    NASA Astrophysics Data System (ADS)

    Couceiro, Micael S.; Martins, Fernando M. L.; Rocha, Rui P.; Ferreira, Nuno M. F.

    2012-11-01

    The Darwinian Particle Swarm Optimization (DPSO) is an evolutionary algorithm that extends the Particle Swarm Optimization using natural selection to enhance the ability to escape from sub-optimal solutions. An extension of the DPSO to multi-robot applications has been recently proposed and denoted as Robotic Darwinian PSO (RDPSO), benefiting from the dynamical partitioning of the whole population of robots, hence decreasing the amount of required information exchange among robots. This paper further extends the previously proposed algorithm using fractional calculus concepts to control the convergence rate, while considering the robot dynamical characteristics. Moreover, to improve the convergence analysis of the RDPSO, an adjustment of the fractional coefficient based on mobile robot constraints is presented and experimentally assessed with 2 real platforms. Afterwards, this novel fractional-order RDPSO is evaluated in 12 physical robots being further explored using a larger population of 100 simulated mobile robots within a larger scenario. Experimental results show that changing the fractional coefficient does not significantly improve the final solution but presents a significant influence in the convergence time because of its inherent memory property.

  6. A Statistical Treatment of Bioassay Pour Fractions

    NASA Technical Reports Server (NTRS)

    Barengoltz, Jack; Hughes, David W.

    2014-01-01

    The binomial probability distribution is used to treat the statistics of a microbiological sample that is split into two parts, with only one part evaluated for spore count. One wishes to estimate the total number of spores in the sample based on the counts obtained from the part that is evaluated (pour fraction). Formally, the binomial distribution is recharacterized as a function of the observed counts (successes), with the total number (trials) an unknown. The pour fraction is the probability of success per spore (trial). This distribution must be renormalized in terms of the total number. Finally, the new renormalized distribution is integrated and mathematically inverted to yield the maximum estimate of the total number as a function of a desired level of confidence ( P(fraction. The extension to recovery efficiency corrections is also presented. Now the product of recovery efficiency and pour fraction may be small enough that the likely value may be much larger than the usual calculation: the number of spores divided by that product. The use of this analysis would not be limited to microbiological data.

  7. Normal and superfluid fractions of inhomogeneous nonequilibrium quantum fluids

    NASA Astrophysics Data System (ADS)

    Gladilin, Vladimir N.; Wouters, Michiel

    2016-04-01

    We present a theoretical analysis of the normal and superfluid fractions of quantum fluids described by a nonequilibrium extension of the Gross-Pitaevskii equation in the presence of an external potential. Both disordered and regular potentials are considered. The normal and superfluid fractions are defined by the response of the nonequilibrium quantum fluid to a vector potential, in analogy with the equilibrium case. We find that the physical meaning of these definitions breaks down out of equilibrium. The normal and superfluid fractions no longer add up to one and for some types of external potentials they can even become negative.

  8. Fractional Schrödinger equation in optics.

    PubMed

    Longhi, Stefano

    2015-03-15

    In quantum mechanics, the space-fractional Schrödinger equation provides a natural extension of the standard Schrödinger equation when the Brownian trajectories in Feynman path integrals are replaced by Levy flights. Here an optical realization of the fractional Schrödinger equation, based on transverse light dynamics in aspherical optical cavities, is proposed. As an example, a laser implementation of the fractional quantum harmonic oscillator is presented in which dual Airy beams can be selectively generated under off-axis longitudinal pumping. PMID:25768196

  9. Quark matter induced extensive air showers

    SciTech Connect

    Lawson, Kyle

    2011-05-15

    If the dark matter of our Galaxy is composed of nuggets of quarks or antiquarks in a color superconducting phase there will be a small but nonzero flux of these objects through the Earth's atmosphere. A nugget of quark matter will deposit only a small fraction of its kinetic energy in the atmosphere and is likely to be undetectable. If however the impacting object is composed of antiquarks, the energy deposited can be quite large. In this case nuclear annihilations within the nugget will trigger an extensive air shower the particle content of which is similar to that produced by an ultrahigh energy cosmic ray. This paper gives a qualitative description of the basic properties of such a shower. Several distinctions from an air shower initiated by a single ultrahigh energy nucleus will be described, allowing these events to be distinguished from the cosmic ray background. The subtlety of these features may mean that some fraction of the high energy cosmic ray spectrum may in fact be due to this type of dark matter interaction. The estimated flux of dark matter nuggets and the energy deposited in the atmosphere are such that the Pierre Auger Observatory may prove an ideal facility to place constraints on the flux of heavy quark matter objects. This paper attempts to highlight the best techniques to search for a quark matter signature through an extensive air shower signal.

  10. The BGAN extension programme

    NASA Astrophysics Data System (ADS)

    Rivera, Juan J.; Trachtman, Eyal; Richharia, Madhavendra

    2005-11-01

    Mobile satellite telecommunications systems have undergone an enormous evolution in the last decades, with the interest in having advanced telecommunications services available on demand, anywhere and at any time, leading to incredible advances. The demand for braodband data is therefore rapidly gathering pace, but current solutions are finding it increasingly difficult to combine large bandwidth with ubiquitous coverage, reliability and portability. The BGAN (Broadband Global Area Network) system, designed to operate with the Inmarsat-4 satellites, provides breakthrough services that meet all of these requirements. It will enable broadband connection on the move, delivering all the key tools of the modern office. Recognising the great impact that Inmarsat's BGAN system will have on the European satellite communications industry, and the benefits that it will bring to a wide range of European industries, in 2003 ESA initiated the "BGAN Extension" project. Its primary goals are to provide the full range of BGAN services to truly mobile platforms, operating in aeronautical, vehicular and maritime environments, and to introduce a multicast service capability. The project is supported by the ARTES Programme which establishes a collaboration agreement between ESA, Inmarsat and a group of key industrial and academic institutions which includes EMS, Logica, Nera and the University of Surrey (UK).

  11. What is a fractional derivative?

    NASA Astrophysics Data System (ADS)

    Ortigueira, Manuel D.; Tenreiro Machado, J. A.

    2015-07-01

    This paper discusses the concepts underlying the formulation of operators capable of being interpreted as fractional derivatives or fractional integrals. Two criteria for required by a fractional operator are formulated. The Grünwald-Letnikov, Riemann-Liouville and Caputo fractional derivatives and the Riesz potential are accessed in the light of the proposed criteria. A Leibniz rule is also obtained for the Riesz potential.

  12. Positive fractional linear electrical circuits

    NASA Astrophysics Data System (ADS)

    Kaczorek, Tadeusz

    2013-10-01

    The positive fractional linear systems and electrical circuits are addressed. New classes of fractional asymptotically stable and unstable electrical circuits are introduced. The Caputo and Riemann-Liouville definitions of fractional derivatives are used to analysis of the positive electrical circuits composed of resistors, capacitors, coils and voltage (current) sources. The positive fractional electrical and specially unstable different types electrical circuits are analyzed. Some open problems are formulated.

  13. Experimental observation of fractional echoes

    NASA Astrophysics Data System (ADS)

    Karras, G.; Hertz, E.; Billard, F.; Lavorel, B.; Siour, G.; Hartmann, J.-M.; Faucher, O.; Gershnabel, Erez; Prior, Yehiam; Averbukh, Ilya Sh.

    2016-09-01

    We report the observation of fractional echoes in a double-pulse excited nonlinear system. Unlike standard echoes, which appear periodically at delays which are integer multiples of the delay between the two exciting pulses, the fractional echoes appear at rational fractions of this delay. We discuss the mechanism leading to this phenomenon, and provide experimental demonstration of fractional echoes by measuring third harmonic generation in a thermal gas of CO2 molecules excited by a pair of femtosecond laser pulses.

  14. Privatising Agricultural Extension: Caveat Emptor.

    ERIC Educational Resources Information Center

    Kidd, A. D.; Lamers, J. P. A.; Ficarelli, P. P.; Hoffmann, V.

    2000-01-01

    Discusses forces promoting privatization of agricultural extension. Discusses experiences of privatization and commercialization of extension and related problems in various countries, particularly developing countries. Suggests that the state will continue to play an important role in agricultural extension in many countries and that…

  15. Fractional diffusion on bounded domains

    SciTech Connect

    Defterli, Ozlem; D'Elia, Marta; Du, Qiang; Gunzburger, Max Donald; Lehoucq, Richard B.; Meerschaert, Mark M.

    2015-03-13

    We found that the mathematically correct specification of a fractional differential equation on a bounded domain requires specification of appropriate boundary conditions, or their fractional analogue. In this paper we discuss the application of nonlocal diffusion theory to specify well-posed fractional diffusion equations on bounded domains.

  16. Autolysis and extension of isolated walls from growing cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.; Durachko, D. M.

    1994-01-01

    Walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, we treated the walls in various ways to enhance or inhibit long-term wall extension ('creep') and measured autolysis as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, we found no correlation between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. We also found that exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). Our results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.

  17. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-01-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised. PMID:26621372

  18. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-12-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised.

  19. Soot Volume Fraction Imaging

    NASA Technical Reports Server (NTRS)

    Greenberg, Paul S.; Ku, Jerry C.

    1994-01-01

    A new technique is described for the full-field determination of soot volume fractions via laser extinction measurements. This technique differs from previously reported point-wise methods in that a two-dimensional array (i.e., image) of data is acquired simultaneously. In this fashion, the net data rate is increased, allowing the study of time-dependent phenomena and the investigation of spatial and temporal correlations. A telecentric imaging configuration is employed to provide depth-invariant magnification and to permit the specification of the collection angle for scattered light. To improve the threshold measurement sensitivity, a method is employed to suppress undesirable coherent imaging effects. A discussion of the tomographic inversion process is provided, including the results obtained from numerical simulation. Results obtained with this method from an ethylene diffusion flame are shown to be in close agreement with those previously obtained by sequential point-wise interrogation.

  20. Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

    SciTech Connect

    Stekhoven, Daniel J.; Omasits, Ulrich; Quebatte, Maxime; Dehio, Christoph; Ahrens, Christian H.

    2014-03-01

    Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled us to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.

  1. Proteomic analysis of human aqueous humor using multidimensional protein identification technology

    PubMed Central

    Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

    2009-01-01

    Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

  2. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  3. Bioassay, isolation and studies on the mechanism of action of neurite extension factor

    NASA Technical Reports Server (NTRS)

    Kligman, D.

    1984-01-01

    The identification and purification of molecules active in promoting neurite outgrowth requires a sensitive reproducible bioassay. A quantitative bioassay was utilized to purify a neurite extension factor (NEF) based on counting the number of phase bright neurons with processes at least equal to one cell body diameter after 20 hrs. in culture is defined, serum free medium. Using a combination of heat treatment DEAE cellulose chromatography and gel filtration, an acidic protein of M sub r = 75,000 was highly purified. Upon reduction, it yields subunits of M sub r = 37,000. Purified fractions are active half maximally at 100 ng/ml in inducing neurite outgrowth in this bioassay. Currently, monoclonal antibodies to NEF are being produced. Female Balb C mice were immunized with the antigen and fusions with mouse myeloma cells will be performed to yield hybridoma cells.

  4. Extension education and the role of university extension departments

    NASA Astrophysics Data System (ADS)

    Musa, Michael B.

    1994-03-01

    The categorisation by Yesufu is important in delineating the several functions and orientations through which extension education can be effected. Extension education as a catalyst in the field of development education is an imperative for all universities and other allied institutions, whether technological, agricultural, liberal or conventionally oriented. Towards this end, appropriately trained extension education personnel are a necessity. Extension departments have peculiar and unique problems due to their dual orientation. The need, therefore, arises for special attention and consideration in the areas of funding, organisational scheduling, and human and infrastructural resources.

  5. Fractionally charged skyrmions in fractional quantum Hall effect

    NASA Astrophysics Data System (ADS)

    Balram, Ajit C.; Wurstbauer, U.; Wójs, A.; Pinczuk, A.; Jain, J. K.

    2015-11-01

    The fractional quantum Hall effect has inspired searches for exotic emergent topological particles, such as fractionally charged excitations, composite fermions, abelian and nonabelian anyons and Majorana fermions. Fractionally charged skyrmions, which support both topological charge and topological vortex-like spin structure, have also been predicted to occur in the vicinity of 1/3 filling of the lowest Landau level. The fractional skyrmions, however, are anticipated to be exceedingly fragile, suppressed by very small Zeeman energies. Here we show that, slightly away from 1/3 filling, the smallest manifestations of the fractional skyrmion exist in the excitation spectrum for a broad range of Zeeman energies, and appear in resonant inelastic light scattering experiments as well-defined resonances slightly below the long wavelength spin wave mode. The spectroscopy of these exotic bound states serves as a sensitive tool for investigating the residual interaction between composite fermions, responsible for delicate new fractional quantum Hall states in this filling factor region.

  6. Fractionally charged skyrmions in fractional quantum Hall effect.

    PubMed

    Balram, Ajit C; Wurstbauer, U; Wójs, A; Pinczuk, A; Jain, J K

    2015-01-01

    The fractional quantum Hall effect has inspired searches for exotic emergent topological particles, such as fractionally charged excitations, composite fermions, abelian and nonabelian anyons and Majorana fermions. Fractionally charged skyrmions, which support both topological charge and topological vortex-like spin structure, have also been predicted to occur in the vicinity of 1/3 filling of the lowest Landau level. The fractional skyrmions, however, are anticipated to be exceedingly fragile, suppressed by very small Zeeman energies. Here we show that, slightly away from 1/3 filling, the smallest manifestations of the fractional skyrmion exist in the excitation spectrum for a broad range of Zeeman energies, and appear in resonant inelastic light scattering experiments as well-defined resonances slightly below the long wavelength spin wave mode. The spectroscopy of these exotic bound states serves as a sensitive tool for investigating the residual interaction between composite fermions, responsible for delicate new fractional quantum Hall states in this filling factor region. PMID:26608906

  7. Fractionally charged skyrmions in fractional quantum Hall effect

    PubMed Central

    Balram, Ajit C.; Wurstbauer, U.; Wójs, A.; Pinczuk, A.; Jain, J. K.

    2015-01-01

    The fractional quantum Hall effect has inspired searches for exotic emergent topological particles, such as fractionally charged excitations, composite fermions, abelian and nonabelian anyons and Majorana fermions. Fractionally charged skyrmions, which support both topological charge and topological vortex-like spin structure, have also been predicted to occur in the vicinity of 1/3 filling of the lowest Landau level. The fractional skyrmions, however, are anticipated to be exceedingly fragile, suppressed by very small Zeeman energies. Here we show that, slightly away from 1/3 filling, the smallest manifestations of the fractional skyrmion exist in the excitation spectrum for a broad range of Zeeman energies, and appear in resonant inelastic light scattering experiments as well-defined resonances slightly below the long wavelength spin wave mode. The spectroscopy of these exotic bound states serves as a sensitive tool for investigating the residual interaction between composite fermions, responsible for delicate new fractional quantum Hall states in this filling factor region. PMID:26608906

  8. Fractionally charged skyrmions in fractional quantum Hall effect.

    PubMed

    Balram, Ajit C; Wurstbauer, U; Wójs, A; Pinczuk, A; Jain, J K

    2015-11-26

    The fractional quantum Hall effect has inspired searches for exotic emergent topological particles, such as fractionally charged excitations, composite fermions, abelian and nonabelian anyons and Majorana fermions. Fractionally charged skyrmions, which support both topological charge and topological vortex-like spin structure, have also been predicted to occur in the vicinity of 1/3 filling of the lowest Landau level. The fractional skyrmions, however, are anticipated to be exceedingly fragile, suppressed by very small Zeeman energies. Here we show that, slightly away from 1/3 filling, the smallest manifestations of the fractional skyrmion exist in the excitation spectrum for a broad range of Zeeman energies, and appear in resonant inelastic light scattering experiments as well-defined resonances slightly below the long wavelength spin wave mode. The spectroscopy of these exotic bound states serves as a sensitive tool for investigating the residual interaction between composite fermions, responsible for delicate new fractional quantum Hall states in this filling factor region.

  9. Fractionally charged skyrmions in fractional quantum Hall effect

    SciTech Connect

    Balram, Ajit C.; Wurstbauer, U.; Wójs, A.; Pinczuk, A.; Jain, J. K.

    2015-11-26

    The fractional quantum Hall effect has inspired searches for exotic emergent topological particles, such as fractionally charged excitations, composite fermions, abelian and nonabelian anyons and Majorana fermions. Fractionally charged skyrmions, which support both topological charge and topological vortex-like spin structure, have also been predicted to occur in the vicinity of 1/3 filling of the lowest Landau level. The fractional skyrmions, however, are anticipated to be exceedingly fragile, suppressed by very small Zeeman energies. Here we show that, slightly away from 1/3 filling, the smallest manifestations of the fractional skyrmion exist in the excitation spectrum for a broad range of Zeeman energies, and appear in resonant inelastic light scattering experiments as well-defined resonances slightly below the long wavelength spin wave mode. The spectroscopy of these exotic bound states serves as a sensitive tool for investigating the residual interaction between composite fermions, responsible for delicate new fractional quantum Hall states in this filling factor region.

  10. Fractional telegrapher's equation from fractional persistent random walks

    NASA Astrophysics Data System (ADS)

    Masoliver, Jaume

    2016-05-01

    We generalize the telegrapher's equation to allow for anomalous transport. We derive the space-time fractional telegrapher's equation using the formalism of the persistent random walk in continuous time. We also obtain the characteristic function of the space-time fractional process and study some particular cases and asymptotic approximations. Similarly to the ordinary telegrapher's equation, the time-fractional equation also presents distinct behaviors for different time scales. Specifically, transitions between different subdiffusive regimes or from superdiffusion to subdiffusion are shown by the fractional equation as time progresses.

  11. Fractional telegrapher's equation from fractional persistent random walks.

    PubMed

    Masoliver, Jaume

    2016-05-01

    We generalize the telegrapher's equation to allow for anomalous transport. We derive the space-time fractional telegrapher's equation using the formalism of the persistent random walk in continuous time. We also obtain the characteristic function of the space-time fractional process and study some particular cases and asymptotic approximations. Similarly to the ordinary telegrapher's equation, the time-fractional equation also presents distinct behaviors for different time scales. Specifically, transitions between different subdiffusive regimes or from superdiffusion to subdiffusion are shown by the fractional equation as time progresses. PMID:27300830

  12. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method extension to quantify simultaneously melamine and cyanuric acid in egg powder and soy protein in addition to milk products.

    PubMed

    Rodriguez Mondal, Ana Mary; Desmarchelier, Aurélien; Konings, Erik; Acheson-Shalom, Ruth; Delatour, Thierry

    2010-11-24

    As a consequence of the adulteration of infant formulas and milk powders with melamine (MEL) in China in 2008, much attention has been devoted to the analysis of MEL [and cyanuric acid (CA)] in dairy products. Several methods based on high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR), or Raman spectroscopy have been described in the literature. However, no method is available for the simultaneous determination of MEL and CA in other raw materials, which are considered as high-risk materials for economically motivated adulteration. The present paper reports the results of an interlaboratory-based performance evaluation conducted with seven laboratories worldwide. The purpose was to demonstrate the ability of a cleanup-free LC-MS/MS method, originally developed for cow's milk and milk-powdered infant formula, to quantify MEL and CA in egg powder and soy protein. Limit of detection (LOD) and limit of quantification (LOQ) were 0.02 and 0.05 mg/kg for MEL in egg powder and soy protein, respectively. For CA, LOD and LOQ were 0.05 and 0.10 mg/kg in egg powder and 1.0 and 1.50 mg/kg in soy protein, respectively. Recoveries ranged within a 97-113% range for both MEL and CA in egg powder and soy protein. Reproducibility values (RSD(R)) from seven laboratories were within a 5.4-11.7% range for both analytes in the considered matrices. Horwitz ratio (HorRat) values between 0.4 and 0.7 indicate acceptable among-laboratory precision for the method described. PMID:21038852

  13. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method extension to quantify simultaneously melamine and cyanuric acid in egg powder and soy protein in addition to milk products.

    PubMed

    Rodriguez Mondal, Ana Mary; Desmarchelier, Aurélien; Konings, Erik; Acheson-Shalom, Ruth; Delatour, Thierry

    2010-11-24

    As a consequence of the adulteration of infant formulas and milk powders with melamine (MEL) in China in 2008, much attention has been devoted to the analysis of MEL [and cyanuric acid (CA)] in dairy products. Several methods based on high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR), or Raman spectroscopy have been described in the literature. However, no method is available for the simultaneous determination of MEL and CA in other raw materials, which are considered as high-risk materials for economically motivated adulteration. The present paper reports the results of an interlaboratory-based performance evaluation conducted with seven laboratories worldwide. The purpose was to demonstrate the ability of a cleanup-free LC-MS/MS method, originally developed for cow's milk and milk-powdered infant formula, to quantify MEL and CA in egg powder and soy protein. Limit of detection (LOD) and limit of quantification (LOQ) were 0.02 and 0.05 mg/kg for MEL in egg powder and soy protein, respectively. For CA, LOD and LOQ were 0.05 and 0.10 mg/kg in egg powder and 1.0 and 1.50 mg/kg in soy protein, respectively. Recoveries ranged within a 97-113% range for both MEL and CA in egg powder and soy protein. Reproducibility values (RSD(R)) from seven laboratories were within a 5.4-11.7% range for both analytes in the considered matrices. Horwitz ratio (HorRat) values between 0.4 and 0.7 indicate acceptable among-laboratory precision for the method described.

  14. Shelf-life extension of refrigerated sea bass slices wrapped with fish protein isolate/fish skin gelatin-ZnO nanocomposite film incorporated with basil leaf essential oil.

    PubMed

    Arfat, Yasir Ali; Benjakul, Soottawat; Vongkamjan, Kitiya; Sumpavapol, Punnanee; Yarnpakdee, Suthasinee

    2015-10-01

    Microbiological, chemical and sensory changes of sea bass slices wrapped with fish protein isolate (FPI)/fish skin gelatin (FSG) films incorporated with 3 % ZnO nanoparticles (ZnONP) (w/w, based on protein content) and 100 % basil leaf essential oil (BEO) (w/w, based on protein content) during storage of 12 days at 4 °C were investigated. Sea bass slices wrapped with FPI/FSG-ZnONP-BEO film had the lowest growth of psychrophilic bacteria, lactic acid bacteria and spoilage microorganisms including Pseudomonas , H2S-producing bacteria and Enterobacteriaceae throughout storage of 12 days in comparison with those wrapped with FPI/FSG-BEO, FPI/FSG-ZnONP, FPI/FSG film, polypropylene film (PP film) and the control (without wrapping), respectively (P < 0.05). Lowered increases in pH, total volatile base, peroxide value and TBARS value were found in FPI/FSG-ZnO-BEO film wrapped samples, compared with others (P < 0.05). Sensory evaluation revealed that shelf-life of sea bass slices was longest for samples wrapped with FPI/FSG-ZnONP-BEO film (12 days), as compared to the control (6 days) (P < 0.05). PMID:26396365

  15. Implementation of molecular dynamics and its extensions with the coarse-grained UNRES force field on massively parallel systems; towards millisecond-scale simulations of protein structure, dynamics, and thermodynamics.

    PubMed

    Liwo, Adam; Ołdziej, Stanisław; Czaplewski, Cezary; Kleinerman, Dana S; Blood, Philip; Scheraga, Harold A

    2010-03-01

    We report the implementation of our united-residue UNRES force field for simulations of protein structure and dynamics with massively parallel architectures. In addition to coarse-grained parallelism already implemented in our previous work, in which each conformation was treated by a different task, we introduce a fine-grained level in which energy and gradient evaluation are split between several tasks. The Message Passing Interface (MPI) libraries have been utilized to construct the parallel code. The parallel performance of the code has been tested on a professional Beowulf cluster (Xeon Quad Core), a Cray XT3 supercomputer, and two IBM BlueGene/P supercomputers with canonical and replica-exchange molecular dynamics. With IBM BlueGene/P, about 50 % efficiency and 120-fold speed-up of the fine-grained part was achieved for a single trajectory of a 767-residue protein with use of 256 processors/trajectory. Because of averaging over the fast degrees of freedom, UNRES provides an effective 1000-fold speed-up compared to the experimental time scale and, therefore, enables us to effectively carry out millisecond-scale simulations of proteins with 500 and more amino-acid residues in days of wall-clock time.

  16. Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

    PubMed

    Ballot, E; Desbos, A; Monier, J C

    1996-09-01

    Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH. PMID:8811044

  17. Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth

    DOE PAGESBeta

    Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea; Hettich, Robert L.

    2015-06-25

    The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, includingmore » membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.« less

  18. Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth

    SciTech Connect

    Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea; Hettich, Robert L.

    2015-06-25

    The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.

  19. 35. WEST END ELEVATION, PROPOSED EXTENSION OF COAL HOUSE, EXTENSIONS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. WEST END ELEVATION, PROPOSED EXTENSION OF COAL HOUSE, EXTENSIONS OF ENGINE AND COAL HOUSES, DEER ISLAND PUMPING STATION, METROPOLITAN WATER AND SEWERAGE BOARD, METROPOLITAN SEWERAGE WORKS, JANUARY 1908, SHEET NO. 7. Aperture card 6498-7. - Deer Island Pumping Station, Boston, Suffolk County, MA

  20. 34. PLAN, PROPOSED EXTENSION OF COAL HOUSE, EXTENSIONS OF ENGINE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    34. PLAN, PROPOSED EXTENSION OF COAL HOUSE, EXTENSIONS OF ENGINE AND COAL HOUSES, DEER ISLAND PUMPING STATION, METROPOLITAN WATER AND SEWERAGE BOARD, METROPOLITAN SEWARAGE WORKS, JANUARY 1909, SHEET NO. 11. Aperture card 6498-11. - Deer Island Pumping Station, Boston, Suffolk County, MA

  1. Welding torch gas cup extension

    NASA Technical Reports Server (NTRS)

    Gordon, Stephen S. (Inventor)

    1988-01-01

    The invention relates to a gas shielded electric arc welding torch having a detachable gas cup extension which may be of any desired configuration or length. The gas cup extension assembly is mounted on a standard electric welding torch gas cup to enable welding in areas with limited access. The gas cup assembly has an upper tubular insert that fits within the gas cup such that its lower portion protrudes thereform and has a lower tubular extension that is screwed into the lower portion. The extension has a rim to define the outer perimeter of the seat edge about its entrance opening so a gasket may be placed to effect an airtight seal between the gas cup and extension. The tubular extension may be made of metal or cermaic material that can be machined. The novelty lies in the use of an extension assembly for a standard gas cup of an electric arc welding torch which extension assembly is detachable permitting the use of a number of extensions which may be of different configurations and materials and yet fit the standard gas cup.

  2. Robotic hand with modular extensions

    SciTech Connect

    Salisbury, Curt Michael; Quigley, Morgan

    2015-01-20

    A robotic device is described herein. The robotic device includes a frame that comprises a plurality of receiving regions that are configured to receive a respective plurality of modular robotic extensions. The modular robotic extensions are removably attachable to the frame at the respective receiving regions by way of respective mechanical fuses. Each mechanical fuse is configured to trip when a respective modular robotic extension experiences a predefined load condition, such that the respective modular robotic extension detaches from the frame when the load condition is met.

  3. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  4. Method development for VOST Fractionator

    SciTech Connect

    St. Germain Wickham, M.E.; Cummins, S.B.; Radolovich, G. )

    1994-01-01

    A VOST Fractionator was designed and tested to fractionate an original VOST sample into two samples: one large and one small sample. The device allows for quantitation of high levels of compounds in the small fraction and trace levels in the large fraction. Several preliminary validation samples were prepared, split, and analyzed to test the feasibility of the VOST Fractionator. These validation samples contained 40,000 ng of three terpene compounds and 100 ng of 42 other volatile target analytes. Analyte recoveries ranged from 70 to 130 percent, except for five water-soluble compounds. Recovery for the terpene compounds was 110 to 118 percent. Precision for triplicate spiked samples was less than 30 percent relative standard deviation (%RSD) for most compounds. Results indicate that the VOST Fractionator accurately splits the sample and allows quantitation of extremely high levels of compounds without sacrificing sensitivity for trace compounds. 3 refs., 1 fig., 3 tabs.

  5. Nonbiological fractionation of iron isotopes

    NASA Technical Reports Server (NTRS)

    Anbar, A. D.; Roe, J. E.; Barling, J.; Nealson, K. H.

    2000-01-01

    Laboratory experiments demonstrate that iron isotopes can be chemically fractionated in the absence of biology. Isotopic variations comparable to those seen during microbially mediated reduction of ferrihydrite are observed. Fractionation may occur in aqueous solution during equilibration between inorganic iron complexes. These findings provide insight into the mechanisms of iron isotope fractionation and suggest that nonbiological processes may contribute to iron isotope variations observed in sediments.

  6. A fucose containing polymer-rich fraction from the brown alga Ascophyllum nodosum mediates lifespan increase and thermal-tolerance in Caenorhabditis elegans, by differential effects on gene and protein expression.

    PubMed

    Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin D; Critchley, Alan T; Zhang, Junzeng; Fitton, J H; Stringer, Damien N; Gardiner, Vicki-Anne; Prithiviraj, Balakrishnan

    2014-02-01

    The extracts of the brown alga, Ascophyllum nodosum, which contains several bioactive compounds, have been shown to impart biotic and abiotic stress tolerance properties when consumed by animals. However, the physiological, biochemical and molecular mechanism underlying such effects remain elusive. We investigated the effect of A. nodosum fucose-containing polymer (FCP) on tolerance to thermally induced stress using the invertebrate animal model, Caenorhabditis elegans. FCP at a concentration of 150 μg mL(-1) significantly improved the life span and tolerance against thermally induced stress in C. elegans. The treatment increased the C. elegans survival by approximately 24%, when the animals were under severe thermally induced stress (i.e. 35 °C) and 27% under mild stress (i.e. 30 °C) conditions. The FCP induced differential expression of genes and proteins is associated with stress response pathways. Under thermal stress, FCP treatment significantly altered the expression of 65 proteins (54 up-regulated & 11 down-regulated). Putative functional analysis of FCP-induced differential proteins signified an association of altered proteins in stress-related molecular and biochemical pathways of the model worm. PMID:24323434

  7. A fucose containing polymer-rich fraction from the brown alga Ascophyllum nodosum mediates lifespan increase and thermal-tolerance in Caenorhabditis elegans, by differential effects on gene and protein expression.

    PubMed

    Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin D; Critchley, Alan T; Zhang, Junzeng; Fitton, J H; Stringer, Damien N; Gardiner, Vicki-Anne; Prithiviraj, Balakrishnan

    2014-02-01

    The extracts of the brown alga, Ascophyllum nodosum, which contains several bioactive compounds, have been shown to impart biotic and abiotic stress tolerance properties when consumed by animals. However, the physiological, biochemical and molecular mechanism underlying such effects remain elusive. We investigated the effect of A. nodosum fucose-containing polymer (FCP) on tolerance to thermally induced stress using the invertebrate animal model, Caenorhabditis elegans. FCP at a concentration of 150 μg mL(-1) significantly improved the life span and tolerance against thermally induced stress in C. elegans. The treatment increased the C. elegans survival by approximately 24%, when the animals were under severe thermally induced stress (i.e. 35 °C) and 27% under mild stress (i.e. 30 °C) conditions. The FCP induced differential expression of genes and proteins is associated with stress response pathways. Under thermal stress, FCP treatment significantly altered the expression of 65 proteins (54 up-regulated & 11 down-regulated). Putative functional analysis of FCP-induced differential proteins signified an association of altered proteins in stress-related molecular and biochemical pathways of the model worm.

  8. Trigonometric Integrals via Partial Fractions

    ERIC Educational Resources Information Center

    Chen, H.; Fulford, M.

    2005-01-01

    Parametric differentiation is used to derive the partial fractions decompositions of certain rational functions. Those decompositions enable us to integrate some new combinations of trigonometric functions.

  9. Carbon isotopic fractionation in heterotrophic microbial metabolism

    NASA Technical Reports Server (NTRS)

    Blair, N.; Leu, A.; Munoz, E.; Olsen, J.; Kwong, E.; Des Marais, D.

    1985-01-01

    Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4 percent depleted in C-13 relative to the glucose used as the carbon source, whereas the acetate was 12.3 percent enriched in C-13. The acetate C-13 enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6 percent depleted in C-13, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7 percent, respectively. Aspartic and glutamic acids were -1.6 and +2.7 percent, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.

  10. Carbon isotopic fractionation in heterotrophic microbial metabolism

    SciTech Connect

    Blair, N.; Leu, A.; Munoz, E.; Olsen, J.; Kwong, E.; Des Marais, D.

    1985-10-01

    Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4 percent depleted in C-13 relative to the glucose used as the carbon source, whereas the acetate was 12.3 percent enriched in C-13. The acetate C-13 enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6 percent depleted in C-13, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7 percent, respectively. Aspartic and glutamic acids were -1.6 and +2.7 percent, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle. 38 references.

  11. Extensive exchange of rat liver microsomal phospholipids.

    PubMed

    Zilversmit, D B; Hughes, M E

    1977-08-15

    Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.

  12. Extensive exchange of rat liver microsomal phospholipids.

    PubMed

    Zilversmit, D B; Hughes, M E

    1977-08-15

    Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer. PMID:889827