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Sample records for acidic isoelectric point

  1. A Study on Amino Acids: Synthesis of Alpha-Aminophenylacetic Acid (Phenylglycine) and Determination of its Isoelectric Point.

    ERIC Educational Resources Information Center

    Barrelle, M.; And Others

    1983-01-01

    Background information and procedures are provided for an experimental study on aminophenylacetic acid (phenylglycine). These include physical chemistry (determination of isoelectric point by pH measurement) and organic chemistry (synthesis of an amino acid in racemic form) experiments. (JN)

  2. Isoelectric Point, Electric Charge, and Nomenclature of the Acid-Base Residues of Proteins

    ERIC Educational Resources Information Center

    Maldonado, Andres A.; Ribeiro, Joao M.; Sillero, Antonio

    2010-01-01

    The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. (Contains 8 figures and 4 tables.)

  3. Isoelectric Point Separations of Peptides and Proteins.

    PubMed

    Pergande, Melissa R; Cologna, Stephanie M

    2017-01-25

    The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies.

  4. Isoelectric Point Separations of Peptides and Proteins

    PubMed Central

    Pergande, Melissa R.; Cologna, Stephanie M.

    2017-01-01

    The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies. PMID:28248255

  5. ISOELECTRIC POINTS FOR THE MYCELIUM OF FUNGI

    PubMed Central

    Robbins, William J.

    1924-01-01

    1. Mycelium of Rhizopus nigricans when stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.0. 2. When grown on potato dextrose agar the reaction of which was varied with phosphoric acid the extent of colony growth of Rhizopus nigricans plotted against the initial Sörensen value of the agar produced a double maximum curve with the minimum between the two maxima at initial pH 5.2. 3. When grown in potato dextrose broth the reaction of which was varied with phosphoric acid the dry matter produced by Rhizopus nigricans plotted against the Sörensen value of the broth produced a double maximum curve with the minimum between the two maxima at initial pH 5.2 or average pH 4.9. 4. Mycelium of Rhizopus nigricans placed in buffer mixtures of 0.01 M phosphoric acid and sodium hydroxide of pH 4.1 to 6.3, changed the reaction in most cases toward greater alkalinity. 5. Mycelium of Fusarium lycopersici stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.5. PMID:19872067

  6. Plant Cuticles Are Polyelectrolytes with Isoelectric Points around Three 1

    PubMed Central

    Schönherr, Jörg; Huber, Rudolf

    1977-01-01

    The isoelectric points of isolated cuticles from Citrus aurantium L. (3.15), Prunus armeniaca L. (3.45), and Pyrus communis L. (2.90) leaves were determined from membrane potentials. At pH values below the isoelectric point, cuticular membranes carry a net positive charge and are permselective to anions (determined using 82Br−). Above the isoelectric point, they carry a net negative charge and are permselective to cations (determined using 24Na+). There are no gradients of fixed charges across the cuticular membranes as indicated by the absence of asymmetry potentials. Positive charges in the membranes originate from residues of basic amino acids of proteins or polypeptides contained in a nonextractable form within the cuticle. The exchange capacity of basic fixed groups in the cuticles of six species (Lycopersicon esculentum Mill., Capsicum annuum L. fruit cuticles, and Brassaia spec. leaf cuticles in addition to the above species) varied between 0.010 and 0.025 meq g−1 cuticle. Fixed acidic groups were donated by residues of acidic amino acids, polygalacturonic acid, and nonesterified -COOH groups of the cutin polymer. At pH 8, total cation exchange capacity as determined using 45Ca2+ varied between 0.26 (Citrus) and 0.30 (apricot) meq g−1. PMID:16659804

  7. Bistable isoelectric point photoswitching in green fluorescent proteins observed by dynamic immunoprobed isoelectric focusing.

    PubMed

    Hughes, Alex J; Tentori, Augusto M; Herr, Amy E

    2012-10-24

    We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.

  8. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    ERIC Educational Resources Information Center

    Browning, Mark; Vanable, Joseph

    2002-01-01

    Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

  9. Proteome-pI: proteome isoelectric point database

    PubMed Central

    Kozlowski, Lukasz P.

    2017-01-01

    Proteome-pI is an online database containing information about predicted isoelectric points for 5029 proteomes calculated using 18 methods. The isoelectric point, the pH at which a particular molecule carries no net electrical charge, is an important parameter for many analytical biochemistry and proteomics techniques, especially for 2D gel electrophoresis (2D-PAGE), capillary isoelectric focusing, liquid chromatography–mass spectrometry and X-ray protein crystallography. The database, available at http://isoelectricpointdb.org allows the retrieval of virtual 2D-PAGE plots and the development of customised fractions of proteome based on isoelectric point and molecular weight. Moreover, Proteome-pI facilitates statistical comparisons of the various prediction methods as well as biological investigation of protein isoelectric point space in all kingdoms of life. For instance, using Proteome-pI data, it is clear that Eukaryotes, which evolved tight control of homeostasis, encode proteins with pI values near the cell pH. In contrast, Archaea living frequently in extreme environments can possess proteins with a wide range of isoelectric points. The database includes various statistics and tools for interactive browsing, searching and sorting. Apart from data for individual proteomes, datasets corresponding to major protein databases such as UniProtKB/TrEMBL and the NCBI non-redundant (nr) database have also been precalculated and made available in CSV format. PMID:27789699

  10. In-capillary enrichment, proteolysis and separation using capillary electrophoresis with discontinuous buffers: application on proteins with moderately acidic and basic isoelectric points.

    PubMed

    Nesbitt, Chandra A; Yeung, Ken K-C

    2009-01-01

    Advances in mass spectrometry and capillary-format separation continue to improve the sensitivity of protein analysis. Of equal importance is the miniaturization of sample pretreatment such as enrichment and proteolysis. In a previous report (Nesbitt et al., Electrophoresis, 2008, 29, 466-474), nanoliter-volume protein enrichment, tryptic digestion, and partial separation was demonstrated in capillary electrophoresis followed by MALDI mass spectral analysis. A discontinuous buffer system, consisting of ammonium (pH 10) and acetate (pH 4), was used to create a pH junction inside the capillary, trapping a protein with a neutral isoelectric point, myoglobin (pI 7.2). Moreover, co-enrichment of myoglobin with trypsin led to an in-capillary digestion. In this paper, the ability of this discontinuous buffer system to perform similar in-capillary sample pretreatment on proteins with moderately acidic and basic pI was studied and reported. Lentil lectin (pI 8.6) and a multi-phosphorylated protein, beta-casein (pI 5.1), were selected as model proteins. In addition to the previously shown tryptic digestion, proteolysis with endoproteinase Asp-N was also performed. Digestion of these acidic and basic pI proteins produced a few peptides with extreme pI values lying outside the trapping range of the discontinuous buffer. An alteration in the peptide trapping procedure was made to accommodate these analytes. Offline MALDI mass spectral analysis confirmed the presence of the expected peptides. The presented miniaturized sample pretreatment methodology was proven to be applicable on proteins with a moderately wide range of pI. Flexibility in the choice of protease was also evident.

  11. Isoelectric point determination of live polioviruses by capillary isoelectric focusing with whole column imaging detection.

    PubMed

    Thomassen, Yvonne E; van Eikenhorst, Gerco; van der Pol, Leo A; Bakker, Wilfried A M

    2013-06-18

    Using a capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) method, the isoelectric points (pI) of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of inactivated polio vaccines (IPV)-Mahoney (type 1), MEF (type 2), and Saukett (type 3)-as well as for attenuated oral polio vaccines (OPV) and Sabin types 1, 2, and 3. A method for analyzing biological hazardous components (biological safety level 2) was set up for the CIEF-WCID analyzer used. This method is based on closed circuits. The determined pI's were 6.2 for Mahoney, 6.7 for MEF-1, and 5.8 for Saukett. The pI's of Sabin types 1, 2, and 3 viruses were 7.4, 7.2, and 6.3, respectively. Resolution of the virus peaks was shown to be reproducible. Using this adjusted CIEF-WCID technique, the pI of biologically hazardous components like toxins or viruses can be determined, which is beneficial for the development of vaccine production methods among others.

  12. The differences in the isoelectric points of biofilm-positive and biofilm-negative Candida parapsilosis strains.

    PubMed

    Ruzicka, Filip; Horka, Marie; Hola, Veronika; Kubesova, Anna; Pavlik, Tomas; Votava, Miroslav

    2010-03-01

    The isoelectric points of 39 Candida parapsilosis strains were determined by means of capillary isoelectric focusing. The value of the isoelectric point corresponded well with cell surface hydrophobicity, as well as with the ability to form biofilm in these yeasts.

  13. Tuning the isoelectric point of graphene by electrochemical functionalization

    PubMed Central

    Zuccaro, Laura; Krieg, Janina; Desideri, Alessandro; Kern, Klaus; Balasubramanian, Kannan

    2015-01-01

    The ability to control the charge-potential landscape at solid-liquid interfaces is pivotal to engineer novel devices for applications in sensing, catalysis and energy conversion. The isoelectric point (pI)/point of zero charge (pzc) of graphene plays a key role in a number of physico-chemical phenomena occurring at the graphene-liquid interface. Supported by theory, we present here a methodology to identify the pI/pzc of (functionalized) graphene, which also allows for estimating the nature and extent of ion adsorption. The pI of bare graphene (as-prepared, chemical vapor deposition (CVD)-grown) is found to be less than 3.3, which we can continuously modify up to 7.5 by non-covalent electrochemical attachment of aromatic amino groups, preserving the favorable electronic properties of graphene throughout. Modelling all the observed results with detailed theory, we also show that specific adsorption of ions and the substrate play only an ancillary role in our capability to tune the pI of graphene. PMID:26134956

  14. pH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) hydrogels with reduced ionic strength loading solutions offer improved oral delivery potential for high isoelectric point-exhibiting therapeutic proteins

    PubMed Central

    Koetting, Michael C.; Peppas, Nicholas A.

    2014-01-01

    pH-Responsive hydrogels comprised of itaconic acid copolymerized with N-vinylpyrrolidone (P(IA-co-NVP)) were synthesized and tested as carriers for the oral delivery of high isoelectric point (pI) exhibiting therapeutic proteins. Swelling studies show that P(IA-co-NVP) hydrogels exhibit significantly greater and faster pH-responsive swelling than previously studied methacrylic acid-based hydrogels, achieving up to 68% greater equilibrium swelling and 10.4 times greater swelling in time-limited experiments. Using salmon calcitonin as a model high pI protein therapeutic, we show that P(IA-co-NVP) hydrogels exhibit significantly greater delivery potential than methacrylic acid-based hydrogels. Additionally, we show that utilizing a lower ionic strength solution during drug loading significantly improves drug delivery potential for high pI therapeutics. By using a 1.5 mM PBS buffer rather than the standard 150 mM PBS buffer during loading, up to 83 times as much calcitonin can be delivered in neutral conditions, with up to a 9.6 fold improvement in percent release. Using P(IA-co-NVP) hydrogel microparticles and a low ionic strength loading solution, up to 48 μg calcitonin/mg hydrogel can be delivered in small intestinal conditions. Based on expected absorption in the small intestine, this is sufficient delivery potential for achieving therapeutic dosage via a single, regularly-sized pill taken daily. PMID:24853463

  15. Occurrence of two phosphorylated forms of yeast fructose-1,6-bisphosphatase with different isoelectric points.

    PubMed

    Burlini, N; Facheris, P; Tortora, P; Guerritore, A

    1988-12-09

    Yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) immunoprecipitated from glucose-derepressed wild-type cells and subjected to isoelectric focusing, appears as a unique peak, essentially homogeneous and devoid of incorporated phosphate. However, after cell incubation with glucose, two phosphorylated forms are detectable. The isoelectric point of one is higher and of the other is lower than that of the native form. In contrast, in the mutant ABYS1 which is deficient in several vacuolar proteinases (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343), only the more acidic phospho form appears after cell incubation with glucose. However, sequence data rule out the possibility that limited proteolysis is the event responsible for the appearance of the more basic form of the phosphoenzyme. Nevertheless, time courses of glucose-induced inactivation of fructose-1,6-bisphosphatase show that the enzyme undergoes a substantially slower inactivation in the ABYS1 mutant as compared to the wild-type. These findings point to a degradative mechanism involving, besides the well-known phosphorylation, an additional as yet unknown modification which probably sensitizes the enzyme to proteolytic attack; furthermore, the enzyme responsible for such a modification seems to require one or more of the vacuolar proteinases missing in the mutant for its maturation.

  16. A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing.

    PubMed

    Lecoeur, Marie; Goossens, Jean-François; Vaccher, Claude; Bonte, Jean-Paul; Foulon, Catherine

    2011-10-01

    Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico-chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV-detectable pI markers using a central composite design. The separation was performed in a fused-silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol-water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.

  17. Determination of isoelectric points of metals and metallic alloys by adhesion of latex particles.

    PubMed

    Lefèvre, Grégory; Cerović, Ljiljana; Milonjić, Slobodan; Fédoroff, Michel; Finne, Jörgen; Jaubertie, Anne

    2009-09-15

    A set-up and a method were developed to determine the isoelectric point of metals and metallic alloys samples (stainless steels, inconel, zircaloy, aluminum and dural) by measuring the adhesion rate of negative latex particles. The concentration of polystyrene spheres with surface carboxylate groups (initially 0.5-1 mg L(-1)) in contact with metallic samples was measured as a function of pH and time by turbidimetry. The simulation of measurements by a model predicting the sticking coefficient based on DLVO theory was used for the determination of the isoelectric point from experimental results. It was found that the isoelectric points of aluminum (8.7) and dural (9.1), treated by boiling water, are close to those of hydrated aluminum oxides powders. For stainless steels, inconel and zircaloy, the values of isoelectric points were found to be between 2.4 and 3.0, far below the isoelectric points measured for metallic oxides constituting the alloy surface layer. This difference was explained by two different charging mechanisms: (1) deprotonation of hydroxyl groups on the surface of the metal oxide in suspension or as a thick layer, (2) adsorption of hydroxide ions on a metal surface covered by a thin oxide layer, as observed on hydrophobic surfaces.

  18. Fluorescence-labeled peptides as isoelectric point (pI) markers in capillary isoelectric focusing with fluorescence detection.

    PubMed

    Shimura, K; Kasai, K

    1995-08-01

    Commercially available peptides, mostly angiotensin derivatives, were labeled at their N-terminal amino group with 5-carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent pI markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversed-phase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their pIs were determined by comigration with protein-pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser-induced fluorescence detection (He-Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 x 10(-12) M (0.8 amol). Tetramethylrhodamine-labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the pIs of the fluorescent derivatives of the lectin were determined by using the fluorescence-labeled peptides as pI markers.

  19. Effect of pH on antigen binding by clonotypic antibodies with different isoelectric points

    SciTech Connect

    Endo, Y.; Miyai, K.; Hata, N.; Iijima, Y.

    1987-02-01

    Polyclonal rabbit antibodies to thyroxine, human myoglobin, human growth hormone, human thyrotropin, human alpha-fetoprotein, and human thyroglobulin were fractionated into clonotypic antibodies with different isoelectric points by agarose isoelectric focusing or chromatofocusing. The effect of pH on the binding of these antigens by their respective clonotypic antibodies was assessed by radioimmunoassay. The profiles of the pH effect differed both for different antigens and for different pI's of the antibodies used. The pH optima in the radioimmunoassays for protein antigens were found to be expressed as a function of pI and molecular weight of both antigen and antibody molecules.

  20. Rat small-intestinal β-galactosidases. Studies on the fractionation of `acid' β-galactosidase with isoelectric focusing, gel filtration and ion-exchange chromatography

    PubMed Central

    Asp, Nils-Georg

    1970-01-01

    1. Different forms of the rat small-intestinal `acid' β-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of `acid' β-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had Kav. 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of `acid' β-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin. PMID:5420050

  1. Isoelectric point of cell-free K99 antigen exhibiting hemagglutinating properties.

    PubMed Central

    Morris, J A; Stevens, A E; Sojka, W J

    1978-01-01

    The isoelectric point of the K99 antigen in partially purified preparations isolated from Escherichia coli B41 was 4.2. Electrofocused K99 antigen hemagglutinated guinea pig and sheep erythrocytes and gave a single precipitin line on diffusion against antisera to E. coli B41 and absorbed factor K99 antisera. PMID:346483

  2. HPLC separation of human serum albumin isoforms based on their isoelectric points.

    PubMed

    Turell, Lucía; Botti, Horacio; Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

  3. HPLC separation of human serum albumin isoforms based on their isoelectric points

    PubMed Central

    Bonilla, Lucía; Torres, María José; Schopfer, Francisco; Freeman, Bruce A.; Armas, Larissa; Ricciardi, Alejandro; Alvarez, Beatriz; Radi, Rafael

    2014-01-01

    Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA–SHg+), HSA with Cys34 oxidized to sulfenic acid (HSA–SOH) and HSA oxidized to sulfinate anion (HSA–SO2−) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3–585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA PMID:24316526

  4. Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

    PubMed

    Poehler, Elisabeth; Herzog, Christin; Lotter, Carsten; Pfeiffer, Simon A; Aigner, Daniel; Mayr, Torsten; Nagl, Stefan

    2015-11-21

    We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and β-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

  5. Approximate calculation and experimental derivation of native isoelectric points of membrane protein complexes of Arabidopsis chloroplasts and mitochondria.

    PubMed

    Behrens, Christof; Hartmann, Kristina; Sunderhaus, Stephanie; Braun, Hans-Peter; Eubel, Holger

    2013-03-01

    Electric charges are important intrinsic properties of proteins. They directly affect functionality and also mediate interactions with other molecules such as cofactors, substrates and regulators of enzymatic activity, with lipids as well as other proteins. As such, analysis of the electric properties of proteins gives rise to improved understanding of the mechanism by which proteins fulfil their specific functions. This is not only true for singular proteins but also applies for defined assemblies of proteins, protein complexes and supercomplexes. Charges in proteins often are a consequence of the presence of basic and acidic amino acid residues within polypeptide chains. In liquid phase, charge distributions of proteins change in response to the pH of their environment. The interdependence of protein charge and the surrounding pH is best described by the isoelectric point, which is notoriously difficult to obtain for native protein complexes. Here, experimentally derived native isoelectric points (npIs) for a range mitochondrial and plastid protein complexes are provided. In addition, for four complexes, npIs were calculated by a novel approach which yields results largely matching the experimental npIs.

  6. Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression

    PubMed Central

    1984-01-01

    Human epidermal keratinocytes express under various growth conditions a total of at least nine keratins that can be divided into two subfamilies. Subfamily A comprises 40-, 46-, 48-, 50-/50'-, and 56.5- kilodalton (kd) keratins which are relatively acidic (pI less than 5.5) and, with the exception of 46-kd keratin, are recognized by AE1 monoclonal antibody. Subfamily B comprises 52-, 56-, 58-, and 65-67-kd keratins which are relatively basic (pI greater than 6) and are recognized by AE3 monoclonal antibody. Within each keratin subfamily, there is a constant member (50-/50'- and 58-kd keratins of the subfamilies A and B, respectively) that is always expressed. The other seven keratins of both subfamilies are variable members whose expression depends upon the cellular differentiated state, which is in turn modulated by the growth environment. The 56.5-kd keratin (subfamily A) and the 65-67-kd keratins (subfamily B) are coordinately expressed during keratinization. In contrast, the 40-, 46-, and 48-kd keratins (subfamily A) and the 52- and 56-kd keratins (subfamily B) are characteristic of cultured epidermal cells forming nonkeratinized colonies. These results demonstrate that human epidermal keratins can be classified according to their reactivity with monoclonal antikeratin antibodies, isoelectric point, and mode of expression. The classification of keratins into various subgroups may have important implications for the mechanisms of epidermal differentiation, the evolution of keratin heterogeneity, and the use of keratin markers for tumor diagnosis. PMID:6201491

  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. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point

    NASA Astrophysics Data System (ADS)

    de Vries, R.

    2004-02-01

    Electrostatic complexation of flexible polyanions with the whey proteins α-lactalbumin and β-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Hückel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that α-lactalbumin complexes much more strongly than β-lactoglobulin. For α-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for β-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches.

  9. Continuous on-chip fluorescence labelling, free-flow isoelectric focusing and marker-free isoelectric point determination of proteins and peptides.

    PubMed

    Herzog, Christin; Poehler, Elisabeth; Peretzki, Andrea J; Borisov, Sergey M; Aigner, Daniel; Mayr, Torsten; Nagl, Stefan

    2016-04-26

    We present a microfluidic platform that contains a micro flow reactor for on-chip biomolecule labelling that is directly followed by a separation bed for continuous free-flow electrophoresis and has an integrated hydrogel-based near-infrared fluorescent pH sensor layer. Using this assembly, labelling of protein and peptide mixtures, their separation via free-flow isoelectric focusing and the determination of the isoelectric point (pI) of the separated products via the integrated sensor layer could be carried out within typically around 5 minutes. Spatially-resolved immobilization of fluidic and sensing structures was carried out via multistep photolithography. The assembly was characterized and optimized with respect to their fluidic and pH sensing properties and applied in the IEF of model proteins, peptides and a tryptic digest from physalaemine. We have therefore realized continuous sample preparation and preparative separation, analyte detection, process observation and analyte assignment capability based on pI on a single platform the size of a microscope slide.

  10. Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins.

    PubMed

    Yang, Chun; Zhang, Weibing; Zhang, Jie; Duan, Jicheng; Zhang, Yukui

    2005-01-01

    A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.

  11. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample

    NASA Astrophysics Data System (ADS)

    Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G. Julius

    2016-09-01

    Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the ‘footprint’ of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

  12. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample.

    PubMed

    Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G Julius

    2016-09-01

    Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the 'footprint' of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

  13. Isoelectric point of fluorite by direct force measurements using atomic force microscopy.

    PubMed

    Assemi, Shoeleh; Nalaskowski, Jakub; Miller, Jan D; Johnson, William P

    2006-02-14

    Interaction forces between a fluorite (CaF2) surface and colloidal silica were measured by atomic force microscopy (AFM) in 1 x 10(-3) M NaNO3 at different pH values. Forces between the silica colloid and fluorite flat were measured at a range of pH values above the isoelectric point (IEP) of silica so that the forces were mainly controlled by the fluorite surface charge. In this way, the IEP of the fluorite surface was deduced from AFM force curves at pH approximately 9.2. Experimental force versus separation distance curves were in good agreement with theoretical predictions based on long-range electrostatic interactions, allowing the potential of the fluorite surface to be estimated from the experimental force curves. AFM-deduced surface potentials were generally lower than the published zeta potentials obtained from electrokinetic methods for powdered samples. Differences in methodology, orientation of the fluorite, surface carbonation, and equilibration time all could have contributed to this difference.

  14. Nanoscale Roughness and Morphology Affect the IsoElectric Point of Titania Surfaces

    PubMed Central

    Borghi, Francesca; Vyas, Varun; Podestà, Alessandro; Milani, Paolo

    2013-01-01

    We report on the systematic investigation of the role of surface nanoscale roughness and morphology on the charging behaviour of nanostructured titania (TiO2) surfaces in aqueous solutions. IsoElectric Points (IEPs) of surfaces have been characterized by direct measurement of the electrostatic double layer interactions between titania surfaces and the micrometer-sized spherical silica probe of an atomic force microscope in NaCl aqueous electrolyte. The use of a colloidal probe provides well-defined interaction geometry and allows effectively probing the overall effect of nanoscale morphology. By using supersonic cluster beam deposition to fabricate nanostructured titania films, we achieved a quantitative control over the surface morphological parameters. We performed a systematical exploration of the electrical double layer properties in different interaction regimes characterized by different ratios of characteristic nanometric lengths of the system: the surface rms roughness Rq, the correlation length ξ and the Debye length λD. We observed a remarkable reduction by several pH units of IEP on rough nanostructured surfaces, with respect to flat crystalline rutile TiO2. In order to explain the observed behavior of IEP, we consider the roughness-induced self-overlap of the electrical double layers as a potential source of deviation from the trend expected for flat surfaces. PMID:23874708

  15. Survival of Listeria innocua in rainbow trout protein recovered by isoelectric solubilization and precipitation with acetic and citric acids.

    PubMed

    Otto, R A; Paker, I; Bane, L; Beamer, S; Jaczynski, J; Matak, K E

    2011-08-01

    During mechanical fish processing, a substantial amount of protein is discarded as by-products. Isoelectric solubilization and precipitation (ISP) is a process that uses extreme pH shifts to solubilize and precipitate protein from by-products to recover previously discarded protein. Typically, strong acids are used for pH reduction, but these acids do not have a pasteurization effect (6 log reduction) on bacterial load; therefore, organic acids were used during ISP processing to test the impact on Listeria innocua concentrations. Headed and gutted rainbow trout (Oncorhynchus mykiss) were inoculated with L. innocua, homogenized, and brought to the target pH with granular citric acid (pH 2.0 and 2.5) or glacial acetic acid (pH 3.0 and 3.5). Proteins were solubilized for 10 min at 4°C, and insoluble components (e.g., skin and insoluble protein) were removed by centrifugation. The remaining solution was pH shifted to the protein isoelectric point (pH 5.5) with sodium hydroxide, and precipitated protein was separated from the water. Microbial cells for each component (proteins, insolubles, and water) were enumerated on modified Oxford agar (MOX) and tryptic soy agar with 6% yeast extract (TSAYE). The sums of the surviving cells from each component were compared with the initial inoculum levels. No significant differences were observed between results obtained from TSAYE and from MOX (P > 0.05). Significant reductions in microbial populations were detected, regardless of pH or acid type (P < 0.05). The greatest reduction was at pH 3.0 with glacial acetic acid, resulting in a mean reduction of 6.41 log CFU/g in the recovered protein and 5.88 log CFU/g in the combined components. These results demonstrate the antimicrobial potential of organic acids in ISP processing.

  16. Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point.

    PubMed

    Dave, Sandeep; Mahajan, Sahil; Chandra, Vemika; Gupta, Pawan

    2011-11-01

    Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.

  17. Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

    PubMed

    Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

    2017-03-16

    The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme.

  18. Application of relative warp analysis to the evaluation of two-dimensional gels in proteomics: studying isoelectric point and relative molecular mass variation.

    PubMed

    Rodríguez-Piñeiro, Ana M; Carvajal-Rodríguez, Antonio; Rolán-Alvarez, Emilio; Rodríguez-Berrocal, Francisco J; Martínez-Fernández, Mónica; Páez de la Cadena, María

    2005-01-01

    We propose a geometric-morphometrics method (relative warp analysis) to be used in proteomic comparisons. This approach was applied to a dataset from a comparison between 5 controls and 5 patients with colorectal cancer disease published elsewhere. The spots in the 2-D maps were used as landmarks in a morphometric study, and the differences in shape (spot distribution) among them were obtained. The shape variables were used to compare controls and patients. These components mostly ignore random or experimental effects affecting all the proteins in any of the two dimensions studied. Furthermore, the method allows the researcher to find those proteins which contributed the most to the local shape component detected. Applying this approach, we detected variations in the position (isoelectric point and/or relative molecular mass) of some spots that may reflect differences in the amino acidic sequence or post-translational modifications.

  19. Initial bioadhesion on dental materials as a function of contact time, pH, surface wettability, and isoelectric point.

    PubMed

    Müller, Christine; Lüders, Anne; Hoth-Hannig, Wiebke; Hannig, Matthias; Ziegler, Christiane

    2010-03-16

    The adsorption of bovine serum albumin (BSA) on surfaces of dental enamel and of dental materials was investigated by scanning force spectroscopy. This method provides adhesion forces which can be measured as a function of contact time between protein and surface, pH, wettability, and isoelectric point of the surface. Whereas the chosen ceramic and composite materials resemble very well the adhesion on natural enamel, a much stronger adhesion was found for the more hydrophobic surfaces, that is, gold, titanium, poly(methyl methacrylate) (PMMA), and poly(tetrafluoroethylene) (PTFE). On hydrophilic surfaces, adhesion is mainly influenced by the electrostatic forces between protein and surface. However, the conformational change of BSA at pH values above pH 8 has to be taken into account. On the very hydrophobic PTFE surface, the special interface structure between PTFE and water plays an important role which governs BSA adhesion.

  20. Analysis of polyamidoamine dendrimers by isoelectric focusing.

    PubMed

    Upadhaya, Samik K; Swanson, Douglas R; Tomalia, Donald A; Sharma, Ajit

    2014-01-01

    Polyamidoamine dendrimers have been studied extensively for their potential applications in nanomedicine. Their uses as imaging, drug, and nucleic acid delivery agents are nearing clinical trials. As such, characterization of polyamidoamine dendrimers and their nano-devices is of immense importance for monitoring the efficiency of their synthesis, purity, and quality control of manufactured products as well as their in vivo behavior. We report here the analysis of polyamidoamine dendrimers possessing various cores and surface groups with a simple and inexpensive isoelectric focusing method. The isoelectric points of the dendrimers were readily determined from a calibration plot generated by running proteins with known pI values. The isoelectric points for various surface-modified polyamidoamine dendrimers ranged from 4 to 9. Polyamidoamine dendrimers possessing terminal hydroxyl groups gave a pI > 7, while those with terminal carboxyl groups exhibit a pI < 7. Generation number and cores of the dendrimers did not significantly affect their isoelectric points. Isoelectric focusing thus offers another important tool for characterizing these nanomolecules.

  1. On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Yang, Ping; Dovichi, Norman J

    2012-10-31

    Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300-1800. Good focusing was achieved for insulin receptor, which produced ~10 s peak width. For 0.1 mg mL(-1) bovine serum albumin (BSA) digests, 24±1 peptides (sequence coverage 47±4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.

  2. Separation of polypeptides by isoelectric point focusing in electrospray-friendly solution using a multiple-junction capillary fractionator.

    PubMed

    Chingin, Konstantin; Astorga-Wells, Juan; Pirmoradian Najafabadi, Mohammad; Lavold, Thorleif; Zubarev, Roman A

    2012-08-07

    We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration.

  3. Effects of peptide fractions with different isoelectric points from wheat gluten hydrolysates on lipid oxidation in pork meat patties.

    PubMed

    Park, Eun Young; Imazu, Hiroko; Matsumura, Yasuki; Nakamura, Yasushi; Sato, Kenji

    2012-08-01

    Wheat gluten hydrolysate (WGH) was fractionated on the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing (autofocusing). Cooked pork patties were stored at 4 and 20 °C in the dark. WGH and autofocusing fractions suppressed the oxidation of lipids in the patties. The acidic (pI < 3.0) and basic (pI > 9.0) autofocusing fractions suppressed lipid oxidation in the cooked patties to a greater extent than other fractions and WGH. Each autofocusing fraction was evaluated by 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical scavenging activities, β-carotene bleaching, oxygen radical absorbance capacity, and Fe(2+) chelating assays; however, none of the in vitro assays predicted the suppressive effect of WGH on lipid oxidation in the cooked patties. These findings suggest that the microdistribution of peptides in food systems and their interaction with food matrix compounds play a significant role in the suppression of lipid oxidation in meat patties rather than radical scavenging activity.

  4. Electrolysis and isoelectric focusing

    NASA Astrophysics Data System (ADS)

    Choi, Y. S.; Lui, Roger; Yu, Xun

    1994-01-01

    This paper consists of two parts. In the first part, the authors prove the existence of steady-state solutions for a three-species electrolyte. The species are subject to both dissociation-association reactions inside the electrolyte and electrochemical reactions at the boundary electrodes. This is a common occurrence in electrolysis. In the second part, the authors investigate how to use this model to describe isoelectric focusing, which is a common technique used to separate large protein molecules. In particular, the isoelectric focusing point for a particular type of protein molecule is calculated using formal perturbation analysis.

  5. Enrichment of the Glycyrrhizic Acid from Licorice Roots (Glycyrrhiza glabra L.) by Isoelectric Focused Adsorptive Bubble Chromatography.

    PubMed

    Karaoğul, Eyyüp; Parlar, Perihan; Parlar, Harun; Alma, M Hakkı

    2016-01-01

    The main aim of this study was to enrich glycyrrhizic acid ammonium salt known as one of the main compounds of licorice roots (Glycyrrhiza glabra L.) by isoelectric focused adsorptive bubble separation technique with different foaming agents. In the experiments, four bubble separation parameters were used with β-lactoglobulin, albumin bovine, and starch (soluble) preferred as foaming agents and without additives. The enrichment of glycyrrhizic acid ammonium salt into the foam was influenced by different additive substances. The results showed that highest enrichment values were obtained from β-lactoglobulin as much as 368.3 times. The lowest enrichment values (5.9 times) were determined for the application without additive. After enrichment, each experiment of glycyrrhizic acid ammonium salt confirmed that these substances could be quantitatively enriched into the collection vessel with isoelectric focused adsorptive bubble separation technique. The transfer of glycyrrhizic acid ammonium salt into the foam from standard solution in the presence of additive was more efficient than aqueous licorice extract.

  6. Enrichment of the Glycyrrhizic Acid from Licorice Roots (Glycyrrhiza glabra L.) by Isoelectric Focused Adsorptive Bubble Chromatography

    PubMed Central

    Karaoğul, Eyyüp; Parlar, Perihan; Parlar, Harun; Alma, M. Hakkı

    2016-01-01

    The main aim of this study was to enrich glycyrrhizic acid ammonium salt known as one of the main compounds of licorice roots (Glycyrrhiza glabra L.) by isoelectric focused adsorptive bubble separation technique with different foaming agents. In the experiments, four bubble separation parameters were used with β-lactoglobulin, albumin bovine, and starch (soluble) preferred as foaming agents and without additives. The enrichment of glycyrrhizic acid ammonium salt into the foam was influenced by different additive substances. The results showed that highest enrichment values were obtained from β-lactoglobulin as much as 368.3 times. The lowest enrichment values (5.9 times) were determined for the application without additive. After enrichment, each experiment of glycyrrhizic acid ammonium salt confirmed that these substances could be quantitatively enriched into the collection vessel with isoelectric focused adsorptive bubble separation technique. The transfer of glycyrrhizic acid ammonium salt into the foam from standard solution in the presence of additive was more efficient than aqueous licorice extract. PMID:26949562

  7. The effect of using citric or acetic acid on survival of Listeria monocytogenes during fish protein recovery by isoelectric solubilization and precipitation process.

    PubMed

    Otto, R A; Beamer, S; Jaczynski, J; Matak, K E

    2011-10-01

    Isoelectric solubilization and precipitation (ISP) is a protein recovery process effective at reducing Listeria innocua, a nonpathogenic bacterium typically used as a surrogate for L. monocytogenes in recovered trout protein. The response of L. monocytogenes to ISP processing was determined and compared to the response of L. innocua. Headed and gutted rainbow trout were inoculated with L. monocytogenes (10.16 log CFU/g), homogenized, and pH-adjusted with granular citric acid (pH 2.0 and 2.5) or glacial acetic acid (pH 3.0 and 3.5). Proteins were solubilized and centrifugation was used to remove insoluble components (skin, insoluble protein, so on). The supernatant was returned to the protein isoelectric point (pH 5.5) with NaOH and centrifuged to remove precipitated protein. Microbial load was enumerated on both growth and selective media; recovery was not significantly different (P > 0.05). Surviving cells from each component (protein, insoluble, and water) were compared to initial inoculum numbers. Significant reductions were detected at all pH (P < 0.05). The greatest reductions were at pH 3.0 with acetic acid, with a mean log reduction of 3.03 in the combined components, and a 3.53 log reduction in the protein portion. Data were compared to results from a previous study using L. innocua. Significant differences (P < 0.05) in recovery were found between the 2 species at pH 2.0 and 3.0 with greater recovery of L. monocytogenes, regardless of processing pH or acid type. These results demonstrate the variability in resistance between species and indicate that L. innocua is not an appropriate surrogate for L. monocytogenes for ISP processing with organic acids.

  8. Modification of interactions among proteins with the lowering of solution pD toward the isoelectric point in presence of different valent ions

    NASA Astrophysics Data System (ADS)

    Das, Kaushik; Kundu, Sarathi; Mehan, S.; Aswal, V. K.

    2014-08-01

    Bovine serum albumins show a short-range attraction and a long-range electrostatic repulsion among them and these interactions are modified depending upon the solution pD and different dissolved counterions in the solution. Small angle neutron scattering study shows that for equal mono-valent (Na+) and di-valent (Ni2+) ion concentrations, both the attractive and repulsive interaction decreases with lowering the solution pD toward the protein isoelectric point, however for the tri-valent (Fe3+) ion attractive interaction increases and repulsive interaction decreases. Interaction variation for the equal ionic strength of the three different valent ions becomes prominent as the pD decreases toward the isoelectric point.

  9. Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing--electrospray ionization mass spectrometry in glycerol-water media.

    PubMed

    Lecoeur, Marie; Gareil, Pierre; Varenne, Anne

    2010-11-12

    On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.

  10. Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver.

    PubMed

    Kubicz, A; Szalewicz, A; Chrambach, A

    1991-01-01

    1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.

  11. Capillary Isoelectric Focusing

    NASA Astrophysics Data System (ADS)

    Markuszewski, Michał J.; Bujak, Renata; Daghir, Emilia

    Capillary isoelectric focusing (CIEF) is a widespread technique for the analysis of peptides and proteins in biological samples. CIEF is used to separate mixtures of compounds on the basis of differences in their isoelectric point. Aspects of sample preparation, capillary selection, zone mobilization procedures as well as various detection modes used have been described and discussed. Moreover CIEF, coupled to various types of detection techniques (MALDI or LIF), has increasingly been applied to the analysis of variety different high-molecular compounds. CIEF is considered as a highly specific analytical method which may be routinely used in the separation of rare hemoglobin variants. In addition, the application of CIEF in proteomic field have been discussed on the examples of analyses of glycoproteins and immunoglobins due to the meaning in clinical diagnostic.

  12. Isoelectric focusing of dansylated amino acids in immobilized pH gradients

    NASA Technical Reports Server (NTRS)

    Bianchi-Bosisio, Adriana; Righetti, Pier Giorgio; Egen, Ned B.; Bier, Milan

    1986-01-01

    The 21 free amino acids commonly encountered in proteins have been transformed into 'carrier ampholyte' species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH interval 3.1 to 4.1, except for arginine, which still retains a pI of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2-4 ng/sq mm. All nearest neighbors are separated except for the following couples: Asn-Gln, Gly-Thr, Val-Ile and Cys-Cys2, with a resolving power, in a Delta(pI) scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids), the order of pI values is well correlated with the pK values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of pK(COOH)-pI values of the different amino acids, the pK of the tertiary amino group in the dansyl label has been calculated to be 5.11 + or - 0.06. Knowing the pK of the amino-dansyl and the pI of the excess, free dansyl label (pI = 3.34), a pK of 1.57 is derived for its sulfonic acid group.

  13. Hydrolytically stable, diaminocarboxylic acid-based membranes buffering in the pH range from 6 to 8.5 for isoelectric trapping separations.

    PubMed

    Fleisher, Helen C; Vigh, Gyula

    2005-06-01

    Diaminocarboxylic acid carrier ampholytes, such as L-histidine, 2,3-diaminopropionic acid, L-ornithine, and L-lysine, were reacted with glycerol-1,3-diglycidyl ether (GDGE) and poly(vinyl alcohol) (PVA) in the presence of sodium hydroxide to produce hydrolytically and mechanically stable hydrogels, supported on a PVA substrate, for use as buffering membranes in isoelectric trapping (IET) separations. The pH values of the DACAPVA membranes were determined with the help of small-molecule pI markers and proteins and were found to be in the 6 < pH < 8.5 range. The membranes were successfully used to isoelectrically trap small ampholytes, desalt ampholyte solutions in IET mode, and effect the binary separation of chicken egg white proteins.

  14. Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point.

    PubMed

    Jeon, Won Bae

    2010-05-01

    Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

  15. Isoelectric focusing in space

    NASA Technical Reports Server (NTRS)

    Bier, M.; Egen, N. B.; Mosher, R. A.; Twitty, G. E.

    1982-01-01

    The potential of space electrophoresis is conditioned by the fact that all electrophoretic techniques require the suppression of gravity-caused convection. Isoelectric focusing (IEF) is a powerful variant of electrophoresis, in which amphoteric substances are separated in a pH gradient according to their isoelectric points. A new apparatus for large scale IEF, utilizing a recycling principle, has been developed. In the ground-based prototype, laminar flow is provided by a series of parallel filter elements. The operation of the apparatus is monitored by an automated array of pH and ultraviolet absorption sensors under control of a desk-top computer. The apparatus has proven to be useful for the purification of a variety of enzymes, snake venom proteins, peptide hormones, and other biologicals, including interferon produced by genetic engineering techniques. In planning for a possible space apparatus, a crucial question regarding electroosmosis needs to be addressed To solve this problem, simple focusing test modules are planned for inclusion in an early Shuttle flight.

  16. Use of a Spreadsheet to Calculate the Net Charge of Peptides and Proteins as a Function of pH: An Alternative to Using "Canned" Programs to Estimate the Isoelectric Point of These Important Biomolecules

    ERIC Educational Resources Information Center

    Sims, Paul A.

    2010-01-01

    An approach is presented that utilizes a spreadsheet to allow students to explore different means of calculating and visualizing how the charge on peptides and proteins varies as a function of pH. In particular, the concept of isoelectric point is developed to allow students to compare the results of their spreadsheet calculations with those of…

  17. Colloidal Properties of Aqueous Fullerenes: Isoelectric Points and Aggregation Kinetics of C60 and C60 Derivatives

    EPA Science Inventory

    Aqueous colloidal suspensions of C-60 (aqu/C-60) and the C-60 derivatives PCBM ([6,6]-phenyl C-61-butyric acid methyl ester) and the corresponding butyl and octyl esters, PCBB and PCBO (aqu/PCB-R, where R is an alkyl group), were produced by stirring in double deionized water for...

  18. Free flow and capillary isoelectric focusing of bacteria from the tomatoes plant tissues.

    PubMed

    Horká, Marie; Horký, Jaroslav; Matousková, Hana; Slais, Karel

    2009-02-06

    The means of the preconcentration and preseparation of selected species or pathovars of bacteria directly from the plant tissue suspension by free flow isoelectric focusing are introduced here. After the focusing, the resulting fraction of microorganisms, native or dynamically modified by the non-ionogenic tenside on the basis of pyrenebutanoate, was separated by capillary isoelectric focusing and/or cultivated and positively identified by gas chromatographic analysis of fatty acid methyl esters. Simultaneously, capillary isoelectric focusing with UV and fluorometric detection was used for the rapid estimation of unknown isoelectric points of the examined plant pathogenic species of genus Clavibacter, Xanthomonas and Pseudomonas prior to the preconcentration and preseparation. The microorganisms were of different origin, native and/or dynamically modified by the non-ionogenic tenside.

  19. Diverse lectin-binding specificity of four ZP3 glycoprotein isoforms with a discrete isoelectric point in chicken egg coat.

    PubMed

    Okumura, Hiroki; Fukushima, Hideaki; Momoda, Masaki; Ima, Yurie; Matsuda, Tsukasa; Ujita, Minoru

    2012-08-03

    The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galβ1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((β-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galβ1-4GlcNAcβ1-2Manα1-6) and RCA I (terminal Galβ1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.

  20. Separation of two forms of rabbit metallothionein by isoelectric focusing.

    PubMed

    Nordberg, G F; Nordberg, M; Piscator, M; Vesterberg, O

    1972-02-01

    Rabbits were given repeated injections of cadmium chloride. Cadmium- and zinc-containing protein fractions were obtained from the livers of these animals by precipitation procedures and Sephadex G-75 chromatography. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further separation by isoelectric focusing showed two main protein peaks with isoelectric points at 3.9 and 4.5 respectively. Amino acid analysis of these two forms showed similar content of most amino acids [residues per cent.: cysteine (28%), aspartate (8%), threonine (5-6%), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13% respectively), proline (9 and 5% respectively) and glutamate (2 and 4% respectively). The two forms of the protein both contained cadmium, but only the one with pI4.5 contained also significant amounts of zinc.

  1. Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase1[C][W

    PubMed Central

    Srivastava, Vaibhav; Srivastava, Manoj Kumar; Chibani, Kamel; Nilsson, Robert; Rouhier, Nicolas; Melzer, Michael; Wingsle, Gunnar

    2009-01-01

    Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays. PMID:19176719

  2. Analysis of low molecular weight acids by monolithic immobilized pH gradient-based capillary isoelectric focusing coupled with mass spectrometry.

    PubMed

    Wang, Tingting; Fekete, Agnes; Gaspar, Andras; Ma, Junfeng; Liang, Zhen; Yuan, Huiming; Zhang, Lihua; Schmitt-Kopplin, Philippe; Zhang, Yukui

    2011-02-01

    A novel method for the separation and detection of low molecular weight (LMW) acids was developed using monolithic immobilized pH gradient-based capillary isoelectric focusing coupled with mass spectrometry. Two main parameters, focusing conditions and delivery buffer conditions, which might affect separation efficiency, were optimized with the focusing time of 7 min at 350 V/cm and the delivery buffer of 50% (v/v) acetonitrile in 10 mmol/L ammonium formate (pH 3.0). Under these conditions, the linear correlation between the volume of delivery solvent and the pK(a) of the model components was observed. In addition, the separation mechanism of LMW acids was proposed as well. We suppose that this method may provide a useful tool for the characterization of LMW components (e.g. natural organic matter of different origins).

  3. Protein profiling and phosphoprotein analysis by isoelectric focusing.

    PubMed

    Maccarrone, Giuseppina; Filiou, Michaela D

    2015-01-01

    Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.

  4. High-buffering capacity, hydrolytically stable, low-pI isoelectric membranes for isoelectric trapping separations.

    PubMed

    Lalwani, Sanjiv; Shave, Evan; Vigh, Gyula

    2004-10-01

    Hydrolytically stable, low-pI isoelectric membranes have been synthesized from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. The acidic functional groups are selected such that the pI value of the ampholytic component is determined by the pK(a) values of the acidic functional groups. When the concentration of the ampholytic component incorporated into the membrane is higher than a required minimum value, the pI of the membrane becomes independent of variations in the actual incorporation rate of the ampholytic compound. The new, low-pI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH < 1.5 anolytes and replaced the hydrolytically less stable polyacrylamide-based isoelectric membranes. The new low-pI isoelectric membranes have excellent mechanical stability, low electric resistance, good buffering capacity, and long life time, even when used with as much as 50 W power and current densities as high as 33 mA/cm(2) during the isoelectric trapping separations.

  5. Production and functional evaluation of a protein concentrate from giant squid (Dosidicus gigas) by acid dissolution and isoelectric precipitation.

    PubMed

    Cortés-Ruiz, Juan A; Pacheco-Aguilar, Ramón; Elena Lugo-Sánchez, M; Gisela Carvallo-Ruiz, M; García-Sánchez, Guillermina

    2008-09-15

    A protein concentrate from giant squid (Dosidicus gigas) was produced under acidic conditions and its functional-technological capability evaluated in terms of its gel-forming ability, water holding capacity and colour attributes. Technological functionality of the concentrate was compared with that of squid muscle and a neutral concentrate. Protein-protein aggregates insoluble at high ionic strength (I=0.5M), were detected in the acidic concentrate as result of processing with no preclusion of its gel-forming ability during the sol-to-gel thermal transition. Even though washing under acidic condition promoted autolysis of the myosin heavy chain, the acidic concentrate displayed an outstanding ability to gel giving samples with a gel strength of 455 and 1160gcm at 75% and 90% compression respectively, and an AA folding test grade indicative of high gel strength, elasticity, and cohesiveness. The process proved to be a good alternative for obtaining a functional protein concentrate from giant squid muscle.

  6. Devices, systems, and methods for microscale isoelectric fractionation

    DOEpatents

    Sommer, Gregory J.; Hatch, Anson V.; Wang, Ying-Chih; Singh, Anup K.

    2016-08-09

    Embodiments of the present invention provide devices, systems, and methods for microscale isoelectric fractionation. Analytes in a sample may be isolated according to their isoelectric point within a fractionation microchannel. A microfluidic device according to an embodiment of the invention includes a substrate at least partially defining a fractionation microchannel. The fractionation microchannel has at least one cross-sectional dimension equal to or less than 1 mm. A plurality of membranes of different pHs are disposed in the microchannel. Analytes having an isoelectric point between the pH of the membranes may be collected in a region of the fractionation channel between the first and second membranes through isoelectric fractionation.

  7. Devices, systems, and methods for microscale isoelectric fractionation

    DOEpatents

    Sommer, Gregory J; Hatch, Anson V; Wang, Ying-Chih; Singh, Anup K

    2015-04-14

    Embodiments of the present invention provide devices, systems, and methods for microscale isoelectric fractionation. Analytes in a sample may be isolated according to their isoelectric point within a fractionation microchannel. A microfluidic device according to an embodiment of the invention includes a substrate at least partially defining a fractionation microchannel. The fractionation microchannel has at least one cross-sectional dimension equal to or less than 1 mm. A plurality of membranes of different pHs are disposed in the microchannel. Analytes having an isoelectric point between the pH of the membranes may be collected in a region of the fractionation channel between the first and second membranes through isoelectric fractionation.

  8. Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

    PubMed

    Ettori, C; Righetti, P G; Chiesa, C; Frigerio, F; Galli, G; Grandi, G

    1992-09-01

    Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

  9. Isoelectric focusing in a silica nanofluidic channel: effects of electromigration and electroosmosis.

    PubMed

    Hsu, Wei-Lun; Inglis, David W; Startsev, Michael A; Goldys, Ewa M; Davidson, Malcolm R; Harvie, Dalton J E

    2014-09-02

    Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.

  10. Microfluidic high-resolution free-flow isoelectric focusing.

    PubMed

    Kohlheyer, Dietrich; Eijkel, Jan C T; Schlautmann, Stefan; van den Berg, Albert; Schasfoort, Richard B M

    2007-11-01

    A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm-1. Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is Delta(pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast

  11. Preparative isoelectric focusing and Joule effect: a purification cell that contains a heat exchanger.

    PubMed

    Araque, A; Jaugey, J; Javet, P

    1996-01-01

    Reproducibility in protein purification by preparative isoelectric focusing depends greatly on temperature control during the separation process. A preparative apparatus is described, including a heat exchanger between compartments with isoelectric membranes. The selectivity of the isoelectric membranes was optimized as a function of isoelectric points of the separated proteins. At 2500 V and 60 W, 0.3 g of horse heart myoglobin from 0.2 g of whale skeletal muscle myoglobin could be separated in 1 h. At a total load of 2 g protein, 97% of bovine hemoglobin (2% initial concentration) was purified from bovine serum albumin (0.15%).

  12. Isoelectric focusing in a drop.

    PubMed

    Weiss, Noah G; Hayes, Mark A; Garcia, Antonio A; Ansari, Rafat R

    2011-01-04

    A novel approach to molecular separations is investigated using a technique termed droplet-based isoelectric focusing. Drops are manipulated discretely on a superhydrophobic surface, subjected to low voltages for isoelectric focusing, and split-resulting in a preparative separation. A universal indicator dye demonstrates the generation of stable, reversible pH gradients (3-10) in ampholyte buffers, and these gradients lead to protein focusing within the drop length. Focusing was visually characterized, spectroscopically verified, and assessed quantitatively by noninvasive light scattering measurements. It was found to correlate with a quantitative model based on 1D steady-state theory. This work illustrates that molecular separations can be deployed within a single open drop, and the differential fractions can be separated into new discrete liquid elements.

  13. CAPILLARY ISOELECTRIC FOCUSING (CIEF) FOR THE CHARACTERIZATION OF HUMIC SUBSTANCES

    EPA Science Inventory

    Preparative solution isoelectric focusing was used to fractionate 50 mg of a soil fulvic acid (FA); the harvested fractions were characterized with UV-Vis spectroscopy, gel permeation chromatography and capillary zone electrophoresis (CZE) and showed a distribution in the created...

  14. Applications of isoelectric focusing in forensic serology.

    PubMed

    Murch, R S; Budowle, B

    1986-07-01

    The typing of certain polymorphic proteins present in human body fluids is an important aspect of the analysis of serological evidence. This is particularly true when dealing with evidence related to violent criminal activity such as homocide, assault, or rape. Until recently, the routine analysis of the genetic polymorphisms of interest relied upon conventional electrophoretic techniques such as horizontal starch or agarose slab gel or both, cellulose acetate, and vertical polyacrylamide gradient gel methods. These techniques adequately separate a limited number of common variants. In some cases, these methods are still those of choice. However, as a result of the nature of the conventional approach, problems with time required for analysis, resolution, diffusion of bands, sensitivity of protein detection, and cost are often encountered. Isoelectric focusing (IEF) offers an effective alternative to conventional electrophoresis for genetic marker typing. This method exploits the isoelectric point of allelic products rather than charge-to-mass ratio in a particular pH environment. The advantages of employing IEF include: reduction of time of analysis, increased resolution of protein bands, the possibility of subtyping existing phenotypes, increased sensitivity of detection, the counteraction of diffusion effects, and reduced cost per sample.

  15. Rotating apparatus for isoelectric focusing

    NASA Technical Reports Server (NTRS)

    Bier, Milan (Inventor)

    1986-01-01

    This disclosure is directed to an isoelectric focusing apparatus, wherein stabilization of the fluid containing the isolated proteins is achieved by carrying out the separation in a rotating cylinder with the separation cavity of the cylinder being segmented by means of filter elements. The filter elements are constituted of a material offering some degree of resistance to fluid convection, but allowing relatively free and unhindered passage of current and transport of proteins. The combined effect of segmentation and rotation has been found to be superior to either segmentation or rotation alone in maintaining the stability of the migrated fractions.

  16. An explanation for the plateau phenomenon in isoelectric focusing

    NASA Technical Reports Server (NTRS)

    Mosher, Richard A.; Thormann, Wolfgang; Bier, Milan

    1986-01-01

    Isoelectric focusing of mixtures of simple ampholytes occurs in two phases, an initial rapid separation phase and a second relatively slow stabilizing phase. Transient and steady-state computer simulation data are shown to predict the development of pH plateaus around neutrality during the stabilizing phase of the focusing of such mixtures. This occurs because a nonzero electrophoretic flux is present in a pure zone of focused ampholyte, which is a function of both its isolectric point (pI) and its Delta-pK value. For an ampholyte with a pI greater than 9 or less than 5, this flux causes the development of a significant concentration gradient within its focused zone which is accompanied by a contraction of this zone along the focusing axis. Acidic and basic ampholytes are thereby displayed toward the anode and cathode respectively, creating a pH plateau in the neutral region. Thus, there will be regions of the focusing column, closer to the electrodes and containing more acidic or basic pH values, within which the resolution of samples will reach a maximum and then decrease.

  17. Hormone Purification by Isoelectric Focusing

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1985-01-01

    Various ground-based research approaches are being applied to a more definitive evaluation of the natures and degrees of electroosmosis effects on the separation capabilities of the Isoelectric Focusing (IEF) process. A primary instrumental system for this work involves rotationally stabilized, horizontal electrophoretic columns specially adapted for the IEF process. Representative adaptations include segmentation, baffles/screens, and surface coatings. Comparative performance and development testing are pursued against the type of column or cell established as an engineering model. Previously developed computer simulation capabilities are used to predict low-gravity behavior patterns and performance for IEF apparatus geometries of direct project interest. Three existing mathematical models plus potential new routines for particular aspects of simulating instrument fluid patterns with varied wall electroosmosis influences are being exercised.

  18. Rotating Apparatus for Isoelectric Focusing

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1986-01-01

    Remixing of separated fractions prevented. Improved isoelectric focusing apparatus helps to prevent electro-osmosis and convection, both of which cause remixing of separated fractions. Fractionating column segmented and rotated about horizontal axis: Only combined effects of both features fully effective in making good separations. Improved apparatus slowly rotated continuously or rocked (at rotational amplitude of at least 180 degrees) about its horizontal axis so average gravitational vector experienced by fluid is zero and convection is therefore suppressed. Electro-osmosis suppressed and convection further suppressed by separating column into disklike compartments along its length with filters. Experiments have shown dimensions of apparatus not critical. Typical compartment and column volumes are 2 and 40 ml, respectively. Rotation speeds lie between 3 and 30 rpm.

  19. Isoelectric focusing of human von Willebrand factor in urea-agarose gels

    SciTech Connect

    Fulcher, C.A.; Ruggeri, Z.M.; Zimmerman, T.S.

    1983-02-01

    An analytical technique has been developed for the isoelectric focusing (IEF) of plasma von Willebrand factor (vWF) in agarose gels containing urea. Under these conditions, vWF freely enters the gel and focuses without artifact. The focused vWF is visualized by staining fixed gels with /sup 125/I-labeled affinity-purified heterologous antibody. Utilizing a pH gradient of 5.0-6.5, normal vWF in plasma or purified preparations focuses into at least three bands with apparent isoelectric points (pI) between pH 5.7 and 5.9. A reproducible difference in the IEF pattern of vWF has been established between normal plasmas and those of individuals with variant von Willebrand's disease (vWd) type IIA and type IIB. In type IIA, vWF has a distinctly lower pI than normal. This difference may be related to the presence of smaller vWF multimers in IIA plasma because forms of vWF of corresponding size contained in normal cryoprecipitate supernatant have a similar pI. Type IIB von Willebrand factor has a pI intermediate between normal and IIA. Neuraminidase treatment of plasma samples before IEF results in an increase in pI in normal, type IIA, and type IIB vWF. The data suggest that none of the 16 type IIA and 9 IIB plasmas studied here contain significantly decreased amounts of sialic acid.

  20. Validation of acid washes as critical control points in hazard analysis and critical control point systems.

    PubMed

    Dormedy, E S; Brashears, M M; Cutter, C N; Burson, D E

    2000-12-01

    A 2% lactic acid wash used in a large meat-processing facility was validated as an effective critical control point (CCP) in a hazard analysis and critical control point (HACCP) plan. We examined the microbial profiles of beef carcasses before the acid wash, beef carcasses immediately after the acid wash, beef carcasses 24 h after the acid wash, beef subprimal cuts from the acid-washed carcasses, and on ground beef made from acid-washed carcasses. Total mesophilic, psychrotrophic, coliforms, generic Escherichia coli, lactic acid bacteria, pseudomonads, and acid-tolerant microorganisms were enumerated on all samples. The presence of Salmonella spp. was also determined. Acid washing significantly reduced all counts except for pseudomonads that were present at very low numbers before acid washing. All other counts continued to stay significantly lower (P < 0.05) than those on pre-acid-washed carcasses throughout all processing steps. Total bacteria, coliforms, and generic E. coli enumerated on ground beef samples were more than 1 log cycle lower than those reported in the U.S. Department of Agriculture Baseline data. This study suggests that acid washes may be effective CCPs in HACCP plans and can significantly reduce the total number of microorganisms present on the carcass and during further processing.

  1. Alkali-stable high-pI isoelectric membranes for isoelectric trapping separations.

    PubMed

    Lalwani, Sanjiv; Shave, Evan; Fleisher, Helen C; Nzeadibe, Kingsley; Busby, M Brent; Vigh, Gyula

    2004-07-01

    Alkali-stable, high-pI isoelectric membranes have been synthesized from quaternary ammonium derivatives of cyclodextrins and poly(vinyl alcohol), and bifunctional cross-linkers, such as glycerol-1,3-diglycidyl ether. The new, high-pI isoelectric membranes were successfully applied as cathodic membranes in isoelectric trapping separations in place of the hydrolytically more labile, polyacrylamide-based cathodic isoelectric membranes, and permitted the use of catholytes as alkaline as 1 M NaOH. The new high-pI isoelectric membranes have shown excellent mechanical stability, low electric resistance and long life times, even when subjected to electrophoresis with current densities as high as 80 mA/cm2.

  2. Use of quasi-isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances.

    PubMed

    Poitevin, Martine; Peltre, Gabriel; Descroix, Stephanie

    2008-04-01

    The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.

  3. Hormone purification by isoelectric focusing in space

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1982-01-01

    The performance of a ground-prototype of an apparatus for recycling isoelectric focusing was evaluated in an effort to provide technology for large scale purification of peptide hormones, proteins, and other biologicals. Special emphasis was given to the effects of gravity on the function of the apparatus and to the determination of potential advantages deriveable from its use in a microgravity environment. A theoretical model of isoelectric focusing sing chemically defined buffer systems for the establishment of the pH gradients was developed. The model was transformed to a form suitable for computer simulations and was used extensively for the design of experimental buffers.

  4. Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis.

    PubMed

    Ruzicka, Filip; Horka, Marie; Hola, Veronika; Votava, Miroslav

    2007-03-01

    The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.

  5. Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea-urea mixtures.

    PubMed

    Gervais, David; King, Darryl

    2014-11-15

    Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pKa values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0-3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.

  6. The automatic use of capillary isoelectric focusing with whole column imaging detection for carbamazepine binding to human serum albumin.

    PubMed

    Maciążek-Jurczyk, Małgorzata; Pawliszyn, Janusz

    2016-08-05

    The binding of the anticonvulsant drug carbamazepine (CBZ) to human serum albumin, both without (dHSA) and in the presence of fatty acids (HSA) was studied in real time by capillary isoelectric focusing with whole column imaging detection (cIEF-WCID). Reaction mixtures at different CBZ:HSA and CBZ:dHSA molar ratios (0:1/25:1) were prepared in phosphate buffer saline (PBS) solution at a physiological pH (7.4), and incubated for 0-72h at 37°C in a water bath. Application of the cIEF-WCID method allowed for observations on the impact of increasing CBZ:serum albumin molar ratios on isoelectric point (pI) shifts, as well as changes in peak area and absorbance, which serve as evidence of structural alterations occurring in the protein in the presence of CBZ. The obtained cIEF-WCID results indicated that the dynamic process of complex formation is not dependent on incubation time. The presented work allowed for recognition of different types of interactions, as well as for the calculation of association constants that demonstrate the stability of the complex. This study was also designed to examine the possible impact of fatty acids (FAs) on protein stability and drug delivery in blood.

  7. A commercial isoelectric focusing apparatus for use in microgravity

    NASA Astrophysics Data System (ADS)

    Johnson, Jerald F.; Dandy, Jonathan S.; Johnson, Terry C.

    2000-01-01

    A series of studies have tested the possibility that the microgravity environment may be superior to laboratories on earth for several biomedical applications. One such application is isoelectric focusing (IEF). The purpose of our research is to design, build, test, and employ an analytical IEF instrument for use in the laboratory on the International Space Station (ISS) and to demonstrate the advantages of space-based IEF. This paper describes IEF in general, discusses the design considerations that arise for IEF in low-gravity, and presents design solutions to some of the systems under development. Isoelectric focusing is a powerful technique that has applications for both analytical analysis the preparative purification of macromolecules. IEF resolves proteins by net charge separation, in either liquid or semi-solid substrates, where the molecules migrate to their isoelectric point (pI). In earth-based IEF, separation media are usually semi-solids such as polyacrylamide and agarose gels. The matrix structure of these media is used to offset the gravity-induced diffusion and convection that occurs in free solutions. With these effects being greatly reduced, a free solution could be used as a superior media. Because diffusion in liquids is reduced in microgravity (Snyder, 1986), a given electrical field should result in more tightly focused bands. This would allow for the separation of proteins that have very closely spaced pI's. If superior results are achieved, there are numerous pharmaceutical and genetic engineering companies that would take advantage of this unique development. The design of the Commercial IsoElectric Focusing Apparatus (CIEFA) presents several significant engineering challenges specific to its operation in the microgravity environment. Three difficulties of particular importance are gases generated through electrolysis, temperature control and verification of protein separation. Gases generated through electrolysis must be isolated from

  8. Mathematical modeling and computer simulation of isoelectric focusing with electrochemically defined ampholytes

    NASA Technical Reports Server (NTRS)

    Palusinski, O. A.; Allgyer, T. T.; Mosher, R. A.; Bier, M.; Saville, D. A.

    1981-01-01

    A mathematical model of isoelectric focusing at the steady state has been developed for an M-component system of electrochemically defined ampholytes. The model is formulated from fundamental principles describing the components' chemical equilibria, mass transfer resulting from diffusion and electromigration, and electroneutrality. The model consists of ordinary differential equations coupled with a system of algebraic equations. The model is implemented on a digital computer using FORTRAN-based simulation software. Computer simulation data are presented for several two-component systems showing the effects of varying the isoelectric points and dissociation constants of the constituents.

  9. Separation of arginase isoforms by capillary zone electrophoresis and isoelectric focusing in density gradient column.

    PubMed

    Pedrosa, M M; Legaz, M E

    1995-04-01

    Four major arginase isoforms, I, II, III and IV, have been detected in Evernia prunastri thallus. They differ in terms of both physical and biochemical properties. The isoelectric point (pI) of these proteins has been determined by both isoelectric focusing in density gradient column and high-performance capillary electrophoresis (HPCE). Isoelectric focusing revealed charge microheterogeneity for isoforms II and IV whereas arginases I and II had the same pI value of 5.8. HPCE separation confirmed this charge microheterogeneity for isoform IV but not for isoform III, and provided evidence of microheterogeneity for isoforms I and II. The effect of various electrolyte buffers and running conditions on the HPCE separation of arginase isoform were investigated. Addition of 0.5 mM spermidine (SPD) to the running buffer reduced the electroosmotic flow (EOF) and permitted discriminating between the native proteins and protein fragments.

  10. Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody.

    PubMed

    Suba, Dávid; Urbányi, Zoltán; Salgó, András

    2015-10-10

    Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some "out-of-the-box" applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs. A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R(2)>0.99) and good resolution between all of the isoform peaks (R=2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers. The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects.

  11. Purification of the Porcine rubulavirus attachment protein by liquid isoelectric focusing.

    PubMed

    Santos-López, Gerardo; Flores, Esmeralda; Baños, Rocío; Herrera-Camacho, Irma; Reyes-Leyva, Julio

    2004-05-01

    Porcine rubulavirus (PoRV) is an emerging virus responsible for meningoencephalitis, respiratory distress, and reproductive alterations in pigs. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PoRV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid hydrolysis) activity has been proposed to be a virulence factor. So, HN is an ideal target for therapeutic treatment and prevention of this viral infection. This work describes a simple, fast, and sensitive method to purify the active form of HN protein based on its isoelectric point. HN was purified at a pH of 4.4, at which a single protein band of 66 kDa was observed on SDS-PAGE. Pure HN showed a maximal enzymatic activity at pH 3.5 and 37 degrees C using bovine fetuin as substrate. However, it retains circa 80% of its activity at a wide temperature range from 30 to 55 degrees C. We also describe improvements of neuraminidase determination method, which permits analysis in a microplate spectrophotometer, thereby increasing the sensitivity and reducing the costs of valuable reagents and biological samples.

  12. Charge-based characterisation of high-molecular-weight glutenin subunits from common wheat by capillary isoelectric focusing.

    PubMed

    Salmanowicz, Bolesław P; Langner, Monika; Franaszek, Sławomir

    2014-11-01

    In this study, the capillary isoelectric focusing (CIEF) method for the separation and charge characterisation of the heterogeneity of high molecular-weight-glutenin subunits (HMW-GS) in common wheat (Triticum aestivum L.) using linear polyacrylamide (LPA) and polyvinyl alcohol (PVA) coated capillaries was developed. Particularly good repeatability and well-resolved charge isoform profiles were obtained by introducing a mixture of carrier ampholytes (pH 3-10 and pH 5-8), a high concentration of urea (6M) and SB3-12 as detergent in a sample solution during separation in a PVA-coated capillary. One major and one or two minor isoforms were observed for the individual HMW-GS. These isoforms were satisfactorily separated using a pH gradient into two groups: y-type isoforms and x-type isoforms encoded by the Glu-B1 locus with shorter migration times and remaining x-type isoforms with longer times. The method produced from eight to twelve isoforms of wheat HMW-GS with pI points in the range of 4.72-6.98. Generally, the minor isoforms were more acidic compared with the major isoform. The y-type subunits had an approximately neutral character (pI 6.70-6.98); however, x-types showed a weakly acidic character (pI 4.72-5.23), with the exception of subunits encoded by the Glu-B1 locus. The isoelectric point peak profiles were compared with capillary zone electrophoresis (CZE) electropherograms. Generally, the number of detected isoforms for the particular HMW-GS detected using both methods were similar.

  13. Crystallization of isoelectrically homogeneous cholera toxin

    SciTech Connect

    Spangler, B.D.; Westbrook, E.M. )

    1989-02-07

    Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. The authors have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. They have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by their procedure readily forms large single crystals. The crystal form has been described previously. They have recorded data from native crystals of cholera toxin to 3.0-{angstrom} resolution with our electronic area detectors. With these data, they have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. They are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.

  14. Mathematical model for steady state, simple ampholyte isoelectric focusing: Development, computer simulation and implementation

    NASA Technical Reports Server (NTRS)

    Palusinski, O. A.; Allgyer, T. T.

    1979-01-01

    The elimination of Ampholine from the system by establishing the pH gradient with simple ampholytes is proposed. A mathematical model was exercised at the level of the two-component system by using values for mobilities, diffusion coefficients, and dissociation constants representative of glutamic acid and histidine. The constants assumed in the calculations are reported. The predictions of the model and computer simulation of isoelectric focusing experiments are in direct importance to obtain Ampholine-free, stable pH gradients.

  15. Focal point analysis of torsional isomers of acrylic acid

    NASA Astrophysics Data System (ADS)

    Alev Çiftçioğlu, Gökçen; Trindle, Carl; Yavuz, Ilhan

    2010-10-01

    The thermochemistry of acrylic acid has presented challenges owing to its high reactivity, tendency to dimerize in the gas phase, and the existence of two very nearly equal energy conformational isomers. Well-tested thermochemical schemes including G2, G3, G4, and CBS-QB3 agree in the prediction that the s-cis syn structure is the most stable of the torsional isomers, with the s-cis anti form lying 3 kJ mol-1 or less higher in energy. Microwave spectra suggest a value of 0.63 kJ mol-1. The energy barrier between these forms is in the neighbourhood of 25 kJ mol-1 according to a MP2/cc-pVDZ calculation. We present estimates of the relative energies of all four torsional isomers and the rotational barrier based on a variant of the Focal Point Analysis developed by Császár and co-workers. These calculations, extending to the CCSD(T)/cc-pV5Z level, predict that the s-cis anti torsional isomer is the most stable form, in contrast to prior estimates. The s-cis syn form lies about 2.9 kJ mol-1 higher, while the s-trans syn and anti forms lie at about 21.7 and 23.3 kJ mol-1, respectively. We estimate the rotational barrier between the s-cis trans and s-cis anti structures to be about 23.9 kJ mol-1. Error ranges derived from the fit to extrapolation forms suggest that our estimates have an uncertainty of about 0.1 kJ mol-1.

  16. A novel prefractionation method combining protein and peptide isoelectric focusing in immobilized pH gradient strips.

    PubMed

    Pernemalm, Maria; Lehtiö, Janne

    2013-02-01

    To increase sensitivity and analytical depth in shotgun proteomics, prefractionation of complex samples is often used. Here we describe a novel prefractionation method, Sandwich high resolution isoelectric focusing, which combines both protein and peptide isoelectric focusing. In the first step, intact proteins are separated on the basis of isoelectric point (pI) using traditional immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing proteins of interest are subsequently cut out and applied to in-strip digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are used as loading bridges. The peptides are thereby directly applied to the peptide isoelectric focusing, without an intermediate elution step, and separated on narrow range IPG strips to reduce the complexity on the peptide level. In the final step, the peptides are eluted into 96-well plates and analyzed with mass spectrometry. In a proof of principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins, with concentrations spanning over 6 orders of magnitude.

  17. Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.

    PubMed

    Hosken, Brian D; Li, Charlene; Mullappally, Berny; Co, Carl; Zhang, Boyan

    2016-06-07

    Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

  18. Isoelectric profiles of human erythropoietin are different in serum and urine.

    PubMed

    Lasne, Françoise; Martin, Laurent; Martin, Jean Antoine; de Ceaurriz, Jacques

    2007-08-01

    By adding a step of immunoaffinity to the method we had previously developed for analysing erythropoietin (EPO) in urine, we were able to study the isoelectric profiles of this hormone in human serum samples. This method was sensitive enough to investigate samples presenting physiological levels of this hormone. Comparison with the corresponding profiles in urine showed that natural EPO was systematically more acidic in urine. The acidification process, which was not patent in the non-human primate Cynomolgus macaque, clearly also affected recombinant EPO when injected into humans. This process was unrelated to any enzymatic activity in urine since the incubation of natural or recombinant EPO in urine induced no transformation of their isoelectric profiles. The nature and mechanism of the structural modifications occurring during the renal handling of this hormone remain to be investigated.

  19. Separation of Recombinant Therapeutic Proteins Using Capillary Gel Electrophoresis and Capillary Isoelectric Focusing.

    PubMed

    De Jong, Caitlyn A G; Risley, Jessica; Lee, Alexis K; Zhao, Shuai Sherry; Chen, David D Y

    2016-01-01

    Detailed step-by-step methods for protein separation techniques based on capillary electrophoresis (CE) are described in this chapter. Focus is placed on two techniques, capillary gel electrophoresis (CGE) and capillary isoelectric focusing (cIEF). CGE is essentially gel electrophoresis, performed in a capillary, where a hydrogel is used as a sieving matrix to separate proteins or peptides based on size. cIEF separates proteins or peptides based on their isoelectric point (pI), the pH at which the protein or peptide bears no charges. Detailed protocols and steps (including capillary preparation, sample preparation, CE separation conditions, and detection) for both CGE and cIEF presented so that readers can follow the described methods in their own labs.

  20. Isoelectric focusing in agarose gel for detection of oligoclonal bands in cerebrospinal and other biological fluids.

    PubMed

    Csako, Gyorgy

    2012-01-01

    Isoelectric focusing (IEF) coupled with immunodetection (immunofixation or immunoblotting) has become the leading technique for the detection and study of oligoclonal bands (OCBs) in cerebrospinal fluid (CSF) and also is increasingly used in other body fluids such as the tear and serum. Limited commercial availability of precast agarose IEF gels for research and a need for customization prompted reporting a detailed general protocol for the preparation and casting of agarose IEF gel along with sample, control, and isoelectric point marker preparation and carrying out the focusing itself for CSF OCBs. However, the method is readily adaptable to the use of other body fluid specimens and, possibly, research specimens such as culture fluids as well.

  1. Capillary isoelectric focusing and fluorometric detection of proteins and microorganisms dynamically modified by poly(ethylene glycol) pyrenebutanoate.

    PubMed

    Horka, Marie; Ruzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

    2006-12-15

    The nonionogenic pyrene-based tenside, poly(ethylene glycol) pyrenebutanoate, was prepared and applied in capillary isoelectric focusing with fluorometric detection. This dye was used here as a buffer additive in capillary isoelectric focusing for a dynamic modification of the sample of proteins and microorganisms. The values of the isoelectric points of the labeled bioanalytes were calculated with use of the fluorescent pI markers and were found comparable with pI of the native compounds. The mixed cultures of proteins and microorganisms, Escherichia coli CCM 3954, Staphylococcus epidermidis CCM 4418, Proteus vulgaris, Enterococcus faecalis CCM 4224, and Stenotrophomonas maltophilia, the strains of the yeast cells, Candida albicans CCM 8180, Candida krusei, Candida parapsilosis, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae were reproducibly focused and separated by the suggested technique. Using UV excitation for the on-column fluorometric detection, the minimum detectable amount was down to 10 cells injected on the separation capillary.

  2. Isoelectric focusing of wound-induced tomato ACC synthase

    SciTech Connect

    White, J.A.; Kende, H. )

    1990-05-01

    Several techniques of electrofocusing have been used to determine whether 1-aminocyclopropane-1-carboxylate (ACC) synthase isolated from wounded tomato pericarp tissue exists in different isoforms, each with its characteristic isoelectric point (pI). The pI of the native enzyme was found to be 6.0 {plus minus} 0.2. When radiolabeled, denatured ACC synthase was electrofocused by non-equilibrium pH gradient electrophoresis (NEpHGE), the enzyme separated into four discernible spots which, upon reaching equilibrium, ranged in pI from 6.6 to 6.9. Immunopurified ACC synthase from four tomato cultivars (Duke, Cornell, Mountain Pride and Pik Red) migrated in each case as a 50-kDa protein on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). We propose that native ACC synthase in extracts of tomato pericarp tissue exists in one single form and that the charge heterogeneities observed upon electrofocusing of denatured enzyme result from modifications of preexisting protein.

  3. Band Dispersion During Isoelectric Focusing in a Microchip

    NASA Astrophysics Data System (ADS)

    Dutta, Prashanta; Shim, Jaesool; Ivory, Cornelius F.

    2008-11-01

    Ampholyte based isoelectric focusing (IEF) is simulated for a two-dimensional horse shoe microchannel. Mobility correction for proteins and ampholytes are considered in the model because the mobility of both large molecules (proteins) and small molecules (ampholytes) varies with ionic strength in the IEF process. Four model proteins are allowed to focus in the presence of 25 biprotic carrier ampholytes in a horse shoe microchannel. Normalized variances of protein bands are calculated from numerical results using moment method. We particularly show dispersion behavior of proteins in IEF and discuss the differences between linear electrophoretic transports and nonlinear IEF in a horse shoe microchannel. Our numerical results show that protein spreading is induced by a turn during gradient formation stage, but the dispersed bands are rearranged and straighten as double peaks of a protein start to focus at the focal point. The rearrangement of spreading band is very unique compared to other linear electrokinetic phenomena (electroosmotic flow and capillary zone electrophoresis) and is independent of channel position and channel shape. Hence, one can perform the IEF to separate proteins in complex geometry without incorporating hyperturns.

  4. Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection.

    PubMed

    Michels, David A; Tu, Andrea W; McElroy, Will; Voehringer, David; Salas-Solano, Oscar

    2012-06-19

    Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.

  5. What Is the Boiling Point and Heat of Vaporization of Sulfuric Acid?

    ERIC Educational Resources Information Center

    Myers, R. Thomas

    1983-01-01

    Discusses the values presented in various handbooks for the boiling point and heat of vaporization of sulfuric acid, noting discrepencies. Analyzes various approaches to data presentation, discussing the data on sulfuric acid in light of the Trouton constant. Points out the need for a more critical use of tables. (JM)

  6. Isoelectric focusing sample injection for capillary electrophoresis of proteins.

    PubMed

    Wu, Xing-Zheng; Zhang, Luo-Hong; Onoda, Koji

    2005-02-01

    Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.

  7. Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants.

    PubMed

    Kristl, Theresa; Stutz, Hanno

    2015-01-01

    One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1-10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3-10 with Pharmalyte 5-6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5-6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.

  8. Isoelectric focusing of proteins and peptides

    NASA Technical Reports Server (NTRS)

    Egen, N.

    1979-01-01

    Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

  9. Highly Sensitive and Multiple Enzyme Activity Assay Using Reagent-release Capillary-Isoelectric Focusing with Rhodamine 110-based Substrates.

    PubMed

    Sueyoshi, Kenji; Nogawa, Yuto; Sugawara, Kasumi; Endo, Tatsuro; Hisamoto, Hideaki

    2015-01-01

    In this study, a simple and highly sensitive enzyme activity assay based on reagent-release capillary-isoelectric focusing is described. Reagent-release capillaries containing a fluorescent substrate, which produces fluorescent products possessing an isoelectric point after reaction with enzymes, provides a simple procedure. This is because it allows to spontaneously inject a sample solution into the capillary by capillary action, mixing reagents, and subsequently concentrating the fluorescent products based on isoelectric focusing. Fluorescent rhodamine 110 and its monoamide derivative, which were generated as a final product and an intermediate, respectively, were then focused and separated by reagent-release capillary-isoelectric focusing. After 30 min of enzyme reactions, two focused fluorescent bands were clearly isolated along the prepared capillaries. Employing the focused band of rhodamine 110 monoamide allowed for highly sensitive detection of enzyme activity in the 10 pg mL(-1) order, while that of the conventional assay using a microplate was in the ng mL(-1) order. Furthermore, arraying reagent-release capillaries of different substrates on a chip allowed for simultaneous multi-assay of enzyme activity with good sensitivity in the pg mL(-1) order for each protein.

  10. Isolation of isoelectrically pure cholera toxin for crystallization

    NASA Astrophysics Data System (ADS)

    Spangler, Brenda D.; Westbrook, Edwin M.

    1991-03-01

    We have determined that the failure of cholera toxin to crystallize well results from its isoelectric heterogeneity, which is probably due to a post-translational process such as deamidation of its B subunit. Every sample of cholera toxin we have examined from commercial or academic suppliers has been heterogeneous; heterogeneous cholera toxin does not crystallize satisfactorily. We have overcome this problem by using ion-exchange fast protein liquid chromatography (FPLC) to obtain an isoelectrically homogeneous species of cholera toxin. Homogeneous cholera toxin crystallizes readily, forming single, nonmosaic crystals suitable for X-ray diffraction studies. For this process, protein was applied to a MonoQ ion-exchange column, then eluted with an isocratic low salt buffer followed by a linear salt gradient (0-100 mM NaCl). Column fractions were analyzed on isoelectric focusing gels, and those fractions containing the desired homogeneous species were pooled and concentrated. Crystals formed within 24 to 48 h in a MOPS/PEG buffer, which made use of slow isoelectric precipitation to induce crystallization.

  11. Isolation of isoelectrically pure cholera toxin for crystallization

    SciTech Connect

    Spangler, B.D.; Westbrook, E.M.

    1989-01-01

    We have determined that the failure of cholera toxin to crystallize well results from its isoelectric heterogeneity, which is probably due to a post-translational process such as deamidation of its B subunit. Every sample of cholera toxin we have examined from commercial or academic suppliers has been heterogeneous; heterogeneous cholera toxin does not crystallize satisfactorily. We have overcome this problem by using ion-exchange fast protein liquid chromatography (FPLC) to obtain an isoelectrically homogeneous species of cholera toxin. Homogeneous cholera toxin crystallizes readily, forming single, nonmosaic crystals suitable for x-ray diffraction studies. For this process, protein was applied to a MonoQ ion-exchange column, then eluted with an isocratic low salt buffer followed by a linear salt gradient (0-100 mM NaCl). Column fractions were analyzed on isoelectric focusing gels, and those fractions containing the desired homogeneous species were pooled and concentrated. Crystals formed within 24 to 48 hours in a MOPS/PEG buffer, which made use of slow isoelectric precipitation to induce crystallization. 23 refs., 6 figs.

  12. Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Dovichi, Norman J

    2017-02-01

    We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH3 H2 O section worked as catholyte. As focusing progressed, the NH3 H2 O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH3 H2 O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH3 H2 O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation ≤0.6% for myoglobin and cytochrome C in triplicate runs.

  13. A chemical test of critical point isomorphism: reactive dissolution of ionic solids in isobutyric acid + water near the consolute point.

    PubMed

    Baird, James K; Baker, Jonathan D; Hu, Baichuan; Lang, Joshua R; Joyce, Karen E; Sides, Alison K; Richey, Randi D

    2015-03-12

    Binary liquid mixtures having a consolute point can be used as solvents for chemical reactions. When excess cerium(IV) oxide is brought into equilibrium with a mixture of isobutyric acid + water, and the concentration of cerium in the liquid phase is plotted in van't Hoff form, a straight line results for temperatures sufficiently in excess of the critical solution temperature. Within 1 K of the critical temperature, however, the concentration becomes substantially suppressed, and the van't Hoff slope diverges toward negative infinity. According to the phase rule, one mole fraction can be fixed. Given this restriction, the temperature behavior of the data is in exact agreement with the predictions of both the principle of critical point isomorphism and the Gibbs-Helmholtz equation. In addition, we have determined the concentration of lead in the liquid phase when crystalline lead(II) sulfate reacts with potassium iodide in isobutyric acid + water. When plotted in van't Hoff form, the data lie on a straight line for all temperatures including the critical region. The phase rule indicates that two mole fractions can be fixed. With this restriction, the data are in exact agreement with the principle of critical point isomorphism.

  14. Human brain activity patterns beyond the isoelectric line of extreme deep coma.

    PubMed

    Kroeger, Daniel; Florea, Bogdan; Amzica, Florin

    2013-01-01

    The electroencephalogram (EEG) reflects brain electrical activity. A flat (isoelectric) EEG, which is usually recorded during very deep coma, is considered to be a turning point between a living brain and a deceased brain. Therefore the isoelectric EEG constitutes, together with evidence of irreversible structural brain damage, one of the criteria for the assessment of brain death. In this study we use EEG recordings for humans on the one hand, and on the other hand double simultaneous intracellular recordings in the cortex and hippocampus, combined with EEG, in cats. They serve to demonstrate that a novel brain phenomenon is observable in both humans and animals during coma that is deeper than the one reflected by the isoelectric EEG, and that this state is characterized by brain activity generated within the hippocampal formation. This new state was induced either by medication applied to postanoxic coma (in human) or by application of high doses of anesthesia (isoflurane in animals) leading to an EEG activity of quasi-rhythmic sharp waves which henceforth we propose to call ν-complexes (Nu-complexes). Using simultaneous intracellular recordings in vivo in the cortex and hippocampus (especially in the CA3 region) we demonstrate that ν-complexes arise in the hippocampus and are subsequently transmitted to the cortex. The genesis of a hippocampal ν-complex depends upon another hippocampal activity, known as ripple activity, which is not overtly detectable at the cortical level. Based on our observations, we propose a scenario of how self-oscillations in hippocampal neurons can lead to a whole brain phenomenon during coma.

  15. Extended Gate Field-Effect Transistor Biosensors for Point-Of-Care Testing of Uric Acid.

    PubMed

    Guan, Weihua; Reed, Mark A

    2017-01-01

    An enzyme-free redox potential sensor using off-chip extended-gate field effect transistor (EGFET) with a ferrocenyl-alkanethiol modified gold electrode has been used to quantify uric acid concentration in human serum and urine. Hexacyanoferrate (II) and (III) ions are used as redox reagent. The potentiometric sensor measures the interface potential on the ferrocene immobilized gold electrode, which is modulated by the redox reaction between uric acid and hexacyanoferrate ions. The device shows a near Nernstian response to uric acid and is highly specific to uric acid in human serum and urine. The interference that comes from glucose, bilirubin, ascorbic acid, and hemoglobin is negligible in the normal concentration range of these interferents. The sensor also exhibits excellent long term reliability and is regenerative. This extended gate field effect transistor based sensor is promising for point-of-care detection of uric acid due to the small size, low cost, and low sample volume consumption.

  16. Implementation of boric acid in the field - Indian Point Unit 3 plant. Final report

    SciTech Connect

    Jusino, B.J.

    1983-05-01

    A corrosion phenomenon termed denting has occurred in some operating PWR nuclear steam generators. A steam-generator boric-acid-conditioning program was implemented at Indian Point Unit 3 to demonstrate the effectiveness of boric acid in reducing or stopping the progression of denting. The program included a four-day low-power, boric-acid soak of the steam generators followed by continuous addition of boric acid at high-power operation. An on-line hydrogen-monitoring technique and steam-generator inspection technique were used to determine the baseline condition of the steam generators and to provide an indication of the rate of denting progression after the addition of boric acid to the steam generators. The results from these tests indicated that, although denting had continued, it had progressed at a slower rate than the immediate period before the boric-acid treatment.

  17. Purification and initial characterization of the 71-kilodalton rat heat-shock protein and its cognate as fatty acid binding proteins.

    PubMed

    Guidon, P T; Hightower, L E

    1986-06-03

    The major rat heat-shock (stress) protein and its cognate were purified to electrophoretic homogeneity from livers of heat-shocked rats. Both proteins exhibited similar behavior on a variety of column chromatography matrices but were separable by preparative isoelectric focusing under nondenaturing conditions by virtue of a 0.2 pH unit difference in isoelectric point. Both purified proteins had similar physical properties, suggesting the possibility that they may have similar biological functions as well. Both proteins were homodimers under nondissociative conditions (Mr 150 000) with isoelectric points of 5.0 (cognate) and 5.2 (major stress protein). After denaturation, both proteins had an increase in isoelectric point of 0.6 pH unit, and the resulting polypeptide chains had apparent molecular weights of 73 000 (cognate) and 71 000 (major stress protein). Similarities in the electrophoretic properties of these two proteins and serum albumin, which also undergoes a large basic shift in isoelectric point due to loss of fatty acids and conformational changes accompanying denaturation, prompted us to search for lipids associated with the purified 71-kilodalton stress protein and its cognate. Thin-layer chromatography of chloroform/methanol extracts of these two proteins revealed nonesterified fatty acids bound to both proteins. Palmitic acid, stearic acid, and a small amount of myristic acid were identified by gas chromatography/mass spectroscopy. Both proteins contained approximately four molecules of fatty acid per dimer with palmitate and stearate present in a one to one molar ratio. Possible roles of the major stress protein and its cognate as fatty acid associated proteins in cellular responses to stress are discussed.

  18. Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing.

    PubMed

    Ferreira, R A; Martins-Dias, S

    2016-09-15

    An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3-10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.

  19. Thermal isoelectric precipitation of alpha-lactalbumin from a whey protein concentrate: Influence of protein-calcium complexation.

    PubMed

    Bramaud, C; Aimar, P; Daufin, G

    1995-07-20

    The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. As precipitation is a phenomenon dependent on the protein hydrophobicity, and as the release of the tightly bound calcium occurring at pH around 4 modifies the alpha-LA hydrophobicity, the specific role of calcium on isoelectric precipitation is investigated. A study of the extent of alpha-LA precipitation in a whey protein concentrate under various operating conditions of pH, temperature, protein concentration, and calcium content is presented. We propose a mechanism for this phenomenon as a combination of a complexation equilibrium and of an irreversible precipitation, to account for the influence of temperature, alpha-LA concentration total ionic content, and calcium concentration, and also to estimate the complexation equilibrium constant. (c) 1995 John Wiley & Sons, Inc.

  20. A rapid flat gel isoelectric focusing method for the determination of apolipoprotein E phenotypes and its application.

    PubMed

    Eto, M; Watanabe, K; Ishii, K

    1985-06-30

    A rapid flat gel isoelectric focusing method has been developed for the determination of apolipoprotein E phenotypes. Isoelectric focusing in 5% polyacrylamide flat gel with 8 mol/l urea and 2.8% pharmalyte (pH 4-6.5) was carried out at 3,000 V and 4 degrees C for 1 h under a constant power of 30 W. The separation of apolipoprotein E isoproteins was good and the isoelectric points were determined. Using this method, the apolipoprotein E phenotype frequency was examined in the Japanese population, and a higher frequency of phenotype E3/3 and a lower frequency of phenotype E3/2 were found in Japanese than those reported for the German, American or English population. In our focusing system the cut-off point of apolipoprotein E2/E3 ratio between the phenotype E3/3 and E3/2 was assumed to be approximately 0.9. These results indicate that this method may be useful for the determination of apolipoprotein E phenotypes.

  1. Classification of wheat varieties by isoelectric focusing patterns of gliadins and neural network.

    PubMed

    Søndergaard, I; Jensen, K; Krath, B N

    1994-05-01

    Classification of wheat varieties, using isoelectric focusing patterns of the gliadins, image processing and neural networks, is described. The method was compared to a statistical classification method, discriminant analysis. The isoelectric point and the area of each band were calculated by image processing. Different methods of presenting the electrophoretic patterns to the neural network were studied. The most effective method was transformation of the electrophoretic pattern to a small (11 x 47 pixels) representation of the original digitized image, which was presented to the neural network as a vector. The neural network was trained with a number of patterns and tested with new patterns from different electrophoretic runs of the same wheat varieties. In this study we used ten different wheat varieties and the neural network was able to classify 95.5% of the patterns correctly. The statistical classification method classified the same data set 91.8% correctly. We conclude that both the neural network and discriminant analysis were able to classify the patterns correctly with a high degree of certainty. The patterns that were misclassified were indistinguishable by visual inspection.

  2. Detection of protein phosphorylation and charge isoforms using vertical one-dimensional isoelectric focusing gels.

    PubMed

    Anderson, Jeffrey C; Peck, Scott C

    2014-01-01

    During many biological responses, changes in protein modifications (e.g., phosphorylation) are often more critical than changes in protein abundance in determining the outcome of cellular responses. These important regulatory changes can alter a protein's location, activity, or binding partners. Monitoring modifications such as phosphorylation is often impeded, or even prevented, because of the need for specialized reagents and equipment that are expensive and/or time-consuming to produce. However, many protein modifications alter the isoelectric point (pI) of a protein. Therefore, we developed a denaturing, one-dimensional isoelectric focusing (IEF) procedure that separates proteins based on their pI to resolve different isoforms, allowing a relatively simple strategy for detecting changes in protein modifications. Although similar results can be achieved by two-dimensional gel electrophoresis, the method described here uses a multi-well SDS-PAGE format that allows many more samples to be assayed within a single gel, thereby greatly decreasing both the time and cost needed to assess modifications of a single protein in response many different treatment conditions. To increase the sensitivity of detection, we also optimized a procedure to transfer proteins from these gels to membranes for subsequent immunodetection. This combination of techniques provides the means of interrogating the number and stoichiometry of isoforms from total protein extracts without a priori knowledge of which modification may occur.

  3. Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

    PubMed

    Choi, Hoseok; Choi, Nakchul; Lim, Butaek; Kim, Tae-Wuk; Song, Simon; Kim, Young-Pil

    2015-11-15

    We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the μIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by μIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.

  4. Effect of Finishing System on Subcutaneous Fat Melting Point and Fatty Acid Composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Angus-cross steers (n = 69) were used to determine the effect of finishing system on subcutaneous fat melting point and fatty acid composition. Three finishing systems were evaluated: 1) mixed pasture for 134 d [MP], 2) mixed pasture for 93 d and alfalfa for 41 d [AL], or 3) concentrate finishing f...

  5. The trace analysis of microorganisms in real samples by combination of a filtration microcartridge and capillary isoelectric focusing.

    PubMed

    Horká, Marie; Horký, Jaroslav; Kubesová, Anna; Zapletalová, Eva; Slais, Karel

    2011-07-01

    Trace analysis of microorganisms in real biological samples needs very sensitive methods for their detection. Most procedures for detecting and quantifying pathogens require a sample preparation step including concentrating microorganisms from large sample volumes with high and reproducible efficiency. Electromigration techniques have great potential to include the preconcentration, separation, and detection of whole cells and therefore they can rapidly indicate the presence of pathogens. The preconcentration and separation of microorganisms from real suspensions utilising a combination of filtration and capillary isoelectric focusing was developed and the possibility for its application to real samples was verified. For our experiments, spores of Monilinia species and of Penicillium expansum were selected as model bioparticles, as they cause major losses in agrosystems. The isoelectric points of the spores of M. laxa, M. fructigena, M. fruticola, and P. expansum were determined and the method was verified using real samples taken directly from infected apples. The coupling of a filtration cartridge with a separation capillary can improve the detection limit of isoelectric focusing with UV detection by at least 4 orders of magnitude. Spores of M. fructigena and of M. laxa in numbers of hundreds of particles per milliliter were detected on a visually noninfected apple surface which was cross-contaminated during handling and storage. The efficiency of preconcentration and a preliminary identification was verified by the phenotyping technique after cultivation of the spores sampled from the apple surface.

  6. Electromechanical properties of dried tendon and isoelectrically focused collagen hydrogels.

    PubMed

    Denning, D; Abu-Rub, M T; Zeugolis, D I; Habelitz, S; Pandit, A; Fertala, A; Rodriguez, B J

    2012-08-01

    Assembling artificial collagenous tissues with structural, functional, and mechanical properties which mimic natural tissues is of vital importance for many tissue engineering applications. While the electro-mechanical properties of collagen are thought to play a role in, for example, bone formation and remodeling, this functional property has not been adequately addressed in engineered tissues. Here the electro-mechanical properties of rat tail tendon are compared with those of dried isoelectrically focused collagen hydrogels using piezoresponse force microscopy under ambient conditions. In both the natural tissue and the engineered hydrogel D-periodic type I collagen fibrils are observed, which exhibit shear piezoelectricity. While both tissues also exhibit fibrils with parallel orientations, Fourier transform analysis has revealed that the degree of parallel alignment of the fibrils in the tendon is three times that of the dried hydrogel. The results obtained demonstrate that isoelectrically focused collagen has similar structural and electro-mechanical properties to that of tendon, which is relevant for tissue engineering applications.

  7. Point mutation of H3/H4 histones affects acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Liu, Xiangyong; Zhang, Xiaohua; Zhang, Zhaojie

    2014-10-10

    The molecular mechanism of acetic acid tolerance in yeast remains unclear despite of its importance for efficient cellulosic ethanol production. In this study, we examined the effects of histone H3/H4 point mutations on yeast acetic acid tolerance by comprehensively screening a histone H3/H4 mutant library. A total of 24 histone H3/H4 mutants (six acetic acid resistant and 18 sensitive) were identified. Compared to the wild-type strain, the histone acetic acid-resistant mutants exhibited improved ethanol fermentation performance under acetic acid stress. Genome-wide transcriptome analysis revealed that changes in the gene expression in the acetic acid-resistant mutants H3 K37A and H4 K16Q were mainly related to energy production, antioxidative stress. Our results provide novel insights into yeast acetic acid tolerance on the basis of histone, and suggest a novel approach to improve ethanol production by altering the histone H3/H4 sequences.

  8. Lab-on-a-chip nucleic-acid analysis towards point-of-care applications

    NASA Astrophysics Data System (ADS)

    Kopparthy, Varun Lingaiah

    Recent infectious disease outbreaks, such as Ebola in 2013, highlight the need for fast and accurate diagnostic tools to combat the global spread of the disease. Detection and identification of the disease-causing viruses and bacteria at the genetic level is required for accurate diagnosis of the disease. Nucleic acid analysis systems have shown promise in identifying diseases such as HIV, anthrax, and Ebola in the past. Conventional nucleic acid analysis systems are still time consuming, and are not suitable for point-ofcare applications. Miniaturized nucleic acid systems has shown great promise for rapid analysis, but they have not been commercialized due to several factors such as footprint, complexity, portability, and power consumption. This dissertation presents the development of technologies and methods for a labon-a-chip nucleic acid analysis towards point-of-care applications. An oscillatory-flow PCR methodology in a thermal gradient is developed which provides real-time analysis of nucleic-acid samples. Oscillating flow PCR was performed in the microfluidic device under thermal gradient in 40 minutes. Reverse transcription PCR (RT-PCR) was achieved in the system without an additional heating element for incubation to perform reverse transcription step. A novel method is developed for the simultaneous pattering and bonding of all-glass microfluidic devices in a microwave oven. Glass microfluidic devices were fabricated in less than 4 minutes. Towards an integrated system for the detection of amplified products, a thermal sensing method is studied for the optimization of the sensor output. Calorimetric sensing method is characterized to identify design considerations and optimal parameters such as placement of the sensor, steady state response, and flow velocity for improved performance. An understanding of these developed technologies and methods will facilitate the development of lab-on-a-chip systems for point-of-care analysis.

  9. Critical-point universality in adsorption: The effect of charcoal on a mixture of isobutyric acid and water near the consolute point

    NASA Astrophysics Data System (ADS)

    Giesy, Timothy J.; Chou, Alan S.; McFeeters, Robert L.; Baird, James K.; Barlow, Douglas A.

    2011-06-01

    The mixture of isobutyric acid and water has a consolute point at a temperature of 25.75 °C and mole fraction 0.1148 isobutyric acid. When charcoal is added to this mixture, the concentration of isobutyric acid is reduced by adsorption. We have measured the action of charcoal on solutions of isobutyric acid and water as a function of isobutyric acid mole fraction at temperatures of 25.85 and 32.50 °C. At the higher temperature, the specific adsorption density (y2α/m) satisfies the Freundlich equation (y2α/m)=KX21/n, where y2α is the mass of isobutyric acid adsorbed, m is the mass of charcoal, X2 is the equilibrium mole fraction of isobutyric acid, n is the Freundlich index, and K=K(T) is an amplitude that depends upon the temperature T. At 25.85 °C, a critical endpoint is located at an isobutyric acid mole fraction X2ce=0.09. When compared with the Freundlich equation at this temperature, a plot of the specific adsorption density as a function of X2 in the vicinity of the critical-endpoint composition assumes a shape which is reminiscent of the derivative of a Dirac delta function. Using critical-point scaling theory, we show that this divergent pattern is consistent with the principle of critical point universality.

  10. Critical-point universality in adsorption: the effect of charcoal on a mixture of isobutyric acid and water near the consolute point.

    PubMed

    Giesy, Timothy J; Chou, Alan S; McFeeters, Robert L; Baird, James K; Barlow, Douglas A

    2011-06-01

    The mixture of isobutyric acid and water has a consolute point at a temperature of 25.75 °C and mole fraction 0.1148 isobutyric acid. When charcoal is added to this mixture, the concentration of isobutyric acid is reduced by adsorption. We have measured the action of charcoal on solutions of isobutyric acid and water as a function of isobutyric acid mole fraction at temperatures of 25.85 and 32.50 °C. At the higher temperature, the specific adsorption density (y(2)(α)/m) satisfies the Freundlich equation (y(2)(α)/m)=KX(2)(1/n), where y(2)(α) is the mass of isobutyric acid adsorbed, m is the mass of charcoal, X(2) is the equilibrium mole fraction of isobutyric acid, n is the Freundlich index, and K=K(T) is an amplitude that depends upon the temperature T. At 25.85 °C, a critical endpoint is located at an isobutyric acid mole fraction X(2)(ce)=0.09. When compared with the Freundlich equation at this temperature, a plot of the specific adsorption density as a function of X(2) in the vicinity of the critical-endpoint composition assumes a shape which is reminiscent of the derivative of a Dirac delta function. Using critical-point scaling theory, we show that this divergent pattern is consistent with the principle of critical point universality.

  11. Equivalence-point electromigration acid-base titration via moving neutralization boundary electrophoresis.

    PubMed

    Yang, Qing; Fan, Liu-Yin; Huang, Shan-Sheng; Zhang, Wei; Cao, Cheng-Xi

    2011-04-01

    In this paper, we developed a novel method of acid-base titration, viz. the electromigration acid-base titration (EABT), via a moving neutralization boundary (MNR). With HCl and NaOH as the model strong acid and base, respectively, we conducted the experiments on the EABT via the method of moving neutralization boundary for the first time. The experiments revealed that (i) the concentration of agarose gel, the voltage used and the content of background electrolyte (KCl) had evident influence on the boundary movement; (ii) the movement length was a function of the running time under the constant acid and base concentrations; and (iii) there was a good linearity between the length and natural logarithmic concentration of HCl under the optimized conditions, and the linearity could be used to detect the concentration of acid. The experiments further manifested that (i) the RSD values of intra-day and inter-day runs were less than 1.59 and 3.76%, respectively, indicating similar precision and stability in capillary electrophoresis or HPLC; (ii) the indicators with different pK(a) values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one; and (iii) the constant equivalence-point titration always existed in the EABT, rather than the classic volumetric analysis. Additionally, the EABT could be put to good use for the determination of actual acid concentrations. The experimental results achieved herein showed a new general guidance for the development of classic volumetric analysis and element (e.g. nitrogen) content analysis in protein chemistry.

  12. Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers.

    PubMed

    Capelli, L; Forlani, F; Perini, F; Guerrieri, N; Cerletti, P; Righetti, P G

    1998-02-01

    A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25 degrees C) high speed separations (10-12 min analysis time at 800 V/cm) could only be elicited in 20 microm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH=pI=2.77) in the presence of 7 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 microm ID), ensuring a five-times higher sensitivity. The gliadin patterns thus obtained are species-specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.

  13. Narrow-range peptide isoelectric focusing as peptide prefractionation method prior to tandem mass spectrometry analysis.

    PubMed

    Pernemalm, Maria

    2013-01-01

    High sample complexity is one of the major challenges in mass spectrometry-based proteomics today. Despite massive improvement in instrumentation, sample prefractionation is still needed to reduce sample complexity and improve proteome coverage. Isoelectric focusing (IEF) has been traditionally used as a first-dimension protein separation technique in two-dimensional gel electrophoresis-based proteomics. Recently, peptide IEF has emerged as appealing alternative for anion exchange chromatography in multidimensional LC-MS/MS workflows. The rationale behind using narrow-range peptide isoelectric focusing as a prefractionation method prior to ms/ms is to reduce the complexity induced by tryptic digestion. This is done by selectively analyzing a sub-fraction of peptides with an acidic pI. The pI range is chosen as it has previously been shown that 96 % of human proteins have at least one tryptic peptide between pH 3.4 and 4.9. This ensures high proteome coverage while reducing the number of peptides with 2/3. In addition the focusing precision is optimal in this range. Therefore, by analyzing this sub-fraction of peptides the complexity of the sample can be reduced without significant loss of proteome coverage. As the theoretical pI of peptides can be calculated, the pI of the identified peptides can be used to validate the peptide sequence (identified peptides with pI outside the pH range 3.4-4.9 are more likely to be false positives). In addition, this approach is compatible with iTRAQ labelling as the different iTRAQ labels migrate similarly in IEF.

  14. Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients.

    PubMed

    Rabilloud, T

    1994-02-01

    A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained.

  15. Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

    SciTech Connect

    Kranz, R.G.; Gennis, R.B.

    1982-04-01

    Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylenediamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a/sub 1/, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.

  16. Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane.

    PubMed Central

    Kranz, R G; Gennis, R B

    1982-01-01

    Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain. Images PMID:6277868

  17. Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

    PubMed

    Fujimoto, S; Murakami, K; Hosoda, T; Yamamoto, Y; Watanabe, K; Morinaka, Y; Ohara, A

    1992-05-01

    Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.

  18. Determination of the Thermal Decomposition Products of Terephthalic Acid by Using Curie-Point Pyrolyzer

    NASA Astrophysics Data System (ADS)

    Begüm Elmas Kimyonok, A.; Ulutürk, Mehmet

    2016-04-01

    The thermal decomposition behavior of terephthalic acid (TA) was investigated by thermogravimetry/differential thermal analysis (TG/DTA) and Curie-point pyrolysis. TG/DTA analysis showed that TA is sublimed at 276°C prior to decomposition. Pyrolysis studies were carried out at various temperatures ranging from 160 to 764°C. Decomposition products were analyzed and their structures were determined by gas chromatography-mass spectrometry (GC-MS). A total of 11 degradation products were identified at 764°C, whereas no peak was observed below 445°C. Benzene, benzoic acid, and 1,1‧-biphenyl were identified as the major decomposition products, and other degradation products such as toluene, benzophenone, diphenylmethane, styrene, benzaldehyde, phenol, 9H-fluorene, and 9-phenyl 9H-fluorene were also detected. A pyrolysis mechanism was proposed based on the findings.

  19. Amino acid and protein changes in tilapia and Spanish mackerel after irradiation and storage

    NASA Astrophysics Data System (ADS)

    Al-Kahtani, Hassan A.; Abu-Tarboush, Hamza M.; Atia, Mohamed; Bajaber, Adnan S.; Ahmed, Mohamed A.; El-Mojaddidi, Mohamed A.

    1998-01-01

    Some amino acids in tilapia decreased while some others increased when subjected to doses up to 10.0 kGy. However, 10 kGy contributed to a significant reduction in all amino acids of Spanish mackerel. Variations in amino acid contents continued during post-irradiation storage with no consistant trend of increase or decrease. SDS-PAGE of protein from both fish showed 27 bands of subunits with MW < 14.0-94.0 KD. Isoelectric focusing patterns of sarcoplasmic protein of unirradiated and irradiated fish showed no charge in the number of bands, while some changes were observed in the intensities of the anodic and cathodic bands depending on isoelectric points (pIs).

  20. A chemical test of the principle of critical point universality: The solubility of nickel (II) oxide in isobutyric acid + water near the consolute point

    NASA Astrophysics Data System (ADS)

    Hu, Baichuan; Baird, James K.; Richey, Randi D.; Reddy, Ramana G.

    2011-04-01

    A mixture of isobutyric acid + water has an upper consolute point at 38.8 mass % isobutyric acid and temperature near 26 °C. Nickel (II) oxide dissolves in this mixture by reacting with the acid to produce water and nickel isobutyrate. The solubility of nickel (II) oxide in isobutyric acid + water has been measured as a function of temperature at compositions, 25, 38.8, and 60 mass % isobutyric acid. For values of the temperature, T, which were at least 2 K in excess of the liquid-liquid phase transition temperature, the measured values of the solubility, s, lie on a straight line when plotted in van't Hoff form with ln s versus 1/T. The slope, (∂ln s/∂(1/T)), of the line is negative indicating that the dissolution reaction is endothermic. When the temperature was within 2 K of the phase transition temperature, however, (∂ln s/∂(1/T)) diverged toward negative infinity. The principle of critical point universality predicts that when excess solid nickel (II) oxide is in dissolution equilibrium with liquid isobutyric acid + water, (∂ln s/∂(1/T)) should diverge upon approaching the consolute point along the critical isopleth at 38.8 mass % isobutyric acid. As determined by the sign of the enthalpy of solution, the sign of this divergence is expected to be negative. Not only do our experiments confirm these predictions, but they also show that identical behavior can be observed at both 25 and 60 mass % isobustyric acid, compositions which lie substantially to either side of the critical composition.

  1. Indirect photomeric detection of anions in capillary electrophoresis using dyes as probes and electrolytes buffered with an isoelectric ampholyte.

    PubMed

    Johns, C; Macka, M; Haddad, P R

    2000-04-01

    The use of highly absorbing anionic dyes as probes and isoelectric ampholytes as buffers in background electrolytes (BGEs) combined with the use of a light emitting diode (LED) as a light source has been studied for ultrasensitive indirect photometric detection in capillary electrophoresis (CE). Potential dyes and buffers were evaluated based on characteristics relevant to indirect photometric detection principles, such as the electrophoretic mobility of the probe dye, its solubility and adsorption behaviour, and the isoelectric point and buffering capacity of the ampholytic buffer. Two dyes, tartrazine and naphthol yellow S, and histidine as the ampholytic buffer, were selected for detailed investigation. Purification of the probes was vital to avoid anionic impurities interfering with the detection. For the electrolytes containing a purified probe (0.5 mM) and histidine as the isoelectric buffer (p/ 7.7), hydroxypropylmethyl cellulose (approximately 0.05%) was effective in suppression of the electroosmotic flow (EOF). Analytical method performance characteristics were determined. For both probes, experimentally determined mobilities were generally close to literature values, excellent peak shapes and separation efficiencies of up to 298 000 theoretical plates were obtained, and detection limits were generally at the sub-microM level. For the naphthol yellow S-histidine BGE, linearity and reproducibility were also evaluated, with excellent linearity being observed over a range of 5-500 microM, and reproducibility (relative standard deviation, RSD) less than 1% for migration times and 2-8% for normalised peak areas. The approach developed was applied successfully to several real samples including tap water, mineral waters, and beer.

  2. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.

    PubMed

    Johlfs, Mary G; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T; Fiscus, Ronald R

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.

  3. Capillary isoelectric focusing-electrospray mass spectrometry for protein analysis

    SciTech Connect

    Tang, Q.; Harrata, A.K.; Lee, C.S.

    1995-10-01

    On-line combination of capillary isoelectric focusing (CIEF) with electrospray mass spectrometry (ESMS) as a two-dimensional separation system is demonstrated. Mixtures of model proteins including cytochrome c (horse heart), myoglobin (horse heart), and carbonic anhydrase II (bovine erythrocyte) are focused and cathodically mobilized in a polyacrylamide-coated capillary. At the end of CIEF capillary, the mobilized protein zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. The effects of carrier ampholyte concentration on the CIEF separation and the protein electrospray ionization mass spectra are presented and discussed. In this study, the focusing effect of CIEF permits analysis of very dilute protein samples. A typical concentration factor of 50-100 times is observed. The concentration detection limit of myoglobin for a full-scan CIEF-ESMS analysis is in the range of 10{sup -7} M, 2 orders of magnitude over that possible with normal capillary zone electrophoresis ESMS. 35 refs., 5 figs., 2 tabs.

  4. Two-dimensional gel electrophoresis: vertical isoelectric focusing.

    PubMed

    Dorri, Yaser

    2012-01-01

    Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

  5. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

    PubMed Central

    Johlfs, Mary G.; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T.; Fiscus, Ronald R.

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems. PMID:26132171

  6. Characterization of Microdialysis Acidification for Capillary Isoelectric Focusing Microelectrospray Ionization Mass Spectrometry

    SciTech Connect

    Yang, Liyu; Lee, Cheng S.; Hofstadler, Steven A. ); Smith, Richard D. )

    1998-01-01

    A microdialysis junction, based on a microdialysis membrane connecting a separate capillary and a short, sharply tapered microelectrospray emitter capillary, is demonstrated for on-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization mass spectrometry (ESI-MS).

  7. A Comprehensive Evaluation of the Melting Points of Fatty Acids and Esters Determined by Differential Scanning Calorimetry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The melting point is one of the most important physical properties of a chemical compound and plays a significant role in determining possible applications. For fatty acid esters the melting point is essential for a variety of food and non-food applications, the latter including biodiesel and its c...

  8. The free fractions of circulating docosahexaenoic acid and eicosapentenoic acid as optimal end-point of measure in bioavailability studies on n-3 fatty acids.

    PubMed

    Scarsi, Claudia; Levesque, Ann; Lisi, Lucia; Navarra, Pierluigi

    2015-05-01

    The high complexity of n-3 fatty acids absorption process, along with the huge amount of endogenous fraction, makes bioavailability studies with these agents very challenging and deserving special consideration. In this paper we report the results of a bioequivalence study between a new formulation of EPA+DHA ethyl esters developed by IBSA Institut Biochimique and reference medicinal product present on the Italian market. Bioequivalence was demonstrated according to the criteria established by the EMA Guideline on the Investigation of Bioequivalence. We found that the free fractions represent a better and more sensitive end-point for bioequivalence investigations on n-3 fatty acids, since: (i) the overall and intra-subject variability of PK parameters was markedly lower compared to the same variability calculated on the total DHA and EPA fractions; (ii) the absorption process was completed within 4h, and the whole PK profile could be drawn within 12-15 h from drug administration.

  9. Ecosystem-scale compensation points of formic and acetic acid in the central Amazon

    NASA Astrophysics Data System (ADS)

    Jardine, K.; Yañez Serrano, A.; Arneth, A.; Abrell, L.; Jardine, A.; Artaxo, P.; Alves, E.; Kesselmeier, J.; Taylor, T.; Saleska, S.; Huxman, T.

    2011-12-01

    Organic acids, central to terrestrial carbon metabolism and atmospheric photochemistry, are ubiquitous in the troposphere in the gas, particle, and aqueous phases. As the dominant organic acids in the atmosphere, formic acid (FA, HCOOH) and acetic acid (AA, CH3COOH) control precipitation acidity in remote regions and may represent a critical link between the terrestrial carbon and water cycles by acting as key intermediates in plant carbon and energy metabolism and aerosol-cloud-precipitation interactions. However, our understanding of the exchange of these acids between terrestrial ecosystems and the atmosphere is limited by a lack of field observations, the existence of biogenic and anthropogenic primary and secondary sources whose relative importance is unclear, and the fact that vegetation can act as both a source and a sink. Here, we first present data obtained from the tropical rainforest mesocosm at Biosphere 2 which isolates primary vegetation sources. Strong light and temperature dependent emissions enriched in FA relative to AA were simultaneously observed from individual branches (FA/AA = 3.0 ± 0.7) and mesocosm ambient air (FA/AA = 1.4 ± 0.3). We also present long-term observations of vertical concentration gradients of FA and AA within and above a primary rainforest canopy in the central Amazon during the 2010 dry and 2011 wet seasons. We observed a seasonal switch from net ecosystem-scale deposition during the dry season to net emissions during the wet season. This switch was associated with reduced ambient concentrations in the wet season (FA < 1.3 nmol mol-1, AA < 2.0 nmol mol-1) relative to the dry season (FA up to 3.3 nmol mol-1, AA up to 6.0 nmol mol-1), and a simultaneous increase in the FA/AA ambient concentration ratios from 0.3-0.8 in the dry season to 1.0-2.1 in the wet season. These observations are consistent with a switch between a biomass burning dominated source in the dry season (FA/AA < 1.0) to a vegetation dominated source in the

  10. Ecosystem-scale compensation points of formic and acetic acid in the central Amazon

    NASA Astrophysics Data System (ADS)

    Jardine, K.; Yañez Serrano, A.; Arneth, A.; Abrell, L.; Jardine, A.; Artaxo, P.; Alves, E.; Kesselmeier, J.; Taylor, T.; Saleska, S.; Huxman, T.

    2011-09-01

    Organic acids, central to terrestrial carbon metabolism and atmospheric photochemistry, are ubiquitous in the troposphere in the gas, particle, and aqueous phases. As the dominant organic acids in the atmosphere, formic acid (FA, HCOOH) and acetic acid (AA, CH3COOH) control precipitation acidity in remote regions and may represent a critical link between the terrestrial carbon and water cycles by acting as key intermediates in plant carbon and energy metabolism and aerosol-cloud-precipitation interactions. However, our understanding of the exchange of these acids between terrestrial ecosystems and the atmosphere is limited by a lack of field observations, the existence of biogenic and anthropogenic primary and secondary sources whose relative importance is unclear, and the fact that vegetation can act as both a source and a sink. Here, we first present data obtained from the tropical rainforest mesocosm at Biosphere 2 which isolates primary vegetation sources. Strong light and temperature dependent emissions enriched in FA relative to AA were simultaneously observed from individual branches (FA/AA = 2.1 ± 0.6) and mesocosm ambient air (FA/AA = 1.4 ± 0.3). We also present long-term observations of vertical concentration gradients of FA and AA within and above a primary rainforest canopy in the central Amazon during the 2010 dry and 2011 wet seasons. We observed a seasonal switch from net ecosystem-scale deposition during the dry season to net emissions during the wet season. This switch was associated with reduced ambient concentrations in the wet season (FA < 1.3 nmol mol-1, AA < 2.0 nmol mol-1) relative to the dry season (FA up to 3.3 nmol mol-1, AA up to 6.0 nmol mol-1), and a simultaneous increase in the FA/AA ambient concentration ratios from 0.3-0.8 in the dry season to 1.0-2.1 in the wet season. These observations are consistent with a switch between a biomass burning dominated source in the dry season (FA/AA < 1.0) to a vegetation dominated source in the

  11. Ecosystem-Scale Compensation Point Analysis of Formic and Acetic Acid in the Central Amazon

    NASA Astrophysics Data System (ADS)

    Yanez-Serrano, A. M.; Jardine, K. J.; Arneth, A.; Abrell, L.; Jardine, A. B.; Artaxo, P.; Gomes, E.; Kesselmeier, J.; Saleska, S. R.; Huxman, T. E.

    2011-12-01

    Organic acids, central to terrestrial carbon metabolism and atmospheric photochemistry, are ubiquitous in the troposphere in the gas, particle, and aqueous phases. As the dominant organic acids in the atmosphere, formic acid (FA, HCOOH) and acetic acid (AA, CH3COOH) control precipitation acidity in remote regions and may represent a critical link between the terrestrial carbon and water cycles by acting as key intermediates in plant carbon and energy metabolism and aerosol-cloud-precipitation interactions. However, our understanding of the exchange of these acids between terrestrial ecosystems and the atmosphere is limited by a lack of field observations, the existence of biogenic and anthropogenic primary and secondary sources whose relative importance is unclear, and the fact that vegetation can act as both a source and a sink. Here, we present results from the tropical rainforest mescosom at Biosphere 2 which isolates primary vegetation sources. Strong light and temperature dependent emissions of FA and AA were simultaneously observed from individual branches and mesocosm ambient air with a strong enrichment in FA (FA/AA = 1.4 +/- 0.3, R2 of 0.89 +/- 0.10). We also present long-term observations of vertical concentration gradients of FA and AA within and above a primary rainforest canopy in central Amazonia during the 2010 dry and 2011 wet seasons. We observed a seasonal switch from net ecosystem-scale deposition during the dry season to net emissions during the wet season. This switch was associated with reduced ambient concentrations in the wet season (FA < 1.3 ppbv, AA < 2.0 ppbv) relative to the dry season (FA up to 3.3 ppbv, AA up to 6.0 ppbv), and a simultaneous increase in the FA/AA ambient concentration ratios from 0.3-0.8 in the dry season to 1.0-2.1 in the wet season. These observations are consistent with a switch between a biomass burning dominated source in the dry season (FA/AA < 1.0) to a vegetation dominated source in the wet season and call into

  12. Enantiomeric 3-chloromandelic acid system: binary melting point phase diagram, ternary solubility phase diagrams and polymorphism.

    PubMed

    Le Minh, Tam; von Langermann, Jan; Lorenz, Heike; Seidel-Morgenstern, Andreas

    2010-09-01

    A systematic study of binary melting point and ternary solubility phase diagrams of the enantiomeric 3-chloromandelic acid (3-ClMA) system was performed under consideration of polymorphism. The melting point phase diagram was measured by means of thermal analysis, that is, using heat-flux differential scanning calorimetry (DSC). The results reveal that 3-ClMA belongs to the racemic compound-forming systems. Polymorphism was found for both the enantiomer and the racemate as confirmed by X-ray powder diffraction analysis. The ternary solubility phase diagram of 3-ClMA in water was determined between 5 and 50 degrees C by the classical isothermal technique. The solubilities of the pure enantiomers are extremely temperature-dependent. The solid-liquid equilibria of racemic 3-ClMA are not trivial due to the existence of polymorphism. The eutectic composition in the chiral system changes as a function of temperature. Further, solubility data in the alternative solvent toluene are also presented.

  13. Cloud point extraction of uranium using H₂DEH[MDP] in acidic conditions.

    PubMed

    Labrecque, Charles; Potvin, Sabrina; Whitty-Léveillé, Laurence; Larivière, Dominic

    2013-03-30

    A procedure has been developed for the cloud point extraction (CPE) of uranium (VI) using H2DEH[MDP] (P,P-di(2-ethylhexyl) methanediphosphonic acid) with inductively coupled plasma coupled to mass spectrometry (ICP-MS). The method is based on the modification of the cloud point temperature using cetyl trimethyl ammonium bromide (CTAB) and KI. Optimal conditions of extraction were found using a cross-optimization of every parameter (non-ionic and ionic surfactant concentrations, chelating agent concentration, pH and the extraction, and phase separation temperatures). Furthermore, the figures of merit of the methodology were assessed (limit of detection, limit of quantification, recovery, sensibility, and linear range) and are reported. Quantitative extraction (99 ± 0.5%) was obtained in drinking water samples over a wide range of uranium concentrations. The approach was also validated using drinking (SCP EP-L-3 and SCP EP-H-3), and wastewater (SCP EU-L-3) certified materials. Interferences from most critical anions and cations were evaluated to determine the reliability of the method. The proposed method showed robustness since its performance is maintained over a wide range of pH and metal ion concentrations.

  14. Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary

    PubMed Central

    Shimura, Kiyohito; Nagai, Toshihiko

    2016-01-01

    For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein. PMID:27976690

  15. Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary.

    PubMed

    Shimura, Kiyohito; Nagai, Toshihiko

    2016-12-15

    For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.

  16. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    PubMed

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins.

  17. Reduction of Acid-Fast and Non-Acid-Fast Bacteria by Point of Use Coagulation-Flocculation-Disinfection

    PubMed Central

    Casanova, Lisa M.; Sobsey, Mark D.

    2015-01-01

    Point of use (POU) household water treatment is increasingly being adopted as a solution for access to safe water. Non-tuberculous Mycobacteria (NTM) are found in water, but there is little research on whether NTM survive POU treatment. Mycobacteria may be removed by multi-barrier treatment systems that combine processes such as coagulation, settling and disinfection. This work evaluated removal of a non-tuberculous Mycobacterium (Mycobaterium terrae) and a Gram-negative non-acid-fast environmental bacterium (Aeromonas hydrophila) by combined coagulation-flocculation disinfection POU treatment. Aeromonas hydrophila showed 7.7 log10 reduction in demand free buffer, 6.8 log10 in natural surface water, and 4 log10 reduction in fecally contaminated surface water. Turbidity after treatment was <1 NTU. There was almost no reduction in levels of viable M. terrae by coagulant-flocculant-disinfectant in natural water after 30 minutes. The lack of Mycobacteria reduction was similar for both combined coagulant-flocculant-disinfectant and hypochlorite alone. A POU coagulant-flocculant-disinfectant treatment effectively reduced A. hydrophila from natural surface waters but not Mycobacteria. These results reinforce previous findings that POU coagulation-flocculation-disinfection is effective against gram-negative enteric bacteria. POU treatment and safe storage interventions may need to take into account risks from viable NTM in treated stored water and consider alternative treatment processes to achieve NTM reductions. PMID:26580632

  18. Steady-state protein focusing in carrier ampholyte based isoelectric focusing: Part I-Analytical solution.

    PubMed

    Shim, Jaesool; Yoo, Kisoo; Dutta, Prashanta

    2017-03-01

    The determination of an analytical solution to find the steady-state protein concentration distribution in IEF is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady-state protein distribution in carrier ampholyte based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I, and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field.

  19. pH transients during salt removal in isoelectric trapping separations: a curse revisited.

    PubMed

    Shave, Evan; Vigh, Gyula

    2007-02-01

    The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH 7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (mu(anion)/mu(cation)) and the pH difference between pH 7 and the pH of the buffering membranes (|pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|). When |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1, mu(anion)/mu(cation)<1 leads to an acidic pH transient, mu(anion)/mu(cation) = 1 eliminates the pH transient and mu(anion)/mu(cation)>1 leads to a basic pH transient. When mu(anion)/mu(cation) = 1, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|<1 leads to a basic pH transient, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1 eliminates the pH transient and |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| value, pH transients caused by dissimilar anion and cation mobilities can be avoided.

  20. Propionic acid fermentation by Propionibacterium freudenreichii CCTCC M207015 in a multi-point fibrous-bed bioreactor.

    PubMed

    Feng, Xiao-Hai; Chen, Fei; Xu, Hong; Wu, Bo; Yao, Jun; Ying, Han-Jie; Ouyang, Ping-Kai

    2010-11-01

    Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l(-1) after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.

  1. Relationship of amino acid composition and molecular weight of antifreeze glycopeptides to non-colligative freezing point depression.

    PubMed

    Schrag, J D; O'Grady, S M; DeVries, A L

    1982-08-06

    Many polar fishes synthesize a group of eight glycopeptides that exhibit a non-colligative lowering of the freezing point of water. These glycopeptides range in molecular weight between 2600 and 33 700. The largest glycopeptides [1-5] lower the freezing point more than the small ones on a weight basis and contain only two amino acids, alanine and threonine, with the disaccharide galactose-N-acetyl-galactosamine attached to threonine. The small glycopeptides, 6, 7, and 8, also lower the freezing point and contain proline, which periodically substitutes for alanine. Glycopeptides with similar antifreeze properties isolated from the saffron cod and the Atlantic tomcod contain an additional amino acid, arginine, which substitutes for threonine in glycopeptide 6. In this study we address the question of whether differences in amino acid composition or molecular weight between large and small glycopeptides are responsible for the reduced freezing point depressing capability of the low molecular weight glycopeptides. The results indicate that the degree of amino acid substitutions that occur in glycopeptides 6-8 do not have a significant effect on the unusual freezing point lowering and that the observed decrease in freezing point depression with smaller glycopeptides can be accounted for on the basis of molecular weight.

  2. Total Acid Value Titration of Hydrotreated Biomass Fast Pyrolysis Oil: Determination of Carboxylic Acids and Phenolics with Multiple End-Point Detection

    SciTech Connect

    Christensen, E.; Alleman, T. L.; McCormick, R. L.

    2013-01-01

    Total acid value titration has long been used to estimate corrosive potential of petroleum crude oil and fuel oil products. The method commonly used for this measurement, ASTM D664, utilizes KOH in isopropanol as the titrant with potentiometric end point determination by pH sensing electrode and Ag/AgCl reference electrode with LiCl electrolyte. A natural application of the D664 method is titration of pyrolysis-derived bio-oil, which is a candidate for refinery upgrading to produce drop in fuels. Determining the total acid value of pyrolysis derived bio-oil has proven challenging and not necessarily amenable to the methodology employed for petroleum products due to the different nature of acids present. We presented an acid value titration for bio-oil products in our previous publication which also utilizes potentiometry using tetrabutylammonium hydroxide in place of KOH as the titrant and tetraethylammonium bromide in place of LiCl as the reference electrolyte to improve the detection of these types of acids. This method was shown to detect numerous end points in samples of bio-oil that were not detected by D664. These end points were attributed to carboxylic acids and phenolics based on the results of HPLC and GC-MS studies. Additional work has led to refinement of the method and it has been established that both carboxylic acids and phenolics can be determined accurately. Use of pH buffer calibration to determine half-neutralization potentials of acids in conjunction with the analysis of model compounds has allowed us to conclude that this titration method is suitable for the determination of total acid value of pyrolysis oil and can be used to differentiate and quantify weak acid species. The measurement of phenolics in bio-oil is subject to a relatively high limit of detection, which may limit the utility of titrimetric methodology for characterizing the acidic potential of pyrolysis oil and products.

  3. Sevoflurane-induced isoelectric EEG and burst suppression: differential and antagonistic effect of added nitrous oxide.

    PubMed

    Niu, B; Xiao, J Y; Fang, Y; Zhou, B Y; Li, J; Cao, F; Tian, Y K; Mei, W

    2017-03-08

    The objective of this study was to investigate whether nitrous oxide influenced the ED50 of sevoflurane for induction of isoelectric electroencephalogram (ED50isoelectric ) differently from its influence on the ED50 of sevoflurane for electroencephalogram burst suppression (ED50burst ). In a prospective, randomised, double-blind, parallel group, up-down sequential allocation study, 77 ASA physical status 1 and 2 patients received sevoflurane induction and, after tracheal intubation, were randomly allocated to receive sevoflurane with either 40% oxygen in air (control group) or 60% nitrous oxide in oxygen mixture (nitrous group). The ED50isoelectric in the two groups was determined using Dixon's up and down method, starting at 2.5% with 0.2% step size of end-tidal sevoflurane. The electroencephalogram was considered as isoelectric when a burst suppression ratio of 100% lasted > 1 min. The subsequent concentrations of sevoflurane administered were determined by the presence or absence of isoelectric electroencephalogram in the previous patient in the same group. The ED50isoelectric in the nitrous group 4.08 (95%CI, 3.95-4.38)% was significantly higher than that in the control group 3.68 (95%CI, 3.50-3.78)% (p < 0.0001). The values for ED50burst were 3.05 (95%CI, 2.66-3.90)% and 3.02 (95%CI, 3.00-3.05)% in nitrous group and control group, respectively (p = 0.52). The addition of 60% nitrous oxide increases ED50isoelectric , but not the ED50burst of sevoflurane. Neither result indicates an additive effect of anaesthetic agents, as might be expected, and possible reasons for this are discussed.

  4. Characterization of hyaluronic acid and synovial fluid in stagnation point elongational flow.

    PubMed

    Haward, Simon J

    2014-03-01

    Hyaluronic acid (HA) is an important biomacromolecule, which fulfils a number of vital physiological functions (especially in the joint synovial fluid) and also has consumer and pharmaceutical applications. HA solution properties have already been quite thoroughly characterized in response to steady shear flows but are less well understood in highly deforming extensional flows. In this study, flow-induced birefringence measurements are made as a function of the strain rate in planar elongational flow at the stagnation point of a cross-slot device using HA solutions of a range of molecular weights (0.9×10(6) g mol(-1)≤Mw≤4.8×10(6) g mol(-1)) and at dilute concentrations. The results provide macromolecular relaxation times, molecular weight distributions and the extensional viscosities and Trouton ratios of the fluids. The HA relaxation time is found to vary as τ∼Mw1.8, which is consistent with a partially solvated, expanded coil. An intrinsic Trouton ratio is defined, which varies as [Tr]∼Mw2. The measurement of birefringence with strain rate is shown to be highly sensitive to the molecular weight distribution and can resolve subtle changes due to macromolecular degradation and the presence of fracture products. Mechanical degradation experiments in the cross-slots indicate midchain scission of HA macromolecules, strongly suggesting near full extension of the high-molecular weight fraction in the stagnation point extensional flow field. Taken together the results suggest a possible method for analysis of the HA in synovial fluid, and this concept is tested using synovial fluid obtained from porcine tarsal joint.

  5. Reaction kinetics and critical phenomena: iodination of acetone in isobutyric acid + water near the consolute point.

    PubMed

    Hu, Baichuan; Baird, James K

    2010-01-14

    The rate of iodination of acetone has been measured as a function of temperature in the binary solvent isobutyric acid (IBA) + water near the upper consolute point. The reaction mixture was prepared by the addition of acetone, iodine, and potassium iodide to IBA + water at its critical composition of 38.8 mass % IBA. The value of the critical temperature determined immediately after mixing was 25.43 degrees C. Aliquots were extracted from the mixture at regular intervals in order to follow the time course of the reaction. After dilution of the aliquot with water to quench the reaction, the concentration of triiodide ion was determined by the measurement of the optical density at a wavelength of 565 nm. These measurements showed that the kinetics were zeroth order. When at the end of 24 h the reaction had come to equilibrium, the critical temperature was determined again and found to be 24.83 degrees C. An Arrhenius plot of the temperature dependence of the observed rate constant, k(obs), was linear over the temperature range 27.00-38.00 degrees C, but between 25.43 and 27.00 degrees C, the values of k(obs) fell below the extrapolation of the Arrhenius line. This behavior is evidence in support of critical slowing down. Our experimental method and results are significant in three ways: (1) In contrast to in situ measurements of optical density, the determination of the optical density of diluted aliquots avoided any interference from critical opalescence. (2) The measured reaction rate exhibited critical slowing down. (3) The rate law was pseudo zeroth order both inside and outside the critical region, indicating that the reaction mechanism was unaffected by the presence of the critical point.

  6. Femtomolar concentration detection limit and zeptomole mass detection limit for protein separation by capillary isoelectric focusing and laser-induced fluorescence detection.

    PubMed

    Ramsay, Lauren M; Dickerson, Jane A; Dada, Oluwatosin; Dovichi, Norman J

    2009-03-01

    Fluorescence tends to produce the lowest detection limits for most forms of capillary electrophoresis. Two issues have discouraged its use in capillary isoelectric focusing. The first issue is fluorescent labeling of proteins. Most labeling reagents react with lysine residues and convert the cationic residue to a neutral or anionic product. At best, these reagents perturb the isoelectric point of the protein. At worse, they convert each protein into hundreds of different fluorescent products that confound analysis. The second issue is the large background signal generated by impurities within commercial ampholytes. This background signal is particularly strong when excited in the blue portion of the spectrum, which is required by many common fluorescent labeling reagents. This paper addresses these issues. For labeling, we employ Chromeo P540, which is a fluorogenic reagent that converts cationic lysine residues to cationic fluorescent products. The reaction products are excited in the green, which reduces the background signal generated by impurities present within the ampholytes. To further reduce the background signal, we photobleach ampholytes with high-power photodiodes. Photobleaching reduced the noise in the ampholyte blank by an order of magnitude. Isoelectric focusing performed with photobleached pH 3-10 ampholytes produced concentration detection limits of 270 +/- 25 fM and mass detection limits of 150 +/- 15 zmol for Chromeo P540 labeled beta-lactoglobulin. Concentration detection limits were 520 +/- 40 fM and mass detection limits were 310 +/- 30 zmol with pH 4-8 ampholytes. A homogenate was prepared from a Barrett's esophagus cell line and separated by capillary isoelectric focusing, reproducibly generating dozens of peaks. The sample taken for the separation was equal to the labeled protein homogenate from three cells.

  7. Investigation of the Melting Point Depression of 12-Hydroxystearic Acid Organogels Using the Flory Diluent Model

    NASA Astrophysics Data System (ADS)

    Cavicchi, Kevin; Lipowski, Brian

    2013-03-01

    This talk will focus on the gelation behavior of 12-hydroxystearic acid (12-HSA) in organic solvents. Thermo-reversible gelation occurs by crystallization of 12-HSA in organic solvent to form 3-D fibrillar networks. The melting point vs. composition for 12-HSA in a range of solvents has been measured. The liquidus lines could be fit with the Flory-diluent model that takes into account the non-ideal free energy of mixing and the disparity in the size of the solvent and 12-HSA molecules. The fits indicated that the effective molar volume of 12-HSA increased as the hydrogen bonding Hansen solubility parameter δh of the solvent decreased. This is attributed to the hydrogen-bonding driven aggregation of the 12-HSA in the liquid state based on previous observations that 12-HSA forms aggregated structures in non-polar solvents (e.g. dimers and tetrameters). These results indicate that the stabilization of the solid phase in 12-HSA solutions has contributions from both variations in the entropy of mixing as well the enthalpy of mixing. The importance of both these factors for designing small molecule gelators will be discussed.

  8. Genome-wide mapping for fatty acid composition and melting point of fat in a purebred Duroc pig population.

    PubMed

    Uemoto, Y; Soma, Y; Sato, S; Ishida, M; Shibata, T; Kadowaki, H; Kobayashi, E; Suzuki, K

    2012-02-01

    The fatty acid composition and melting point of fatty tissue are among the most important economic traits in pig breeding because of their influence on the eating quality of meat. Identifying the quantitative trait locus (QTL) of these traits may help reveal the genetic structure of fatty acid composition and the melting point of fatty tissue and improve meat-quality traits by marker-assisted selection. We conducted whole-genome QTL analysis for fatty acid composition and melting point of inner and outer subcutaneous fat and inter- and intramuscular fat in a purebred Duroc population. A total of 129 markers were genotyped and used for QTL analysis. For fatty acid compositions of inner and outer subcutaneous fat, three significant QTL and 17 suggestive QTL were detected on SSC2, 4, 6, 8, 9, 10, 11, 12, 14 and 18. For the melting point of inner and outer subcutaneous fat, two significant QTL were detected on the same region of SSC14. For fatty acid compositions of inter- and intramuscular fat, five significant QTL and 13 suggestive QTL were detected on SSC2, 4, 6, 8, 9, 10, 14 and 15. On SSC14, significant QTL for C18:0 and C18:1 of outer subcutaneous fat and intramuscular fat, and melting point of subcutaneous fat, which had high likelihood of odds (LOD) scores (2.67-5.78), were detected in the same region. This study determined QTL affecting fatty acid composition and melting point of different fat tissues in purebred Duroc pigs.

  9. A Multi-Point Sensor Based on Optical Fiber for the Measurement of Electrolyte Density in Lead-Acid Batteries

    PubMed Central

    Cao-Paz, Ana M.; Marcos-Acevedo, Jorge; del Río-Vázquez, Alfredo; Martínez-Peñalver, Carlos; Lago-Ferreiro, Alfonso; Nogueiras-Meléndez, Andrés A.; Doval-Gandoy, Jesús

    2010-01-01

    This article describes a multi-point optical fiber-based sensor for the measurement of electrolyte density in lead-acid batteries. It is known that the battery charging process creates stratification, due to the different densities of sulphuric acid and water. In order to study this process, density measurements should be obtained at different depths. The sensor we describe in this paper, unlike traditional sensors, consists of several measurement points, allowing density measurements at different depths inside the battery. The obtained set of measurements helps in determining the charge (SoC) and state of health (SoH) of the battery. PMID:22319262

  10. Development of a novel semi-quantitative analysis system for ultramicroscale samples by fluorescent capillary isoelectric focusing.

    PubMed

    Kami, Daisuke; Takahashi, Makoto; Gojo, Satoshi; Toyoda, Masashi; Aso, Ryoko; Sekizawa, Ryuichi; Watanabe, Masatoshi

    2014-04-15

    Microchemistry provides methods to analyze small quantities of chemical substances, including proteins, nucleic acids, and carbohydrates in various fields such as biomedical research, tissue engineering, molecular biology, and regeneration medicine. We therefore developed a fluorescent capillary isoelectric focusing (fluorescent cIEF) system for protein detection at an ultramicroscale volume, which aimed to isolate and identify, from a heterogeneous mixture of transduced cells, induced pluripotent stem cells (iPSCs) that could be fully reprogrammed. In addition, we demonstrated that the SOX2 protein, which is indispensable for the acquisition of pluripotency, could be detected by this new fluorescent cIEF system to identify iPSCs in the early phase of complete reprogramming. This method took less than 1 h for completion, including the time required for the antibody-antigen (Ab-Ag) reaction, and required as few as approximately three cells. Thus, this system could help improve iPSC generation as well as cut costs and reduce workloads.

  11. The use of analytical isoelectric focusing for detection and identification of beta-lactamases.

    PubMed

    Mathew, A; Harris, A M; Marshall, M J; Ross, G W

    1975-05-01

    BETA-Lactamases (EC. 3.5.2.6) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of beta-lactamases carried by R factors.

  12. Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients.

    PubMed

    Görg, A; Postel, W; Weiss, W

    1992-01-01

    Two cultivars ("Alexis" and "Lenka") of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band "B" (isoelectric point approximately 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this "B" isoenzyme band, but exhibit the pronounced "A" isoenzyme band (isoelectric point approximately 6.5) instead, suggesting that these isoenzymes (which we suppose to be beta-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, "final attenuation" is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar "Lenka", may also have a role in determining quality.

  13. Protein recovery from rainbow trout (Oncorhynchus mykiss) processing byproducts via isoelectric solubilization/precipitation and its gelation properties as affected by functional additives.

    PubMed

    Chen, Yi-Chen; Jaczynski, Jacek

    2007-10-31

    Solubility of rainbow trout proteins was determined between pH 1.5 and 13.0 and various ionic strengths (IS). Minimum solubility occurred at pH 5.5; however, when IS = 0.2, the minimum solubility shifted toward more acidic pH. Isoelectric solubilization/precipitation was applied to trout processing byproducts (fish meat left over on bones, head, skin, etc.), resulting in protein recovery yields (Kjeldahl, dry basis) between 77.7% and 89.0%, depending of the pH used for solubilization and precipitation. The recovered protein contained 1.4-2.1% ash (dry basis), while the trout processing byproducts (i.e., starting material) 13.9%. Typical boneless and skinless trout fillets contain 5.5% ash, and therefore, the isoelectric solubilization/precipitation effectively removed impurities such as bones, scales, skin, etc., from the trout processing byproducts. The recovered proteins retained gel-forming ability as assessed with dynamic rheology, torsion test, and texture profile analysis (TPA). However, the recovered proteins failed to gel unless beef plasma protein (BPP) was added. Even with BPP, the recovered protein showed some proteolysis between 40 and 55 degrees C. Addition of potato starch, transglutaminase, and phosphate to the recovered proteins resulted in good texture of trout gels as confirmed by torsion test and TPA. Higher ( P < 0.05) shear stress and strain were measured for gels developed from basic pH treatments than the acidic counterparts. However, proteins recovered from acidic treatments had higher ( P < 0.05) lipid content than the basic treatments. This is probably why the gels from acidic treatments were whiter ( L* - 3 b*) ( P < 0.05) than those from the basic ones. Our study demonstrates that functional proteins can be efficiently recovered from low-value fish processing byproducts using isoelectric solubilization/precipitation and subsequently be used in value-added human foods.

  14. Quality assurance of monoclonal antibody pharmaceuticals based on their charge variants using microchip isoelectric focusing method.

    PubMed

    Kinoshita, Mitsuhiro; Nakatsuji, Yuki; Suzuki, Shigeo; Hayakawa, Takao; Kakehi, Kazuaki

    2013-09-27

    Monoclonal antibody (mAb) pharmaceuticals are much more complex than small-molecule drugs. Such complex characteristics raise challenging questions for regulatory evaluation. Although heterogeneity in mAbs based on their charge variants has been mainly evaluated using gel-based isoelectric focusing (IEF) method, recent development in capillary electrophoresis and microchip electrophoresis has made it possible to assure their heterogeneities in more easy and rapid manner. In the present paper, we customized the imaged microchip isoelectric focusing (mIEF) for the analysis of mAbs, and compared the customized version with the conventional capillary isoelectric focusing (cIEF) method, and found that mIEF has much higher performance in operations, and its resolving powers are comparable with those obtained by cIEF.

  15. SIMULTANEOUS DETERMINATION OF IONIZATION CONSTANTS AND ISOELECTRIC POINTS OF 12 HYDROXY-S-TRIAZINES BY CAPILLARY ZONE ELECTROPHORESIS AND CAPILLARY ISOELECTRIC FOCUSING

    EPA Science Inventory

    Capillary zone electrophoresis (CZE) was used to separate and determine simultaneously the pK1,pK2, and pI values of 12 environmentally relevant hydroxytriazines (hydroxymetabolities of atrazine, terbuthylazine, simazine, and propazine and four (arylamion)-s-triazines) and observ...

  16. Isoelectric focusing of basic proteins: the problem of oxidation of cysteines.

    PubMed

    Altland, K; Becher, P; Rossmann, U; Bjellqvist, B

    1988-09-01

    Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.

  17. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  18. Effect of Joule heating on isoelectric focusing of proteins in a microchannel.

    PubMed

    Yoo, Kisoo; Shim, Jaesool; Dutta, Prashanta

    2014-11-01

    Electric field-driven separation and purification techniques, such as isoelectric focusing (IEF) and isotachophoresis, generate heat in the system that can affect the performance of the separation process. In this study, a new mathematical model is presented for IEF that considers the temperature rise due to Joule heating. We used the model to study focusing phenomena and separation performance in a microchannel. A finite volume-based numerical technique is developed to study temperature-dependent IEF. Numerical simulation for narrow range IEF (6 < pH < 10) is performed in a straight microchannel for 100 ampholytes and two model proteins: staphylococcal nuclease and pancreatic ribonuclease. Separation results of the two proteins are obtained with and without considering the temperature rise due to Joule heating in the system for a nominal electric field of 100 V/cm. For the no Joule heating case, constant properties are used, while for the Joule heating case, temperature-dependent titration curves and thermo-physical properties are used. Our numerical results show that the temperature change due to Joule heating has a significant impact on the final focusing points of proteins, which can lower the separation performance considerably. In the absence of advection and any active cooling mechanism, the temperature increase is the highest at the mid-section of a microchannel. We also found that the maximum temperature in the system is a strong function of the [Formula: see text] value of the carrier ampholytes. Simulation results are also obtained for different values of applied electric fields in order to find the optimum working range considering the simulation time and buffer temperature. Moreover, the model is extended to study IEF in a straight microchip where pH is formed by supplying H(+) and OH(-), and the thermal analysis shows that the heat generation is negligible in ion supplied IEF.

  19. Effect of Joule heating on isoelectric focusing of proteins in a microchannel

    PubMed Central

    Yoo, Kisoo; Shim, Jaesool; Dutta, Prashanta

    2014-01-01

    Electric field-driven separation and purification techniques, such as isoelectric focusing (IEF) and isotachophoresis, generate heat in the system that can affect the performance of the separation process. In this study, a new mathematical model is presented for IEF that considers the temperature rise due to Joule heating. We used the model to study focusing phenomena and separation performance in a microchannel. A finite volume-based numerical technique is developed to study temperature-dependent IEF. Numerical simulation for narrow range IEF (6 < pH < 10) is performed in a straight microchannel for 100 ampholytes and two model proteins: staphylococcal nuclease and pancreatic ribonuclease. Separation results of the two proteins are obtained with and without considering the temperature rise due to Joule heating in the system for a nominal electric field of 100 V/cm. For the no Joule heating case, constant properties are used, while for the Joule heating case, temperature-dependent titration curves and thermo-physical properties are used. Our numerical results show that the temperature change due to Joule heating has a significant impact on the final focusing points of proteins, which can lower the separation performance considerably. In the absence of advection and any active cooling mechanism, the temperature increase is the highest at the mid-section of a microchannel. We also found that the maximum temperature in the system is a strong function of the ΔpK  value of the carrier ampholytes. Simulation results are also obtained for different values of applied electric fields in order to find the optimum working range considering the simulation time and buffer temperature. Moreover, the model is extended to study IEF in a straight microchip where pH is formed by supplying H+ and OH−, and the thermal analysis shows that the heat generation is negligible in ion supplied IEF. PMID:25553199

  20. Measurement of total acid number (TAN) and TAN boiling point distribution in petroleum products by electrospray ionization mass spectrometry.

    PubMed

    Qian, Kuangnan; Edwards, Kathleen E; Dechert, Gary J; Jaffe, Stephen B; Green, Larry A; Olmstead, William N

    2008-02-01

    We report a new method for rapid measurement of total acid number (TAN) and TAN boiling point (BP) distribution for petroleum crude and products. The technology is based on negative ion electrospray ionization mass spectrometry (ESI-MS) for selective ionization of petroleum acid and quantification of acid structures and molecular weight distributions. A chip-based nanoelectrospray system enables microscale (<200 mg) and higher throughput (20 samples/h) measurement. Naphthenic acid structures were assigned based on nominal masses of a set of predefined acid structures. Stearic acid is used as an internal standard to calibrate ESI-MS response factors for quantification purposes. With the use of structure-property correlations, boiling point distributions of TAN values can be calculated from the composition. The rapid measurement of TAN BP distributions by ESI is demonstrated for a series of high-TAN crudes and distillation cuts. TAN values determined by the technique agree well with those by the titration method. The distributed properties compare favorably with those measured by distillation and measurement of TAN of corresponding cuts.

  1. Methods for point-of-care detection of nucleic acid in a sample

    DOEpatents

    Bearinger, Jane P.; Dugan, Lawrence C.

    2015-12-29

    Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

  2. Apparatus for point-of-care detection of nucleic acid in a sample

    DOEpatents

    Bearinger, Jane P.; Dugan, Lawrence C.

    2016-04-19

    Provided herein are methods and apparatus for detecting a target nucleic acid in a sample and related methods and apparatus for diagnosing a condition in an individual. The condition is associated with presence of nucleic acid produced by certain pathogens in the individual.

  3. Increase of Unsaturated Fatty Acids (Low Melting Point) of Broiler Fatty Waste Obtained Through Staphylococcus xylosus Fermentation.

    PubMed

    Marques, Roger V; Duval, Eduarda H; Corrêa, Luciara B; Corrêa, Érico K

    2015-11-01

    The increasing rise in the production of meat around the world causes a significant generation of agro-industrial waste--most of it with a low value added. Fatty wastes have the potential of being converted into biodiesel, given the overcome of technological and economical barriers, as well as its presentation in solid form. Therefore, the aim of this work was to investigate the capacity of Staphylococcus xylosus strains to modify the chemical structure of chicken fatty wastes intending to reduce the melting points of the wastes to mild temperatures, thereby breaking new ground in the production of biodiesel from these sources in an economically attractive and sustainable manner. The effects in time of fermentation and concentration of the fat in the medium were investigated, assessing the melting point and profile of fatty acids. The melting temperature showed a decrease of approximately 22 °C in the best operational conditions, due to reduction in the content of saturated fatty acids (high melting point) and increase of unsaturated fatty acids (low melting point).

  4. Chromatofocusing purification of CD1b-antigen complexes and their analysis by isoelectric focusing.

    PubMed

    Garcia-Alles, Luis Fernando; de la Salle, Henri

    2013-01-01

    The presentation of lipid antigens to T cells is mediated by the CD1 proteins. Purified functional CD1/lipid complexes are valuable tools to investigate such immune processes. Here, we describe how these complexes can be prepared in vitro, how they can be purified by chromatofocusing and how to control their antigen-loading status by isoelectric focusing.

  5. Scale-up of isoelectric focusing. [for large scale protein fracionation

    NASA Technical Reports Server (NTRS)

    Bier, Milan

    1986-01-01

    The paper describes some applications to large scale protein fractionation using a recycling isoelectric focusing apparatus. Separation is achieved in free solution without the use of supporting media. Various alternatives for the formation of the pH gradient are discussed and results of a computer simulation are presented.

  6. Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Lang, Frank J., Jr.; Hochstein, Lawrence I.

    1989-01-01

    The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

  7. Peptide and amino acid separation with nanofiltration membranes

    SciTech Connect

    Tsuru, Toshinori; Shutou, Takatoshi; Nakao, Shin-Ichi; Kimura, Shoji )

    1994-05-01

    Several nanofiltration membranes [UTC-20, 60 (Toray Industries), NF-40 (Film-Tech Corporation), Desal-5, G-20 (Desalination Systems), and NTR-7450 (Nitto Electric Industrial Co.)] were applied to separate amino acids and peptides on the basis of charge interaction with the membranes since most of them contain charged functional groups. Nanofiltration membranes having a molecular weight cutoff (MWCO) below 300 (UTC-20, 60, NF-40 and Desal-5) were not suitable for separation of amino acids. On the other hand, separation of amino acids and peptides with nanofiltration membranes having a MWCO around 2000-3000 (NTR-7450 and G-20) was satisfactory based on a charge effect mechanism; charged amino acids and peptides were rejected while neutral amino acids and peptides permeated through the membranes. Separation of peptides having different isoelectric points with nanofiltration membranes was possible by adjusting the pH. 15 refs., 11 figs., 4 tabs.

  8. Investigating the significance of zero-point motion in small molecular clusters of sulphuric acid and water

    SciTech Connect

    Stinson, Jake L. Ford, Ian J.; Kathmann, Shawn M.

    2014-01-14

    The nucleation of particles from trace gases in the atmosphere is an important source of cloud condensation nuclei, and these are vital for the formation of clouds in view of the high supersaturations required for homogeneous water droplet nucleation. The methods of quantum chemistry have increasingly been employed to model nucleation due to their high accuracy and efficiency in calculating configurational energies; and nucleation rates can be obtained from the associated free energies of particle formation. However, even in such advanced approaches, it is typically assumed that the nuclei have a classical nature, which is questionable for some systems. The importance of zero-point motion (also known as quantum nuclear dynamics) in modelling small clusters of sulphuric acid and water is tested here using the path integral molecular dynamics method at the density functional level of theory. The general effect of zero-point motion is to distort the mean structure slightly, and to promote the extent of proton transfer with respect to classical behaviour. In a particular configuration of one sulphuric acid molecule with three waters, the range of positions explored by a proton between a sulphuric acid and a water molecule at 300 K (a broad range in contrast to the confinement suggested by geometry optimisation at 0 K) is clearly affected by the inclusion of zero point motion, and similar effects are observed for other configurations.

  9. Investigating the significance of zero-point motion in small molecular clusters of sulphuric acid and water.

    PubMed

    Stinson, Jake L; Kathmann, Shawn M; Ford, Ian J

    2014-01-14

    The nucleation of particles from trace gases in the atmosphere is an important source of cloud condensation nuclei, and these are vital for the formation of clouds in view of the high supersaturations required for homogeneous water droplet nucleation. The methods of quantum chemistry have increasingly been employed to model nucleation due to their high accuracy and efficiency in calculating configurational energies; and nucleation rates can be obtained from the associated free energies of particle formation. However, even in such advanced approaches, it is typically assumed that the nuclei have a classical nature, which is questionable for some systems. The importance of zero-point motion (also known as quantum nuclear dynamics) in modelling small clusters of sulphuric acid and water is tested here using the path integral molecular dynamics method at the density functional level of theory. The general effect of zero-point motion is to distort the mean structure slightly, and to promote the extent of proton transfer with respect to classical behaviour. In a particular configuration of one sulphuric acid molecule with three waters, the range of positions explored by a proton between a sulphuric acid and a water molecule at 300 K (a broad range in contrast to the confinement suggested by geometry optimisation at 0 K) is clearly affected by the inclusion of zero point motion, and similar effects are observed for other configurations.

  10. Voltammetric iodometric titration of ascorbic acid with dead-stop end-point detection in fresh vegetables and fruit samples.

    PubMed

    Verdini, R A; Lagier, C M

    2000-07-01

    The present work describes a method for determining ascorbic acid, which combines iodometry with a voltammetric technique to detect the end point of the titration. In addition, the validity of the method applied to natural vegetable or fruit samples was assessed. The results were compared with those obtained by an accurate method such as HPLC using UV detection. Similar values of ascorbic acid for different natural samples were obtained by means of this approach (p > 0.05). The limit of quantification was 0.1 mg. This technique presents the advantage of other electroanalytical methods such as avoiding filtration or ultracentrifugation steps, with the additional benefit of using the platinum electrodes, which are routinely used in the laboratory. These facts allow a rapid and efficient quantification of ascorbic acid with very low cost of reagents and equipment.

  11. Colloidal 3-mercaptopropionic acid Capped Lead Sulfide Quantum Dots in a Low Boiling Point Solvent.

    PubMed

    Reinhart, Chase C; Johansson, Erik

    2017-04-10

    Colloidal 3-mercaptopropionic acid (3-MPA) capped lead sulfide quantum dots were prepared in a variety of organic solvents stabilized with a quaternary ammonium halide salt. The stabilized colloids' optical properties were studied through optical absorption and emission spectroscopy and found to be dependent on both the concentration of new ligand and stabilizer, and sample age. Nanocrystal ligand chemistry was studied through a combination of (1)H-NMR and 2-dimensional Nuclear Overhauser Effect Spectroscopy (NOESY) which revealed full displacement of the original oleate ligand to form a dynamically exchanging ligand shell. The colloids were studied optically and via NMR as they aged and revealed a quantitative conversion of monomeric 3-mercaptopropionic acid to its dimer, dithiodipropionic acid (dTdPA).

  12. Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations.

    PubMed

    North, Robert; Hwang, Ann; Lalwani, Sanjiv; Shave, Evan; Vigh, Gyula

    2006-10-20

    Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3isoelectric trapping separations that used the buffering membrane to be characterized. The pI value of the most basic UV-absorbing or fluorescent carrier ampholyte collected in the anodic separation compartment, determined by full-column imaging capillary isoelectric focusing analysis, indicates the operational pH value of the separation membrane.

  13. Capillary isoelectric focusing of microorganisms in the pH range 2-5 in a dynamically modified FS capillary with UV detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Holá, Veronika; Slais, Karel

    2006-07-01

    The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2-5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10(3).

  14. High speed two-dimensional protein separation without gel by isoelectric focusing-asymmetrical flow field flow fractionation: application to urinary proteome.

    PubMed

    Kim, Ki Hun; Moon, Myeong Hee

    2009-09-01

    An online multilane channel system for isoelectric focusing and asymmetrical flow field-flow fractionation (IEF-AF4) is utilized for the two-dimensional separation (2D: isoelectric point, pI, and hydrodynamic diameter, d(s)) of a human proteome sample followed by the shotgun proteomic analysis using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). IEF-AF4 was recently developed to carry out nongel-based high speed two-dimensional protein separation [ Kim , K. , et al. Anal. Chem. 2009, 81 , 1715 ]. In IEF-AF4, proteins are separated according to pI along an IEF channel located at the head of six AF4 channels, and then the fractionated protein bands are directed to multilane AF4 channels for size-based separation. In this report, the original IEF-AF4 system has been modified to avoid the possible adsorption of proteins onto the membrane wall of IEF segments during isoelectric focusing by isolating the IEF channel segments from the multilane AF4 channels. The performance of the modified IEF-AF4 system was tested with protein standards and was further applied for the 2D fractionation of the human urinary proteome sample under two ampholyte solutions with different pH ranges (pH 3-10 and 3-6). The entire 2D separation was achieved in less than 30 min. The collected protein fractions were digested for peptide analysis using nLC-ESI-MS-MS, resulting in the identification of 245 total urinary proteins, including 110 unique proteins that are not yet reported in literature. Our experiments also showed a higher efficiency in the identification of urine proteins using ampholyte solution in the narrower pH range.

  15. Analytical isoelectric focusing of apolipoprotein B of human plasma low-density lipoproteins in the presence of a nonionic and a zwitterionic detergent.

    PubMed

    Melnik, B C; Melnik, S F

    1988-06-01

    A method for the analytical isoelectric focusing of Nonidet-P40-delipidated apolipoprotein B of human plasma low-density lipoproteins has been developed. Isoelectric focusing was performed in the presence of the zwitterionic nondenaturing detergent Chaps, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, and the nonionic surfactant Nonidet-P40, polyoxyethyleneglycol p-t-octylphenol with a mean of 9.0 ethylene oxide units per molecule. Low-density lipoprotein (LDL) apolipoprotein B (apo-B) entered 3.75% polyacrylamide gels without precipitation at the sites of sample application, permitting apoprotein recoveries of greater than 90% in the migrating bands. LDL apo-B exhibited 10 distinguishable bands with apparent isoelectric points of 7.34 (band 1), 7.27 (band 2), 7.16 (band 3), 7.02 (band 4), 6.88 (band 5), 6.70 (band 6), 6.61 (band 7), 6.48 (band 8), 6.40 (band 9), and 6.24 (band 10), respectively. Bands 3 and 4, 6 and 7, as well as 8 and 9 could be identified as major double bands. When the focused apo-B was run in a second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same relative molecular weight of B-100 was obtained for all focused bands. After electrotransfer to nitrocellulose paper, all bands reacted with polyclonal anti-human LDL antibody. Furthermore, the detergent-solubilized apo-B retained the immunological properties of native low-density lipoproteins when tested by double immunodiffusion against polyvalent anti-human LDL sera.

  16. Nucleic-acid testing, new platforms and nanotechnology for point-of-decision diagnosis of animal pathogens.

    PubMed

    Teles, Fernando; Fonseca, Luís

    2015-01-01

    Accurate disease diagnosis in animals is crucial for animal well-being but also for preventing zoonosis transmission to humans. In particular, livestock diseases may constitute severe threats to humans due to the particularly high physical contact and exposure and, also, be the cause of important economic losses, even in non-endemic countries, where they often arise in the form of rapid and devastating epidemics. Rapid diagnostic tests have been used for a long time in field situations, particularly during outbreaks. However, they mostly rely on serological approaches, which may confirm the exposure to a particular pathogen but may be inappropriate for point-of-decision (point-of-care) settings when emergency responses supported on early and accurate diagnosis are required. Moreover, they often exhibit modest sensitivity and hence significantly depend on later result confirmation in central or reference laboratories. The impressive advances observed in recent years in materials sciences and in nanotechnology, as well as in nucleic-acid synthesis and engineering, have led to an outburst of new in-the-bench and prototype tests for nucleic-acid testing towards point-of-care diagnosis of genetic and infectious diseases. Manufacturing, commercial, regulatory, and technical nature issues for field applicability more likely have hindered their wider entrance into veterinary medicine and practice than have fundamental science gaps. This chapter begins by outlining the current situation, requirements, difficulties, and perspectives of point-of-care tests for diagnosing diseases of veterinary interest. Nucleic-acid testing, particularly for the point of care, is addressed subsequently. A range of valuable signal transduction mechanisms commonly employed in proof-of-concept schemes and techniques born on the analytical chemistry laboratories are also described. As the essential core of this chapter, sections dedicated to the principles and applications of microfluidics, lab

  17. Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples.

    PubMed

    Besong Agbo, D; Klafki, H; Poschmann, G; Seyfarth, K; Genius, J; Janßen, C; Stühler, K; Wurst, W; Meyer, H E; Klingenspor, M; Wiltfang, J

    2013-12-15

    We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson's disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.

  18. Phophoglucomutase first locus polymorphism as revealed by isoelectric focusing in Southern Africa.

    PubMed

    Tipler, T D; Dunn, D S; Jenkins, T

    1982-01-01

    Eleven Southern African populations (representing European, Asian and Negroid populations) have been typed for the first locus phosphoglucomutase (PGM1) using isoelectric focusing (pH range 5.0-8.0) in acrylamide gels. The gene frequencies of the four common alleles at this locus in these populations were compared to those found previously in European and Negroid populations. Marked differences in gene frequencies were observed: Negroes have a lower PGM1(2-) compared with Caucasoids due to a lower PGM1(2-) frequency, Indians a relatively high PGM1(2) due to a higher frequency of the PGM1(2+) allele. The Afrikaans and Ashkenazim do not differ appreciably from their European counterparts. The appearances of the rarer PGM1(6) and PGM1(7) alleles on isoelectric focusing are described and some kinetic properties examined. The PGM2(2-1), or 'Atkinson' phenotype, can also be detected with this technique.

  19. Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes

    PubMed Central

    Fonslow, Bryan R.; Kang, Seong A.; Gestaut, Daniel R.; Graczyk, Beth; Davis, Trisha N.; Sabatini, David M.; Yates, John R.

    2010-01-01

    Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15°C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (< 45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25°C) and eventually its individual protein components (30°C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4 – 6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions. PMID:20614870

  20. Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.

    PubMed

    Fonslow, Bryan R; Kang, Seong A; Gestaut, Daniel R; Graczyk, Beth; Davis, Trisha N; Sabatini, David M; Yates, John R

    2010-08-01

    Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

  1. Protein post-translational modification analyses using on-chip immunoprobed isoelectric focusing.

    PubMed

    Tia, Samuel Q; Brown, Katharine; Chen, Danica; Herr, Amy E

    2013-03-05

    Post-translational modifications play a critical role in regulating protein function. Increasingly, determination of protein identity, estimation of abundance, and characterization of post-translational modifications are required for analysis of protein-mediated cell signaling networks. As such, we report an integrated and rapid multispectral immunoprobed isoelectric focusing technique for identifying specific proteins bearing post-translational modifications. Immunoprobed isoelectric focusing is composed of isoelectric focusing in a large pore-size polyacrylamide gel to determine protein pI followed by immobilization of pI-resolved proteins. Proteins are immobilized via covalent attachment to a channel-filling benzophenone-functionalized polyacrylamide gel via brief UV exposure (photoblot), followed by multispectral antibody-based detection. The assay correlates observed post-translational modifications to pI shifts relative to the unmodified protein of interest. During the electrokinetically driven antibody probing stage, we observed nonuniform electrophoretic probe mobility along the channel axis. The spatially varying mobility is attributed to nonuniform charge arising from covalent attachment of ampholytes to the benzophenone-functionalized gel matrix during the photoblotting step. Using the multistep microfluidic assay, phosphorylated and acetylated forms of heat shock protein 27 and superoxide dismutase 2 were detected, respectively. The assay reported protein isoforms in immune-purified sample and raw cell lysate in 2 hours with sample volume requirements of 2 μL. This new assay is well-matched to systems biology frameworks for study of protein post-translational modifications.

  2. Coupling isoelectric focusing gel electrophoresis to mass spectrometry by electrostatic spray ionization.

    PubMed

    Qiao, Liang; Tobolkina, Elena; Liu, Baohong; Girault, Hubert H

    2013-05-07

    Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers but has been limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionization strategy, electrostatic spray ionization, for in situ ionization of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in situ detected by scanning the gel with the electrostatic spray ionization mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionized for MS detection. This method for protein/peptide spots visualization is sensitive, providing sample molecular weight information while avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionization can produce sample ions from a complex background, and with a spatial resolution matching the isoelectric focusing, it is therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry.

  3. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones.

    PubMed

    Lasne, Françoise; Martin, Laurent; Crepin, Nathalie; de Ceaurriz, Jacques

    2002-12-15

    Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.

  4. A new mixed-mode model for interpreting and predicting protein elution during isoelectric chromatofocusing.

    PubMed

    Choy, Derek Y C; Creagh, A Louise; von Lieres, Eric; Haynes, Charles

    2014-05-01

    Experimental data are combined with classic theories describing electrolytes in solution and at surfaces to define the primary mechanisms influencing protein retention and elution during isoelectric chromatofocusing (ICF) of proteins and protein mixtures. Those fundamental findings are used to derive a new model to understand and predict elution times of proteins during ICF. The model uses a modified form of the steric mass action (SMA) isotherm to account for both ion exchange and isoelectric focusing contributions to protein partitioning. The dependence of partitioning on pH is accounted for through the characteristic charge parameter m of the SMA isotherm and the application of Gouy-Chapman theory to define the dependence of the equilibrium binding constant Kbi on both m and ionic strength. Finally, the effects of changes in matrix surface pH on protein retention are quantified through a Donnan equilibrium type model. By accounting for isoelectric focusing, ion binding and exchange, and surface pH contributions to protein retention and elution, the model is shown to accurately capture the dependence of protein elution times on column operating conditions.

  5. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    SciTech Connect

    He, Yi; Scheraga, Harold A.; Liwo, Adam

    2015-12-28

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field.

  6. Optimization of a Nucleic Acids united-RESidue 2-Point model (NARES-2P) with a maximum-likelihood approach

    PubMed Central

    He, Yi; Liwo, Adam; Scheraga, Harold A.

    2015-01-01

    Coarse-grained models are useful tools to investigate the structural and thermodynamic properties of biomolecules. They are obtained by merging several atoms into one interaction site. Such simplified models try to capture as much as possible information of the original biomolecular system in all-atom representation but the resulting parameters of these coarse-grained force fields still need further optimization. In this paper, a force field optimization method, which is based on maximum-likelihood fitting of the simulated to the experimental conformational ensembles and least-squares fitting of the simulated to the experimental heat-capacity curves, is applied to optimize the Nucleic Acid united-RESidue 2-point (NARES-2P) model for coarse-grained simulations of nucleic acids recently developed in our laboratory. The optimized NARES-2P force field reproduces the structural and thermodynamic data of small DNA molecules much better than the original force field. PMID:26723596

  7. Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block Copolymers for Enhanced Brain Delivery

    PubMed Central

    Batrakova, Elena V.; Vinogradov, Serguei V.; Robinson, Sandra M.; Niehoff, Michael L.; Banks, William A.; Kabanov, Alexander V.

    2009-01-01

    There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood—brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain. PMID:16029020

  8. Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries

    PubMed Central

    Niemz, Angelika; Boyle, David S

    2013-01-01

    Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients. PMID:23153237

  9. Acidic resin-catalysed conversion of fructose into furan derivatives in low boiling point solvents.

    PubMed

    Zhu, Hong; Cao, Quan; Li, Chunhu; Mu, Xindong

    2011-09-27

    Conversion of fructose into furan derivatives 5-hydroxymethylfurfural (HMF) and 5-methoxymethylfurfural (MMF) is performed in tetrahydrofuran (THF) and methanol-organic solvent systems, catalysed by an acidic resin Amberlyst-15. The melted fructose can be converted into HMF on the surface of the solid resin catalyst in the presence of THF as an extracting phase, which is a good solvent for HMF and other by-products. The solid resin catalyst can be reused eleven times without losing its catalytic ability, with an average HMF yield of approximately 50%. Upon the addition of methanol, the generated HMF can further react with methanol to form MMF, and the total yield of HMF and MMF could be promoted to 65%. GC-MS analysis confirms the formation of a small amount of methyl levulinate in methanolorganic solvent system.

  10. Online Capillary IsoElectric Focusing-ElectroSpray Ionization Mass Spectrometry (CIEF-ESI MS) in Glycerol-Water Media for the Separation and Characterization of Hydrophilic and Hydrophobic Proteins.

    PubMed

    Mokaddem, Meriem; d'Orlyé, Fanny; Varenne, Anne

    2016-01-01

    Capillary isoelectric focusing (CIEF) is a high-resolution technique for the separation of ampholytes, such as proteins, according to their isoelectric point. CIEF coupled online with MS is regarded as a promising alternative to 2-D PAGE for fast proteome analysis with high-resolving capabilities and enhanced structural information without the drawbacks of conventional slab-gel electrophoresis. However, online coupling has been rarely described, as it presents some difficulties. A new methodology for the online coupling of CIEF with electrospray ionization mass spectrometry (ESI-MS) has been developed in glycerol-water media. This new integrated methodology provides a mean for the characterization of a large number of hydrophilic and hydrophobic proteins.

  11. An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care.

    PubMed

    Choi, Jane Ru; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Li, XiuJun; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-02-07

    With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

  12. Combination of capillary isoelectric focusing in a tapered capillary with MALDI-TOF MS for rapid and reliable identification of Dickeya species from plant samples.

    PubMed

    Horká, Marie; Salplachta, Jiří; Karásek, Pavel; Kubesová, Anna; Horký, Jaroslav; Matoušková, Hana; Slais, Karel; Roth, Michal

    2013-07-16

    This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.

  13. At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysis.

    PubMed

    Dou, Peng; Liu, Zhen

    2011-04-01

    Sample preparation is a crucial step for protein analysis. Functionalized magnetic nanoparticle (MNP)-based extraction has been developed to be a useful sample preparation technique for proteomic analysis. In this paper, we present a strategy for at-line coupling of MNP-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use an anolyte or a catholyte as the desorbing agent. Thus, functionalized MNPs can be facilely at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added to the sample well, which can function as a natural adapter. Once a focusing electric field has been applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as a desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals their pI values. The at-line combination was well demonstrated with three types of functionalized nanoparticles: (1) phenylboronic acid functionalized MNPs for extracting glycoproteins through boronate affinity; (2) carboxyl-functionalized MNPs for extracting positively charged proteins through a weak cation exchange mechanism; and (3) amino-functionalized MNPs for extracting negatively charged proteins through a weak anion exchange mechanism. The at-line combination exhibited several significant advantages, including selectivity, sensitivity, and speed.

  14. Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation.

    PubMed

    Jensen, Hanne B; Poulsen, Nina A; Møller, Hanne S; Stensballe, Allan; Larsen, Lotte B

    2012-11-01

    Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (α(s1)-, α(s2),-, β- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

  15. Quantifying diffuse and point inputs of perfluoroalkyl acids in a nonindustrial river catchment.

    PubMed

    Müller, Claudia E; Spiess, Nora; Gerecke, Andreas C; Scheringer, Martin; Hungerbühler, Konrad

    2011-12-01

    Recently, the role of diffuse inputs of perfluoroalkyl acids (PFAAs) into surface waters has been investigated. It has been observed that river loads increased during rain and that street runoff contained considerable loads of PFAAs. This study aims at quantifying these diffuse inputs and identifying the initial sources in a small nonindustrial river catchment. The river was sampled in three distinct subcatchments (rural, urban, and wastewater treatment plant) at high temporal resolution during two rain events and samples were analyzed for perfluorocarboxylates and perfluorosulfonates. Additionally, rain, stormwater runoff, wastewater effluent, and drinking water were sampled. PFAA concentrations in river water were all low (e.g., < 10 ng/L for perfluorooctanoate, PFOA), but increased during rainfall. PFAA concentrations and water discharge data were integrated into a mass balance assessment that shows that 30-60% of PFAA loads can be attributed to diffuse inputs. Rain contributed 10-50% of the overall loads, mobilization of dry deposition and outdoor release of PFAA from products with 20-60%. We estimated that within a year 2.5-5 g of PFOA originating from rain and surface runoff are emitted into this small catchment (6 km(2), 12,500 persons).

  16. Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.

    PubMed

    Dickerson, Jane A; Ramsay, Lauren M; Dada, Oluwatosin O; Cermak, Nathan; Dovichi, Norman J

    2010-08-01

    CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2-D separation method for proteins. In this method, two capillaries are joined through a buffer-filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first-dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second-dimension separation. A fraction was transferred to the second-dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.

  17. A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

    PubMed Central

    Kulkarni, Aditya; Zeng, Yu; Zhou, Wei; Van Lanen, Steven; Zhang, Weiwen

    2015-01-01

    Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development. PMID:26519385

  18. A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin.

    PubMed

    Kulkarni, Aditya; Zeng, Yu; Zhou, Wei; Van Lanen, Steven; Zhang, Weiwen; Chen, Shawn

    2015-10-30

    Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

  19. Maternal–Fetal Transfer of Domoic Acid in Rats at Two Gestational Time Points

    PubMed Central

    Maucher, Jennifer M.; Ramsdell, John S.

    2007-01-01

    Background and objectives Prenatal exposure to asymptomatic doses of domoic acid (DA) causes learning and memory deficits later in life; therefore, we sought to measure distribution of DA in maternal plasma and brain, prenatal brain, and amniotic fluid 1 hr after exposure, a time frame that normally encompasses acute seizure behavior. Methods Pregnant rats were given a single intravenous dose of DA (0.6 or 1.6 mg/kg body weight) at either gestational day (GD) 13 or GD20, which correspond to the beginning of rat embryo neurogenesis and the last day of gestation, respectively. Using a direct ELISA, dose-dependent levels of DA were detected in each sample matrix tested. Results An average of 6.6 and 14 ng DA/g brain tissue was found in GD13 and GD20 prenatal rats, respectively. Brain concentrations of DA in the GD13 prenates were identical to amniotic fluid levels, consistent with no restriction for DA to enter the GD13 prenatal brain. At GD20 the prenatal brain contained half the concentration of DA in the amniotic fluid, and was approximately half that found in the brain of the dams. After 1 hr, fetal brain and amniotic fluid contained between 1 and 5% of DA found in the maternal circulation. The amniotic fluid levels of DA in this study were also within the same range measured in stranded California sea lions that showed reproductive failure. Conclusions DA crosses the placenta, enters brain tissue of prenates, and accumulates in the amniotic fluid. Amniotic fluid appears to be a useful fluid to monitor DA exposure. PMID:18087593

  20. Determination of protonation macro- and microconstants and octanol/water partition coefficient of the antiinflammatory drug niflumic acid.

    PubMed

    Takács-Novák, K; Avdeef, A; Box, K J; Podányi, B; Szász, G

    1994-11-01

    The drug niflumic acid is an amphoteric substance with overlapping pKa values. The acid-base chemistry of the molecule has been characterized in terms of protonation macroconstants (with reference to stoichiometric ionizations) and microconstants (with reference to ionizations of individual species). The proton-binding sites were assigned using 1H and 13C NMR spectroscopy. Due to the very poor water solubility of niflumic acid, the aqueous pKa values were determined from the apparent ionization constants in methanol-water solutions of various proportions by extrapolation to zero co-solvent using the Yasuda-Shedlovsky procedure. The kz tautomerization microconstant of the equilibrium unionized form<-->zwitterionic form was determined from mixtures of organic solvent (dioxane or methanol) with aqueous buffer (at the pH of isoelectric point) by UV spectroscopy, and used for calculation of the other protonation microconstants. The zwitterionic form of the molecule predominates over the uncharged form, the concentration being maximal at the isoelectric pH. The apparent partition coefficients (Papp) of niflumic acid were measured in octanol/water solution by the shake-flask method over a wide pH range. The lipophilicity profile (logPapp vs pH) shows a parabolic shape near its maximum at the isoelectric point. A relationship derived between Papp, PXH0(micropartition coefficient of the uncharged microspecies) and PX-(partition coefficient of the anion) is valid for amphoteric drugs, in cases where the partition of the unionized form and the ion-pair partition of anion can be confirmed. The logP values of microspecies indicate the high lipophilicity of niflumic acid, which is consistent with its good skin penetration and absorption.

  1. Purification, characterization and amino-acid sequence analysis of a thermostable, low molecular mass endo-beta-1,4-glucanase from blue mussel, Mytilus edulis.

    PubMed

    Xu, B; Hellman, U; Ersson, B; Janson, J C

    2000-08-01

    A cellulase (endo-beta-1,4-D-glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 degrees C. Another unusual feature is that the enzyme retains 55-60% of its maximum activity at 0 degrees C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 degrees C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication).

  2. Experimental and theoretical dynamics of isoelectric focusing - Elucidation of a general separation mechanism

    NASA Technical Reports Server (NTRS)

    Thormann, W.; Mosher, R. A.; Bier, M.

    1986-01-01

    The transient states in isoelectric focusing were monitored using a potential gradient array detector. Electric field profiles are presented which show the formation of transient moving boundaries, as well as the approach of the steady-state distribution, during the focusing of two- and three-component systems. The results are completely consistent with corresponding computer-simulation data. The focusing process is comprised of two sequential phases, a relatively rapid separation phase and a much slower stabilizing phase. It was found that the second phase can last as much as 20 times longer than the separation of the constituents.

  3. Theoretical studies in isoelectric focusing. [mathematical modeling and computer simulation for biologicals purification process

    NASA Technical Reports Server (NTRS)

    Mosher, R. A.; Palusinski, O. A.; Bier, M.

    1982-01-01

    A mathematical model has been developed which describes the steady state in an isoelectric focusing (IEF) system with ampholytes or monovalent buffers. The model is based on the fundamental equations describing the component dissociation equilibria, mass transport due to diffusion and electromigration, electroneutrality, and the conservation of charge. The validity and usefulness of the model has been confirmed by using it to formulate buffer systems in actual laboratory experiments. The model has been recently extended to include the evolution of transient states not only in IEF but also in other modes of electrophoresis.

  4. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  5. On-line capillary isoelectric focusing-electrospray mass spectrometry for protein characterization

    SciTech Connect

    Tang, Qing; Harrata, K.A.; Lee, C.S.

    1996-12-31

    The integration of capillary isoelectric focusing (CIEF) with electrospray mass spectrometry (ESMS) as a two-dimensional separation system for protein characterization will be presented. Mixtures of protein variants are focused and cathodically mobilized in a polyacrylamide coated capillary. At the end of CIEF capillary, the mobilized protein zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. The effects of carrier ampholyte concentration on the CIEF separation and the protein electrospray mass spectra will be discussed. On-line CIEF-ESMS with superior resolving power, speed, and sensitivity will be demonstrated for the analysis of hemoglobin and glycoprotein variants.

  6. Computer simulation and experimental validation of isoelectric focusing in ampholine-free systems

    NASA Technical Reports Server (NTRS)

    Bier, M.; Mosher, R. A.; Palusinski, O. A.

    1981-01-01

    An abbreviated version of a mathematical model of the steady state in isoelectric focusing is presented. Details of the mathematical transformations leading to a model suitable for computer implementation and numerical solution are given in the Appendix. The model describes the structure of concentration, pH, conductivity and potential gradients arising from the focusing of electrochemically defined ampholytes. Some of the results of computer simulations of two and three component systems are of particular interest to the experimentalist and are presented together with experimental validation of this model.

  7. Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

    NASA Technical Reports Server (NTRS)

    Knisley, Keith A.; Rodkey, L. Scott

    1986-01-01

    A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

  8. Point-of-service, quantitative analysis of ascorbic acid in aqueous humor for evaluating anterior globe integrity

    PubMed Central

    Gartia, Manas R.; Misra, Santosh K.; Ye, Mao; Schwartz-Duval, Aaron; Plucinski, Lisa; Zhou, Xiangfei; Kellner, David; Labriola, Leanne T.; Pan, Dipanjan

    2015-01-01

    Limited training, high cost, and low equipment mobility leads to inaccuracies in decision making and is concerning with serious ocular injuries such as suspected ruptured globe or post-operative infections. Here, we present a novel point-of-service (POS) quantitative ascorbic acid (AA) assay with use of the OcuCheck Biosensor. The present work describes the development and clinical testing of the paper-based biosensor that measures the changes in electrical resistance of the enzyme-plated interdigitated electrodes to quantify the level of AA present in ocular fluid. We have demonstrated the proof-of-concept of the biosensor testing 16 clinical samples collected from aqueous humor of patients undergoing therapeutic anterior chamber paracentesis. Comparing with gold standard colorimetric assay for AA concentration, OcuCheck showed accuracy of >80%, sensitivity of >88% and specificity of >71%. At present, there are no FDA-approved POS tests that can directly measures AA concentration levels in ocular fluid. We envisage that the device can be realized as a handheld, battery powered instrument that will have high impact on glaucoma care and point-of-care diagnostics of penetrating ocular globe injuries. PMID:26525715

  9. Point-of-service, quantitative analysis of ascorbic acid in aqueous humor for evaluating anterior globe integrity

    NASA Astrophysics Data System (ADS)

    Gartia, Manas R.; Misra, Santosh K.; Ye, Mao; Schwartz-Duval, Aaron; Plucinski, Lisa; Zhou, Xiangfei; Kellner, David; Labriola, Leanne T.; Pan, Dipanjan

    2015-11-01

    Limited training, high cost, and low equipment mobility leads to inaccuracies in decision making and is concerning with serious ocular injuries such as suspected ruptured globe or post-operative infections. Here, we present a novel point-of-service (POS) quantitative ascorbic acid (AA) assay with use of the OcuCheck Biosensor. The present work describes the development and clinical testing of the paper-based biosensor that measures the changes in electrical resistance of the enzyme-plated interdigitated electrodes to quantify the level of AA present in ocular fluid. We have demonstrated the proof-of-concept of the biosensor testing 16 clinical samples collected from aqueous humor of patients undergoing therapeutic anterior chamber paracentesis. Comparing with gold standard colorimetric assay for AA concentration, OcuCheck showed accuracy of >80%, sensitivity of >88% and specificity of >71%. At present, there are no FDA-approved POS tests that can directly measures AA concentration levels in ocular fluid. We envisage that the device can be realized as a handheld, battery powered instrument that will have high impact on glaucoma care and point-of-care diagnostics of penetrating ocular globe injuries.

  10. Initiation points for cellular deoxyribonucleic acid replication in human lymphoid cells converted by Epstein-Barr virus

    SciTech Connect

    Oppenheim, A.; Shlomai, Z.; Ben-Bassat, H.

    1981-08-01

    Replicon size was estimated in two Epstein-Barr virus (EBV)-negative human lymphoma lines, BJAB and Ramos, and four EBV-positive lines derived from the former ones by infection (conversion) with two viral strains, B95-8 and P3HR-1. Logarithmic cultures were pulse-labeled with (/sup -3/H)thymidine, and the deoxyribonucleic acid was spread on microscopic slides and autoradiographed by the method of Huberman and Riggs. Three of the four EBV-converted cell lines, BJAB/B95-8, Ra/B95-8, and Ra/HRIK, were found to have significantly shorter replicons (41, 21, 54% shorter, respectively), i.e., more initiation points, than their EBV-negative parents. BJAB/HRIK had replicons which were only slightly shorter (11%) than those of BJAB. However, analysis of track length demonstrated that extensive track fusion occurred during the labeling of BJAB/HRIK, implying that its true average replicon size is shorter than the observed value. The results indicate that in analogy to simian virus 40, EBV activates new initiation points for cellular DNA replication in EBV-transformed cells.

  11. Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

    PubMed

    Weyda, István; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S

    2014-04-01

    Aspergillus carbonarius accumulates xylitol when it grows on D-xylose. In fungi, D-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD(+)-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using D-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

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

    PubMed

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

    2012-06-01

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

  13. Reciprocating free-flow isoelectric focusing device for preparative separation of proteins.

    PubMed

    Kong, Fan-Zhi; Yang, Ying; Wang, Yi; Li, Guo-Qing; Li, Shan; Xiao, Hua; Fan, Liu-Yin; Liu, Shao-Rong; Cao, Cheng-Xi

    2015-11-27

    The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost.

  14. A novel reagent significantly improved assay robustness in imaged capillary isoelectric focusing.

    PubMed

    Zhang, Xin; Voronov, Sergey; Mussa, Nesredin; Li, Zhengjian

    2017-03-15

    Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.

  15. [Analysis of gingival crevicular fluid. Relation of isoelectric focusing protein patterns to clinical evaluation].

    PubMed

    Aoki, Y; Yoshinaga, E; Tamazawa, O

    1988-12-01

    The purpose of this study was to determine the protein patterns in gingival crevicular fluid relation to the isoelectric focusing protein patterns of GCF and to clinical evaluations. GCF was collected with filter paper from 105 subjects. The probing depth, the gingival index (Löe & Silness) and the plaque index (Silness & Löe) as clinical evaluations The results follow: 1. The main isoelectric focusing protein patterns of GCF were between pH 5.5 and 7.5. In comparison, the GCF and the serum from the same patients showed patterns to similar serum albumin. 2. Between of GCF pH 5.5 and 7.5 the protein patterns that ranged over 60% was pI 5.65, 6.45, 6.55, 6.75 and 7.00. The frequencies of the ranges of protein patterns and clinical evaluation were compared by the X2 test. pI 5.65, 6.45, 6.55 and 6.75 and PD were significant different, as were pI 6.45, 6.55 and 6.75 and GI. But each pI and PIl. were not significantly different.

  16. Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry.

    PubMed

    Mikkonen, Saara; Jacksén, Johan; Roeraade, Johan; Thormann, Wolfgang; Emmer, Åsa

    2016-10-18

    A novel method for preconcentration and purification of the Alzheimer's disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.

  17. Investigating the significance of zero-point motion in small molecular clusters of sulphuric acid and water

    SciTech Connect

    Stinson, Jake L.; Kathmann, Shawn M.; Ford, Ian J.

    2014-01-14

    The nucleation of particles from trace gases in the atmosphere is an important source of cloud condensation nuclei (CCN), and these are vital for the formation of clouds in view of the high supersaturations required for homogeneous water droplet nucleation. The methods of quantum chemistry have increasingly been employed to model nucleation due to their high accuracy and efficiency in calculating configurational energies; and nucleation rates can be obtained from the associated free energies of particle formation. However, even in such advanced approaches, it is typically assumed that the nuclei have a classical nature, which is questionable for some systems. The importance of zero-point motion (also known as quantum nuclear dynamics) in modelling small clusters of sulphuric acid and water is tested here using the path integral molecular dynamics (PIMD) method at the density functional theory (DFT) level of theory. We observe a small zero-point effect on the the equilibrium structures of certain clusters. One configuration is found to display a bimodal behaviour at 300 K in contrast to the stable ionised state suggested from a zero temperature classical geometry optimisation. The general effect of zero-point motion is to promote the extent of proton transfer with respect to classical behaviour. We thank Prof. Angelos Michaelides and his group in University College London (UCL) for practical advice and helpful discussions. This work benefited from interactions with the Thomas Young Centre through seminar and discussions involving the PIMD method. SMK was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences. JLS and IJF were supported by the IMPACT scheme at UCL and by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences. We are grateful for use of the UCL Legion High Performance Computing Facility and the

  18. Computer simulation of two electrophoretic columns coupled for isoelectric focusing in simple buffers

    NASA Technical Reports Server (NTRS)

    Tsai, Amos; Mosher, Richard A.; Bier, Milan

    1986-01-01

    Computer simulation is used to analyze a system of two electrophoretic columns coupled by mixing the anolyte of one with the catholyte of the other. A mathematical model is presented which is used to predict the pH gradients formed by monovalent buffers in this system, when the currents in the columns are unequal. In the column with the higher current a pH gradient is created which increases from anode to cathode and is potentially useful for isoelectric focusing. The breadth of this gradient is dependent upon the ratio of the currents. The function of the second column is the compensation of buffer migration which occurs in the first column, thereby maintaining constant electrolyte composition. The effects of buffer pKs and mobilities are evaluated.

  19. A high-throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation.

    PubMed

    Markely, Lam Raga Anggara; Cheung, Lila; Choi, Young Jun; Ryll, Thomas; Estes, Scott; Prajapati, Shashi; Turyan, Iva; Frenkel, Ruth; Sosic, Zoran; Lambropoulos, James; Tescione, Lia; Ryll, Thomas; Berman, Melissa

    2016-01-01

    The serum half-life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high-throughput method for semi-quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry.

  20. Isoelectric focusing and ELISA for detecting adulteration of donkey milk with cow milk.

    PubMed

    Pizzano, Rosa; Salimei, Elisabetta

    2014-06-25

    Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey-cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1-28 sequence stretch of cow β-casein was also developed for an accurate definition of composition of donkey-cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey-cow milk mixtures prepared at laboratory scale and assayed by ELISA.

  1. High levels of DJ-1 protein and isoelectric point 6.3 isoform in sera of breast cancer patients

    PubMed Central

    Kawate, Takahiko; Iwaya, Keiichi; Koshikawa, Kayoko; Moriya, Tomoyuki; Yamasaki, Tamio; Hasegawa, Sho; Kaise, Hiroshi; Fujita, Tomoyuki; Matsuo, Hirotaka; Nakamura, Takahiro; Ishikawa, Takashi; Hiroi, Sadayuki; Iguchi-Ariga, Sanae MM; Ariga, Hiroyoshi; Murota, Keiichi; Fujimori, Minoru; Yamamoto, Junji; Matsubara, Osamu; Kohno, Norio

    2015-01-01

    In patients with cancer and Parkinson’s disease, the DJ-1 protein may be secreted into the serum during the impaired response of the underlying cell-protective mechanisms. In order to determine the clinical significance of DJ-1 protein in the sera of breast cancer patients, we examined blood samples from a breast cancer group (n = 180) and a non-cancerous control group (n = 300). Higher levels of DJ-1 were detected in the breast cancer group (mean level, 42.7 ng/mL) than the control group (28.3 ng/mL) by ELISA (P = 0.019). Higher DJ-1 levels were significantly associated with advanced clinical grade, according to the TNM classification, negative hormone receptor status, and high Ki-67 labeling index, of biopsied materials; samples showed low DJ-1 protein expression despite upregulated DJ-1 mRNA. DJ-1 isoforms could be detected clearly in 17 blood samples (from 11 breast cancer patients, and 6 non-cancerous controls) by 2-D gel electrophoresis and immunoblot analysis. The isoform at the pI of 6.3 showed the highest intensity in all 11 cancer cases. Conversely, in the 6 non-cancerous cases, isoforms other than the pI 6.3 isoform were highly expressed, and there was a significant difference in the isoform pattern between breast cancer cases and controls (P = 0.00025). These data indicate that high levels of DJ-1, probably of isoform at pI 6.3, is a candidate serum marker of breast cancer. PMID:25867058

  2. High levels of DJ-1 protein and isoelectric point 6.3 isoform in sera of breast cancer patients.

    PubMed

    Kawate, Takahiko; Iwaya, Keiichi; Koshikawa, Kayoko; Moriya, Tomoyuki; Yamasaki, Tamio; Hasegawa, Sho; Kaise, Hiroshi; Fujita, Tomoyuki; Matsuo, Hirotaka; Nakamura, Takahiro; Ishikawa, Takashi; Hiroi, Sadayuki; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi; Murota, Keiichi; Fujimori, Minoru; Yamamoto, Junji; Matsubara, Osamu; Kohno, Norio

    2015-07-01

    In patients with cancer and Parkinson's disease, the DJ-1 protein may be secreted into the serum during the impaired response of the underlying cell-protective mechanisms. In order to determine the clinical significance of DJ-1 protein in the sera of breast cancer patients, we examined blood samples from a breast cancer group (n = 180) and a non-cancerous control group (n = 300). Higher levels of DJ-1 were detected in the breast cancer group (mean level, 42.7 ng/mL) than the control group (28.3 ng/mL) by ELISA (P = 0.019). Higher DJ-1 levels were significantly associated with advanced clinical grade, according to the TNM classification, negative hormone receptor status, and high Ki-67 labeling index, of biopsied materials; samples showed low DJ-1 protein expression despite upregulated DJ-1 mRNA. DJ-1 isoforms could be detected clearly in 17 blood samples (from 11 breast cancer patients, and 6 non-cancerous controls) by 2-D gel electrophoresis and immunoblot analysis. The isoform at the pI of 6.3 showed the highest intensity in all 11 cancer cases. Conversely, in the 6 non-cancerous cases, isoforms other than the pI 6.3 isoform were highly expressed, and there was a significant difference in the isoform pattern between breast cancer cases and controls (P = 0.00025). These data indicate that high levels of DJ-1, probably of isoform at pI 6.3, is a candidate serum marker of breast cancer.

  3. Fine mapping of porcine SSC14 QTL and SCD gene effects on fatty acid composition and melting point of fat in a Duroc purebred population.

    PubMed

    Uemoto, Y; Nakano, H; Kikuchi, T; Sato, S; Ishida, M; Shibata, T; Kadowaki, H; Kobayashi, E; Suzuki, K

    2012-04-01

    The stearoyl-CoA desaturase (delta-9-desaturase; SCD) gene is a candidate gene for fatty acid composition. It is located on pig SSC14 in a region where quantitative trait loci (QTL) for fatty acid composition were previously detected in a Duroc purebred population. The objective of the present study was to fine map the QTL, to identify polymorphisms of the pig SCD gene and to examine the effects of SCD polymorphisms on fatty acid composition and melting point of fat in the population. The pigs were examined for fatty acid composition and melting point of inner and outer subcutaneous fat and inter- and intramuscular fat; the number of pigs examined was 479-521. Two SNPs (g.-353C>T and g.-233T>C) were identified in the promoter region of the SCD gene and were completely linked in the pigs from the base generation. In all pigs, 19 microsatellite markers and SCD haplotypes were then genotyped. Different statistical models were applied to evaluate the effects of QTL and the possible causality of the SCD gene variants with respect to the QTL. The results show that all significant QTL for C14:0, C18:0, C18:1 and melting point of fat were detected in the same region, located near the SCD gene. The results also show a significant association between SCD haplotypes and fatty acid composition and fat melting point in this population. These results indicate that the haplotype of the SCD gene has a strong effect on fatty acid composition and melting point of fat.

  4. Thermodynamics of the dissociation of aminomethanesulfonic acid and its N-substituted derivatives in aqueous solutions at 293-313 K

    NASA Astrophysics Data System (ADS)

    Khoma, R. E.

    2017-01-01

    Δ G, Δ H, and Δ S of the second dissociation step of aminomethanesulfonic acid and its N-methyl, N-hydroxyethyl, N-( tert-butyl), N-benzyl derivatives in aqueous solutions (at the isoelectric point where the ionic strength is 4.75 × 10-4 M) are determined on basis of pH measurements at 293-313 K. It is found that an increase of the electron-acceptor properties of the substituents at nitrogen atom reduces the effect temperature has on the thermodynamic functions of dissociation. Enthalpy-enthropy compensation at an isothermodynamic temperature of 303 K is recorded.

  5. The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing.

    PubMed

    Poyart, C F; Guesnon, P; Bohn, B M

    1981-05-01

    We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the DeltapI(deox.-ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are ;stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (DeltalogP(50)/DeltapH) in solutions, the DeltapI(deox.-ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the DeltapI(deox.-ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A(0) (alpha(2)beta(2)) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5 degrees C and very low concentration of KCl. The variation of the DeltapH(deox.-ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of DeltapI(deox.-ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A(0). In haemoglobin A(0) the DeltapI(deox.-ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The DeltapI(deox.-ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the alpha-chains are decreased by 30% compared with the DeltapI value measured in haemoglobin A(0). When the C-terminus of the beta-chains is altered, as in Hb Nancy (alpha(2)beta(Tyr-145-->Asp) (2)), we observed a 70% decrease in the DeltapI value compared

  6. SpeB proteolysis with imaged capillary isoelectric focusing for the characterization of domain-specific charge heterogeneities of reference and biosimilar Rituximab.

    PubMed

    Zhang, Zichuan; Perrault, Ronel; Zhao, Yun; Ding, Julia

    2016-05-01

    The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2-26.6% Fc/2, 28.9-29.3% LC and 44.4-44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product's basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments.

  7. Enzymatic modification by point mutation and functional analysis of an omega-6 fatty acid desaturase from Arctic Chlamydomonas sp.

    PubMed

    Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Kang, Sung-Ho; Choi, Han-Gu; Kim, Sanghee

    2017-02-07

    Arctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD6) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI culture collection of polar micro-organisms. Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6-transformed cells increased more than 3-fold [6.73 ± 0.13 mg g(-1) dry cell weight (DCW)] compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g(-1) DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase. These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.

  8. Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

    PubMed

    Gannoun-Zaki, Laila; Alibaud, Laeticia; Kremer, Laurent

    2013-01-01

    The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

  9. Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection.

    PubMed

    Horká, Marie; Růzicka, Filip; Horký, Jaroslav; Holá, Veronika; Slais, Karel

    2006-09-01

    We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3-10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5x10(2) to 1x10(3) with on-column UV detection at 280 nm.

  10. Isoelectric focusing and titration curves in biomedicine and in agrofood industries: a multimedia teaching program.

    PubMed

    Cade-Treyer, D; Cade, A; Darjo, A; Jouvion-Moreno, M

    1996-03-01

    The aim of this 45 min, 60 megabyte, modular program is to initiate students, scientists and engineers of biotechnology, biomedicine and agrofood industries into isoelectric focusing (IEF) and titration curves for analytical (e.g. IEF, zone electrophoresis, isotachophoresis, electrotransfer) and preparative (e.g. ion-exchange chromatography, chromatofocusing) application of charge-dependent methods. For advanced teaching, the following theoretical and practical aspects may be of interest: pH gradient engineering, IEF resolving power, generation of pH gradient, sample-ampholyte interactions, pH gradient drift, immobilized pH gradients (IPG), IPG-two-dimensional (2-D) electrophoresis, preparative methods with multi-compartments and IPG membranes, capillary IEF, isozyme analysis, etc.). The program associates fixed and animated drawings, and computer-assisted simulations, with spoken and written commentaries (in English). It is illustrated with numerous IEF gel patterns and titration curves and some video sequences to be run on a multimedia PC with MS Windows 3.1 (or later releases) as the only software. The linear presentation of the program may be used directly on the PC, or may be projected on a screen from the PC, for small classes or for a larger audience (200 persons). Its development as an interactive multimedia program is in progress and will soon be available on the Internet.

  11. A versatile semi-permanent sequential bilayer/diblock polymer coating for capillary isoelectric focusing.

    PubMed

    Bahnasy, Mahmoud F; Lucy, Charles A

    2012-12-07

    A sequential surfactant bilayer/diblock copolymer coating was previously developed for the separation of proteins. The coating is formed by flushing the capillary with the cationic surfactant dioctadecyldimethylammonium bromide (DODAB) followed by the neutral polymer poly-oxyethylene (POE) stearate. Herein we show the method development and optimization for capillary isoelectric focusing (cIEF) separations based on the developed sequential coating. Electroosmotic flow can be tuned by varying the POE chain length which allows optimization of resolution and analysis time. DODAB/POE 40 stearate can be used to perform single-step cIEF, while both DODAB/POE 40 and DODAB/POE 100 stearate allow performing two-step cIEF methodologies. A set of peptide markers is used to assess the coating performance. The sequential coating has been applied successfully to cIEF separations using different capillary lengths and inner diameters. A linear pH gradient is established only in two-step CIEF methodology using 3-10 pH 2.5% (v/v) carrier ampholyte. Hemoglobin A(0) and S variants are successfully resolved on DODAB/POE 40 stearate sequentially coated capillaries.

  12. On-chip protein isoelectric focusing using a photoimmobilized pH gradient.

    PubMed

    Xia, Lin; Lin, FengMing; Wu, Xin; Liu, Chuanli; Wang, Jianshe; Tang, Qi; Yu, Shiyong; Huang, Kunjie; Deng, Yulin; Geng, Lina

    2014-11-01

    An immobilized pH gradient was directly constructed on the inner wall of a microfluidic chip channel by photoimmobilizing focused carrier ampholytes onto the wall. A mixture of carbonic anhydrase, myoglobin, and trypsin inhibitor was successfully isoelectric-focused and separated with good linearity between the pI values of proteins and the location of the focused bands. Furthermore, coating methods for the resistance of protein nonselective adsorption and simultaneously for pH gradient photocoupling were screened. The PEG-silane coating method was found to be better than the cross-linked polyacrylamide coating and aminosilane modification methods. Finally, based on the open tubular column mode of carrier ampholytes' immobilization and effective antiadsorption coating, the immobilized pH gradient was reused and the chip was recycled for the first time. By virtue of its remarkable features including simplicity, convenience, high efficiency of protein enrichment and separation, and potential for coupling site-selective IEF with other analytical or separation techniques, this novel method promises to be useful in several applications related with zwitterionic biomolecules.

  13. Detection of thyroglobulin in antithyroglobulin antibody-positive sera by isoelectric focusing.

    PubMed

    Malvoisin, Etienne; Makhloufi, Djamila; Livrozet, Jean-Michel

    2015-07-20

    Circulating antibodies have the potential to interfere with the measurement of thyroglobulin (Tg) in sera of patients. Here, we determined Tg concentration by isoelectric focusing (IEF) on agarose gel using for detection a rabbit antiserum to human Tg termed FLX. Tg was determined in sera of thyroid patients and HIV-infected patients under antiviral therapy. We showed that Tg IEF was not affected by the presence of anti-Tg antibodies (TgAb). Tg concentrations measured by IEF in TgAb-negative sera were in most of the cases, similar to those obtained by IRMA (immunoradiometric assay). However, in 5 of the 96 thyroid patients, and none of the 46 healthy subjects, Tg was undetectable by antiserum FLX and measurable by IRMA. In HIV-infected patients (64 men and 60 women), Tg was not recognized by FLX in 23 men and 9 women and this was related to abnormal CD4. We hypothesize that the decreased binding of FLX to Tg may be the result of conformational change on the Tg molecule, a phenomenon apparently related to immunodeficiency in HIV-infected patients. For thyroid patients, Tg IEF may be very useful for the interpretation of results when Tg measurements by IRMA and automated immunoassays are affected by interferences.

  14. Sequential injection setup for capillary isoelectric focusing combined with MS detection.

    PubMed

    Páger, Csilla; Dörnyei, Agnes; Kilár, Ferenc

    2011-07-01

    Capillary isoelectric focusing in the presence of electroosmosis with sequential injection of carrier ampholytes and sample was found to be suitable for MS detection. The separate injection of the sample and the ampholytes provides good condition to suppress and overcome the undesirable effect of the presence of ampholytes in MS. By the appropriate selection of ampholyte solutions, whose pH range not necessarily covers the pI values of the analytes, the migration of the components can be controlled, and the impact of the ampholytes on MS detection is decreased. The unique applicability of this setup is shown by testing several parameters, such as the application of volatile electrolyte solutions, the type of the ampholytes, the order and the number of the ampholyte and sample zones. Broad and narrow pH range ampholytes were applied in experiments using uncoated capillaries with different lengths for the analyses of substituted nitrophenol dyes to achieve optimal conditions for the MS detection. Although the sample components are not leaving the pH gradient, due to the decrease in the ampholyte concentration at the position of the components, and because the sample components migrate in charged state, the ionisation is more effective for MS detection.

  15. Laser micromachined isoelectric focusing devices on polymer substrate for electrospray mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lin, Yuehe; Wen, Jenny; Fan, Xiang; Matson, Dean W.; Smith, Richard D.

    1999-08-01

    A microfabricated device for isoelectric focusing (IEF) incorporating an optimized electrospray ionization (ESI) tip was constructed on polycarbonate plates using a laser micromachining technique. The separation channels on an IEF chip were 16 cm long, 50 micrometers wide and 30 micrometers deep. Electrical potentials used for IEF focusing and electrospray were applied through platinum electrodes placed in the buffer reservoirs, and which were isolated from the separation channel by molecular porous membranes. On-line ESI produced directly from a sharp `tip' on the microchip was evaluated. The results indicate that this design can produce a stable electrospray that is further improved and made more flexible with the assistance of sheath gas and sheath liquid. Error analysis of the spectral data shows that the standard deviation in signal intensity for an analyte peak was less than approximately 5% over 3 hours. The production of stable electrosprays directly from microchip IEF devices represents a step towards easily- fabricated microanalytical devices. IEF separations of protein mixtures were demonstrated for uncoated polycarbonate microchips. On-line IEF/ESI-MS was demonstrated using the microfabricated chip with an ion-trap ESI mass spectrometer for characterization of protein mixtures.

  16. Divergent-flow isoelectric focusing for separation and preparative analysis of peptides.

    PubMed

    Duša, Filip; Křenková, Jana; Moravcová, Dana; Kahle, Vladislav; Slais, Karel

    2012-07-01

    A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.

  17. Use of imaged capillary isoelectric focusing technique in development of diphtheria toxin mutant CRM197.

    PubMed

    Rustandi, Richard R; Peklansky, Brian; Anderson, Carrie L

    2014-04-01

    Polysaccharide based-vaccines have been successful in providing protection in adults from bacterial infections, however they are not as effective in infants or young children. To enhance the immune response in these high risk groups, the polysaccharide is conjugated with a carrier protein such as cross-reacting material 197 (CRM197). The CRM197 protein has been well-characterized biochemically and biophysically using various analytical techniques however, none of these have been CE-based methods. Of the various CE techniques, imaged capillary isoelectric focusing (icIEF) is a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. Applications of icIEF technique using Convergent Bioscience icIEF instrumentation with whole-field imaging technology are presented and discussed in this paper. These applications include rapid method development to establish a CRM197 identity test for product release, a concentration assay for upstream and downstream in-process product development, and CRM197 stability with respect to its charge heterogeneity under accelerated temperature stress. The data presented demonstrates the utility of the icIEF method as a multifunctional assay because it can screen for better product candidates during early stage clonal selection as well as support in-process and final product characterization throughout CRM197 development.

  18. High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis

    SciTech Connect

    Tang, Q.; Harrata, A.K.; Lee, C.S. |

    1996-08-01

    On-line capillary isoelectric focusing (CIEF)-electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF-ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10{sup -8} M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits. 9 refs., 6 figs., 2 tabs.

  19. Analysis of amino-terminal variants of amyloid-β peptides by capillary isoelectric focusing immunoassay.

    PubMed

    Haußmann, Ute; Jahn, Olaf; Linning, Philipp; Janßen, Christin; Liepold, Thomas; Portelius, Erik; Zetterberg, Henrik; Bauer, Chris; Schuchhardt, Johannes; Knölker, Hans-Joachim; Klafki, Hans; Wiltfang, Jens

    2013-09-03

    Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβ(x-10), Aβ(x-16), and Aβ(5-38(D23S)) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay.

  20. Optimization of the isoelectric precipitation method to obtain protein isolates from amaranth (Amaranthus cruentus) seeds.

    PubMed

    Salcedo-Chávez, Beatriz; Osuna-Castro, Juan A; Guevara-Lara, Fidel; Domínguez-Domínguez, Jorge; Paredes-López, Octavio

    2002-10-23

    This research was conducted to evaluate the effect of extraction pH (7.8-9.2) and precipitation pH (4.3-5.7) on four selected quality attributes of protein isolates from amaranth seeds (Amaranthus cruentus) such as protein content (PC), whiteness index (WI), enthalpy of transition (EN), and denaturation temperature (DT). Ten different treatments involving extraction and precipitation pH combinations were analyzed by a central composite design; the experimental data were fitted by a second-order model using a least-squares method for each one of the four dependent variables. Response surface methodology was used for the optimization process; in addition, a common optimum value for the four dependent variables was obtained utilizing the desirability method. A confirmatory test showed that the generated regression equations could adequately predict performance of this isoelectric precipitation method. The results indicate that extraction pH and precipitation pH showed an important effect on PC, WI, and EN. However, the different combinations did not significantly affect the DT. Values of 9.2 and 8.0 for extraction pH and 5.7 for precipitation pH produced the best overall result for all responses. Finally, the results have shown that it is possible to obtain protein isolates from A. cruentus seeds at optimized values of extraction pH and precipitation pH, which presented a high protein content and good physicochemical properties.

  1. Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.

    PubMed

    Wang, Wei; Lu, Joann J; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-07-16

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed.

  2. Chlorates induce pitting corrosion of iron in sulfuric acid solutions: An analysis based on current oscillations and a point defect model

    NASA Astrophysics Data System (ADS)

    Pagitsas, M.; Pavlidou, M.; Papadopoulou, S.; Sazou, D.

    2007-01-01

    Current oscillatory phenomena were used to investigate the effect of chlorates on the passive state of iron in sulfuric acid solutions. Experimental results show that chlorates cause pitting corrosion, besides general corrosion. It is shown that pitting is not due to the chlorate ion itself, but to chlorides produced via the reduction of chlorates by ferrous ions. General and pitting corrosion are explained in terms of a point defect model proposed to describe the oxide growth and breakdown.

  3. Bioinformatic and biochemical studies point to AAGR-1 as the ortholog of human acid alpha-glucosidase in Caenorhabditis elegans.

    PubMed

    Sikora, Jakub; Urinovská, Jana; Majer, Filip; Poupetová, Helena; Hlavatá, Jitka; Kostrouchová, Marta; Ledvinová, Jana; Hrebícek, Martin

    2010-08-01

    Human acid alpha-glucosidase (GAA, EC 3.2.1.20) is a lysosomal enzyme that belongs to the glycoside hydrolase family 31 (GH31) and catalyses the hydrolysis of alpha-1,4- and alpha-1,6-glucosidic linkages at acid pH. Hereditary deficiency of GAA results in lysosomal glycogen storage disease type II (GSDII, Pompe disease). The aim of this study was to assess GH31 proteins in Caenorhabditis elegans (C. elegans) to identify the ortholog of human GAA. Bioinformatic searches for GAA ortholog in C. elegans genome revealed four acid alpha-glucosidase-related (aagr-1-4) genes. Multiple sequence alignment of AAGRs with other GH31 proteins demonstrated their evolutionary conservation. Phylogenetic analyses suggested clustering of AAGR-1 and -2 with acid-active and AAGR-3 and -4 with neutral-active GH31 enzymes. In order to prove the AAGRs' predicted alpha-glucosidase activity, we performed RNA interference of all four aagr genes. The impact on the alpha-glucosidase activity was evaluated at pH 4.0 (acid) and pH 6.5 (neutral), with or without the inhibitor acarbose. AAGR-1 and -2 expressed acidic alpha-glucosidase activity; on the contrary, AAGR-3 not -4 represented the predominant neutral alpha-glucosidase activity in C. elegans. Similar results were obtained in each of aagr-1 and -4 deletion mutants. Moreover, based on our structural models of AAGRs and these biochemical experiments, we hypothesize that the enzymatic sensitivity of AAGR-2 and human maltase-glucoamylase to the inhibitor acarbose is associated with a tyrosine residue in the GH31 active site, whereas acarbose resistance of AAGR-1 and human GAA is associated with the corresponding tryptophane in the active site. Acid-active AAGR-1 may thus represent the ortholog of human GAA in C. elegans.

  4. Rapid visual identification of PCR amplified nucleic acids by centrifugal gel separation: Potential use for molecular point-of-care tests.

    PubMed

    Hwang, Sang-Hyun; Kim, Dong-Eun; Im, Ji-Hyun; Kang, Su-Jin; Lee, Do-Hoon; Son, Sang Jun

    2016-05-15

    Recently, nucleic acid amplification and detection techniques have progressed based on advances in in microfluidics, microelectronics, and optical systems. Nucleic acids amplification based point-of-care test (POCT) in resource-limited settings requires simple visual detection methods. Several biosensing methods including lateral flow immunoassays (LFIA) were previously used to visually detect nucleic acids. However, prolonged assay time, several washing steps, and a need for specific antibodies limited their use. Here we developed a novel, rapid method to visualize amplified nucleic acids with naked eyes in clinical samples. First, we optimized conditions based on separation using very low centrifugal force and a density medium to detect human papillomavirus (HPV)-16 DNA in cervical specimens. After DNA extraction, HPV16 PCR was performed with biotin-labeled forward primer and Cy3-labeled reverse primer. PCR amplicon was mixed with streptavidin-magnetic beads, introduced into the density medium. After two-minute centrifugation, the result was visually identified. This system showed identical results with commercial HPV real-time PCR for 30 clinical samples and could detect up to 10(2)copies/mL of HPV DNA without any optical instruments. This robust and sensitive visual detection system is suitable for non-specialist personnel and point-of-care diagnosis in low-resource settings.

  5. Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

    PubMed Central

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T.; Phan, Tony; Pilz, Renate B.; Boss, Gerry R.

    2014-01-01

    SUMMARY The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mTORC2 and IκB kinase regulate Akt activity, and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the non-oxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the non-oxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for two days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a new mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  6. An ab initio Study of the Crystalline Structure of Sulfuric Acid (H2SO4)- The Point Charge Model.

    DTIC Science & Technology

    1987-12-01

    2 ... 8 1.81. 5 111 .4 1111 . Pj LH~ H I Lp ’V. 1 4% % %4"~4 % 4’°" 111’, f LE AN AB INITIO STUDY OF THE CRYSTALLINE STRUCTURE OF SULFURIC ACID...first child .5 .5 4 S. S. S. ni-Ic A I’ J a ~-, ., I ,I/p - ~ ~SJ. ~ >4" h AN AB INITIO STUDY OF THE CRYSTALLINE STRUCTURE OF SULFURIC ACID (H2SO4)- THE

  7. Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

    NASA Astrophysics Data System (ADS)

    Kudo, Norio; Ataka, Mitsuo; Sasaki, Hiroshi; Muramatsu, Tomonari; Katsura, Tatsuo; Tanokura, Masaru

    1996-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

  8. GC/MS determination of fatty acid picolinyl esters by direct curie-point pyrolysis of whole bacterial cells.

    PubMed

    Kurkiewicz, Slawomir; Dzierzewicz, Zofia; Wilczok, Tadeusz; Dworzanski, Jacek P

    2003-01-01

    A single-step method suitable for cellular fatty acid derivatization to picolinyl esters with the use of a pyrolyzer as a thermochemical micro-reactor was developed for whole bacterial cells. This reduced the preparation time from several hours to less than two minutes. In addition, the minimal bacterial mass required for analysis was reduced from several milligrams to micrograms. The profiling of cellular fatty acids of gram-positive and gram-negative bacteria was achieved using three derivatization methods: preparation of methyl esters, beta-picolinyl esters by Harvey's method and a new method based on pyrolytic derivatization to beta-picolinyl esters. It was shown that there are great similarities between profiles of bacterial fatty acids determined by the pyrolytic derivatization method and traditional preparation methods of picolinyl and methyl esters prior to GC analysis. Results obtained by application of the new technique have immense diagnostic value due to vast similarities between profiles of fatty acids derivatized to either picolinyl and methyl esters. Although the latter are referred to in the literature most often, mass spectra of picolinyl esters contain fragment ions that provide structural information about the chain branching, position of unsaturation, and other substituents.

  9. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue*

    PubMed Central

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J.

    2016-01-01

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr308 and Ser473 upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser473-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons. PMID:26945062

  10. Optically directed molecular transport and 3D isoelectric positioning of amphoteric biomolecules

    PubMed Central

    Hafeman, Dean G.; Harkins, James B.; Witkowski, Charles E.; Lewis, Nathan S.; Warmack, Robert J.; Brown, Gilbert M.; Thundat, Thomas

    2006-01-01

    We demonstrate the formation of charged molecular packets and their transport within optically created electrical force-field traps in a pH-buffered electrolyte. We call this process photoelectrophoretic localization and transport (PELT). The electrolyte is in contact with a photoconductive semiconductor electrode and a counterelectrode that are connected through an external circuit. A light beam directed to coordinates on the photoconductive electrode surface produces a photocurrent within the circuit and electrolyte. Within the electrolyte, the photocurrent creates localized force-field traps centered at the illuminated coordinates. Charged molecules, including polypeptides and proteins, electrophoretically accumulate into the traps and subsequently can be transported in the electrolyte by moving the traps over the photoconductive electrode in response to movement of the light beam. The molecules in a single trap can be divided into aliquots, and the aliquots can be directed along multiple routes simultaneously by using multiple light beams. This photoelectrophoretic transport of charged molecules by PELT resembles the electrostatic transport of electrons within force-field wells of solid-state charge-coupled devices. The molecules, however, travel in a liquid electrolyte rather than a solid. Furthermore, we have used PELT to position amphoteric biomolecules in three dimensions. A 3D pH gradient was created in an electrolyte medium by controlling the illumination position on a photoconductive anode where protons were generated electrolytically. Photoelectrophoretic transport of amphoteric molecules through the pH gradient resulted in accumulation of the molecules at their apparent 3D isoelectric coordinates in the medium. PMID:16618926

  11. Broadening of analyte streams due to a transverse pressure gradient in free-flow isoelectric focusing.

    PubMed

    Dutta, Debashis

    2017-02-10

    Pressure-driven cross-flows can arise in free-flow isoelectric focusing systems (FFIEF) due to a non-uniform electroosmotic flow velocity along the channel width induced by the pH gradient in this direction. In addition, variations in the channel cross-section as well as unwanted differences in hydrostatic heads at the buffer/sample inlet ports can also lead to such pressure-gradients which besides altering the equilibrium position of the sample zones have a tendency to substantially broaden their widths deteriorating the separations. In this situation, a thorough assessment of stream broadening due to transverse pressure-gradients in FFIEF devices is necessary in order to establish accurate design rules for the assay. The present article describes a mathematical framework to estimate the noted zone dispersion in FFIEF separations based on the method-of-moments approach under laminar flow conditions. A closed-form expression has been derived for the spatial variance of the analyte streams at their equilibrium positions as a function of the various operating parameters governing the assay performance. This expression predicts the normalized stream variance under the chosen conditions to be determined by two dimensionless Péclet numbers evaluated based on the transverse pressure-driven and electrophoretic solute velocities in the separation chamber, respectively. Moreover, the analysis shows that while the stream width can be expected to increase with an increase in the value of the first Péclet number, the opposite trend will be followed with respect to the latter. The noted results have been validated using Monte Carlo simulations that also establish a time/length scale over which the predicted equilibrium stream width is attained in the system.

  12. Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing.

    PubMed

    Qiu, Fanghua; Hou, Tieying; Huang, Dehong; Xue, Zhifeng; Liang, Dongyan; Li, Qiuming; Lin, Weimiao

    2015-11-01

    Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high‑abundance proteins. Therefore, removing high‑abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high‑abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High‑resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 µg serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix‑assisted laser desorption ionization time‑of‑flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high‑abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research.

  13. Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.

    PubMed

    Schrötter, Sandra; Leondaritis, George; Eickholt, Britta J

    2016-05-06

    The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.

  14. Interfacing microchip isoelectric focusing with on-chip electrospray ionization mass spectrometry.

    PubMed

    Nordman, Nina; Laurén, Susanna; Kotiaho, Tapio; Franssila, Sami; Kostiainen, Risto; Sikanen, Tiina

    2015-06-12

    In this work, we demonstrate the interfacing of microchip capillary isoelectric focusing (cIEF) with online mass spectrometric (MS) detection via a fully integrated, on-chip sheath flow electrospray ionization (ESI) emitter. Thanks to the pH-dependent surface charge of the SU-8 polymer cIEF can be successfully run in native SU-8 microchannels without need for surface pretreatment prior to analysis. On the other hand, the inherent electroosmotic flow (EOF) taking place in SU-8 microchannels at high pH can be exploited to electrokinetic mobilization of the focused pH gradient toward the MS and no external pumps are required. In addition to direct coupling of a cIEF separation channel to an ESI emitter, we developed a two-dimensional separation chip for two-step, multiplex cIEF-transient-isotachophoretic (tITP) separation. In this case, cIEF is performed in the first dimension (effective L=20mm) and tITP in the second dimension (L=35mm) followed by ESI/MS. As a result, the migration order is affected by both the pI values (cIEF) and the intrinsic electrophoretic mobilities (tITP) of the sample components. The selectivity of the separation system was shown to be different from pure cIEF or pure ITP, which allows at best for baseline separation of two compounds with nearly identical pI values. The repeatabilities of the migration times of the two-step cIEF-tITP separation were 3.1-6.8% RSD (n=3). Thanks to the short separation channel, relatively short focusing times of 60-270s (depending on the applied focusing potential) were sufficient for establishment of the pH gradient and cIEF separation of the sample components, yielding total analysis times (including loading, focusing, and mobilization) well below 10min.

  15. The Biflow: an instrument for transfer-loop mediated, continuous, preparative-scale isoelectric trapping separations.

    PubMed

    Shave, Evan; Vigh, Gyula

    2007-07-01

    The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4

  16. Capillary isoelectric focusing-tandem mass spectrometry and reversed-phase liquid chromatography-tandem mass spectrometry for quantitative proteomic analysis of differentiating PC12 cells by eight-plex isobaric tags for relative and absolute quantification.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Keithley, Richard B; Dovichi, Norman J

    2013-08-06

    We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.

  17. The chitosan-gelatin (bio)polyelectrolyte complexes formation in an acidic medium.

    PubMed

    Voron'ko, Nicolay G; Derkach, Svetlana R; Kuchina, Yuliya A; Sokolan, Nina I

    2016-03-15

    The interaction of cationic polysaccharide chitosan and gelatin accompanied by the stoichiometric (bio)polyelectrolyte complexes formation has been studied by the methods of capillary viscometry, UV and FTIR spectroscopy and dispersion of light scattering. Complexes were formed in the aqueous phase, with pH being less than the isoelectric point of gelatin (pIgel). The particle size of the disperse phase increases along with the growth of the relative viscosity in comparison with sols of the individual components-polysaccharide and gelatin. Possible models and mechanism of (bio)polyelectrolyte complexes formation have been discussed. It was shown that the complex formation takes place not only due to the hydrogen bonds, but also due to the electrostatic interactions between the positively charged amino-groups of chitosan and negatively charged amino acid residues (glutamic Glu and aspartic Asp acids) of gelatin.

  18. Simultaneous Separation of Acidic and Basic Isoperoxidases in Wounded Potato Tissue by Acrylamide Gel Electrophoresis 1

    PubMed Central

    Borchert, Rolf; Decedue, Charles J.

    1978-01-01

    Preparation and use of a newly developed pH 4.3 horizontal thin layer acrylamide gel which permits the simultaneous separation of acidic and basic isoperoxidases in up to 30 samples is described. Use of cytochrome c, horseradish peroxidase, and a purified potato isoperoxidase as internal standards for a range in isoelectric points of peroxidases from pH 3 to 11 is introduced to facilitate comparison of results obtained with different materials and different methods. Distribution of tissue-specific isoperoxidases in different cell layers of wounded potato (Solanum tuberosum L.) tissue is shown and their purification described. Evidence for the in vitro degradation of basic potato isoperoxidases resulting in more acidic forms similar to isoperoxidases occurring in wounded potato tissue is presented. The significance of this observation for the postulated differential function of different isoperoxidases is discussed. ImagesFig. 1-3 PMID:16660608

  19. Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification.

    PubMed

    Horká, Marie; Karásek, Pavel; Salplachta, Jiří; Růžička, Filip; Vykydalová, Marie; Kubesová, Anna; Dráb, Vladimír; Roth, Michal; Slais, Karel

    2013-07-25

    In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.

  20. Optical absorption and DFT calculations in L-aspartic acid anhydrous crystals: Charge carrier effective masses point to semiconducting behavior

    NASA Astrophysics Data System (ADS)

    Silva, A. M.; Silva, B. P.; Sales, F. A. M.; Freire, V. N.; Moreira, E.; Fulco, U. L.; Albuquerque, E. L.; Maia, F. F., Jr.; Caetano, E. W. S.

    2012-11-01

    Density functional theory (DFT) computations within the local-density approximation and generalized gradient approximation in pure form and with dispersion correction (GGA+D) were carried out to investigate the structural, electronic, and optical properties of L-aspartic acid anhydrous crystals. The electronic (band structure and density of states) and optical absorption properties were used to interpret the light absorption measurements we have performed in L-aspartic acid anhydrous crystalline powder at room temperature. We show the important role of the layered spatial disposition of L-aspartic acid molecules in anhydrous L-aspartic crystals to explain the observed electronic and optical properties. There is good agreement between the GGA+D calculated and experimental lattice parameters, with (Δa, Δb, Δc) deviations of (0.029,-0.023,-0.024) (units in Å). Mulliken [J. Chem. Phys.JCPSA60021-960610.1063/1.1740588 23, 1833 (1955)] and Hirshfeld [Theor. Chim. ActaTCHAAM0040-574410.1007/BF00549096 44, 129 (1977)] population analyses were also performed to assess the degree of charge polarization in the zwitterion state of the L-aspartic acid molecules in the DFT converged crystal. The lowest-energy optical absorption peaks related to transitions between the top of the valence band and the bottom of the conduction band involve O 2p valence states and C 1p and O 2p conduction states, with the carboxyl and COOH lateral chain group contributing significantly to the energy band gap. Among the calculated band gaps, the lowest GGA+D (4.49-eV) gap is smaller than the experimental estimate of 5.02 eV, as obtained by optical absorption. Such a wide-band-gap energy together with the small carrier effective masses estimated from band curvatures allows us to suggest that an L-aspartic acid anhydrous crystal can behave as a wide-gap semiconductor. A comparison of effective masses among directions parallel and perpendicular to the L-aspartic molecules layers reveals that charge

  1. Low molecular weight bioactive peptides derived from the enzymatic hydrolysis of collagen after isoelectric solubilization/precipitation process of turkey by-products.

    PubMed

    Khiari, Zied; Ndagijimana, Maurice; Betti, Mirko

    2014-09-01

    A process based on the isoelectric solubilization/precipitation (ISP) method was developed to recover collagen from low value poultry by-products. The application of the ISP process to turkey heads generated protein isolates and an insoluble biomass that was used to extract collagen. Isolated turkey head collagen was then enzymatically hydrolyzed for different time periods using alcalase, flavorzyme, and trypsin. The enzymatic hydrolysis approaches consisted of digesting collagen with each one of the 3 enzymes alone (alcalase, flavorzyme, or trypsin), or one of the 3 combinations of 2 enzymes (alcalase/flavorzyme, alcalase/trypsin, or flavorzyme/trypsin), or a cocktail of all 3 enzymes together (alcalase/flavorzyme/trypsin). The molecular weight distribution of turkey head collagen hydrolysates was determined using size exclusion chromatography and matrix-assisted laser desorption ionization-time of flight-mass spectrometry. The enzyme cocktail produced collagen hydrolysates with the greatest amount of low molecular weight peptides ranging from 555.26 to 2,093.74 Da. These collagen peptides showed excellent solubility over a wide pH range (2 -: 8) and were able to bind cholic and deoxycholic acids and significantly (P < 0.05) inhibited plasma amine oxidase in a dose- and time-dependent manner. The ISP process combined with enzyme cocktail hydrolysis represents a potential new way to produce low molecular weight bioactive collagen peptides from low value poultry by-products.

  2. A multiplexed nucleic acid microsystem for point-of-care detection of HIV co-infection with MTB and PCP.

    PubMed

    Xu, Lingjia; Kong, Jilie

    2013-12-15

    Many individuals infected with the human immunodeficiency virus (HIV), especially children in African countries, die of co-infections with Mycobacterium tuberculosis (MTB) (coinfection rate: 50%) or Pneumocystis carinii pneumonia (PCP) (coinfection rate: 81%). The present proposal describes a rapid, portable, low-cost, multiplexed point-of-care diagnostic technique for simultaneously detecting HIV, MTB, and PCP. This technique incorporates a creative micro-device (hardware) and a loop-mediated isothermal amplification strategy (software).

  3. Systemic administration of 3-nitropropionic acid points out a different role for active caspase-3 in neurons and astrocytes.

    PubMed

    Duran-Vilaregut, Joaquim; Del Valle, Jaume; Manich, Gemma; Junyent, Fèlix; Camins, Antoni; Pallàs, Mercè; Pelegrí, Carme; Vilaplana, Jordi

    2010-02-01

    The intraperitoneal administration of 3-nitropropionic acid, which is commonly used to induce toxicity models of Huntington's disease in experimental animals, produces severe brain injury in the lateral part of the striatum. We studied the presence of active caspase-3 in neurons and astrocytes from brains of rats treated with 3-nitropropionic acid following a subacute administration protocol. Active caspase-3 was almost absent in the core of the striatal lesion. However, it was expressed, albeit weakly, in the neurons present in the rim of the lesion. In cortex and non-injured striatal areas, and in the cortex and striatum of control animals, active caspase-3 staining was widely distributed and vivid, but localized in the cell bodies of astrocytes rather than in neurons. In treated animals, some of the active caspase-3 positive neurons localized in the rim of the lesion were also positive for TUNEL staining. This indicates the presence of a caspase-mediated apoptotic process. TUNEL was not present in control animals or in the astrocytes of treated animals. Thus, the presence of active caspase-3 in astrocytes may be merely constitutive.

  4. A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics.

    PubMed

    Byrnes, Samantha; Fan, Andy; Trueb, Jacob; Jareczek, Francis; Mazzochette, Mark; Sharon, Andre; Sauer-Budge, Alexis F; Klapperich, Catherine M

    2013-07-07

    Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure.

  5. A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics

    PubMed Central

    Byrnes, Samantha; Fan, Andy; Trueb, Jacob; Jareczek, Francis; Mazzochette, Mark; Sharon, Andre; Sauer-Budge, Alexis F.; Klapperich, Catherine M.

    2013-01-01

    Many new and exciting portable HIV viral load testing technologies are emerging for use in global medicine. While the potential to provide fast, isothermal, and quantitative molecular diagnostic information to clinicians in the field will soon be a reality, many of these technologies lack a robust front end for sample clean up and nucleic acid preparation. Such a technology would enable many different downstream molecular assays. Here, we present a portable system for centrifuge-free room temperature nucleic acid extraction from small volumes of whole blood (70 µL), using only thermally stable reagents compatible with storage and transport in low resource settings. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of simulated samples demonstrate a lower limit of detection of 1000 copies/ml, with the ability to detect differences in viral load across four orders of magnitude. The system can also be used to store extracted RNA on detachable cartridges for up to one week at ambient temperature, and can be operated using only hand generated air pressure. PMID:23914255

  6. No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus

    SciTech Connect

    Oppenheim, A.; Horowitz, A.T.

    1981-08-01

    BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were stimated by the method of fiber-autoradiography and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcome virus-transformed BALB/c 3T3 cells.

  7. Influence of hydrophilic amino acids and GC-content on expression of recombinant proteins used in vaccines against foot-and-mouth disease virus in Escherichia coli.

    PubMed

    Wei, Hongfei; Fang, Mingli; Wan, Min; Wang, Hua; Zhang, Peiyin; Hu, Xiaoping; Wu, Xiuli; Yang, Ming; Zhang, Yongsheng; Zhou, Lei; Jiao, Chengfeng; Hua, Li; Diao, Wenzhen; Xiao, Yue; Yu, Yongli; Wang, Liying

    2014-04-01

    Epitope-based protein expression in Escherichia coli can be improved by adjusting its amino acid composition and encoding genes. To that end, we analyzed 24 recombinant epitope proteins (rEPs) that carry multiple epitopes derived from VP1 protein of foot-and-mouth disease virus. High level expression of the rEPs was attributed to a high content of Arg, Asn, Asp and Thr, a low content of Gln, Pro and Lys, a high content of hydrophilic amino acids and a higher isoelectric point value resulting from abundant Arg. It is also attributed to the appropriate guanine and cytosine content in the encoding genes. The data provide a reference for adjusting the amino acid composition in designing epitope-based proteins used in vaccines and for adjusting the synonymous codons to improve their expressions in E. coli.

  8. Enzymatic formation of a resorcylic acid by creating a structure-guided single-point mutation in stilbene synthase.

    PubMed

    Bhan, Namita; Li, Lingyun; Cai, Chao; Xu, Peng; Linhardt, Robert J; Koffas, Mattheos A G

    2015-02-01

    A novel C17 resorcylic acid was synthesized by a structure-guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl-CoA, to p-coumaroyl-CoA. The resulting novel pentaketide can be produced strictly by STS-like enzymes and not by Chalcone synthase-like type III polyketide synthases; due to the unique thioesterase like activity of STS-like enzymes. We utilized a liquid chromatography mass spectrometry-based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor-directed biosynthesis and identification of unnatural polyketides by structure-guided mutation of STS-like enzymes.

  9. Isoelectric focusing of Tamm-Horsfall glycoproteins: a simple tool for recognizing recurrent calcium oxalate renal stone formers.

    PubMed

    Schnierle, P; Hering, F; Seiler, H

    1996-01-01

    Tamm-Horsfall glycoproteins (THPs) from healthy probands and a majority of recurrent calcium oxalate renal stone formers reveal different physicochemical properties when analyzed using isoelectric focusing (IEF). The pI values of THPs from healthy probands are approximately 3.5 while THPs from recurrent renal stone formers have pI values of between 4.5 and 6. The two groups of THPs exhibit completely different protein patterns. The differences in IEF analysis allow differentiation between THPs from healthy probands and recurrent calcium oxalate stone formers and may possibly be used as a simple diagnostic method for the recognition of recurrent calcium oxalate renal stone formers.

  10. The formation of stable pH gradients with weak monovalent buffers for isoelectric focusing in free solution

    NASA Technical Reports Server (NTRS)

    Mosher, Richard A.; Thormann, Wolfgang; Graham, Aly; Bier, Milan

    1985-01-01

    Two methods which utilize simple buffers for the generation of stable pH gradients (useful for preparative isoelectric focusing) are compared and contrasted. The first employs preformed gradients comprised of two simple buffers in density-stabilized free solution. The second method utilizes neutral membranes to isolate electrolyte reservoirs of constant composition from the separation column. It is shown by computer simulation that steady-state gradients can be formed at any pH range with any number of components in such a system.

  11. Development of non-denaturing off-gel isoelectric focusing for the separation of uranium-protein complexes in fish.

    PubMed

    Bucher, Guillaume; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-05-01

    An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium-biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium-bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium-protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip.

  12. Recycling isoelectric focusing with computer controlled data acquisition system. [for high resolution electrophoretic separation and purification of biomolecules

    NASA Technical Reports Server (NTRS)

    Egen, N. B.; Twitty, G. E.; Bier, M.

    1979-01-01

    Isoelectric focusing is a high-resolution technique for separating and purifying large peptides, proteins, and other biomolecules. The apparatus described in the present paper constitutes a new approach to fluid stabilization and increased throughput. Stabilization is achieved by flowing the process fluid uniformly through an array of closely spaced filter elements oriented parallel both to the electrodes and the direction of the flow. This seems to overcome the major difficulties of parabolic flow and electroosmosis at the walls, while limiting the convection to chamber compartments defined by adjacent spacers. Increased throughput is achieved by recirculating the process fluid through external heat exchange reservoirs, where the Joule heat is dissipated.

  13. Effect of halide and acid additives on the direct synthesis of hydrogen peroxide using supported gold-palladium catalysts.

    PubMed

    Ntainjua N, Edwin; Piccinini, Marco; Pritchard, James C; Edwards, Jennifer K; Carley, Albert F; Moulijn, Jacob A; Hutchings, Graham J

    2009-01-01

    The effect of halide and acid addition on the direct synthesis of hydrogen peroxide is studied for magnesium oxide- and carbon-supported bimetallic gold-palladium catalysts. The addition of acids decreases the hydrogenation/decomposition of hydrogen peroxide, and the effect is particularly pronounced for the magnesium oxide-supported catalysts whilst for carbon-supported catalysts the pH requires close control to optimize hydrogen peroxide synthesis. The addition of bromide leads to a marked decrease in the hydrogenation/decomposition of hydrogen peroxide with either catalyst. These effects are discussed in terms of the structure of the gold-palladium alloy nanoparticles and the isoelectric point of the support. We conclude that with the highly active carbon-supported gold-palladium catalysts these additives are not required and that therefore this system presents the potential for the direct synthesis of hydrogen peroxide to be operated using green process technology.

  14. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    NASA Astrophysics Data System (ADS)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  15. Disposable dry-reagent cotton thread-based point-of-care diagnosis devices for protein and nucleic acid test.

    PubMed

    Mao, Xun; Du, Ting-E; Wang, Yiyun; Meng, Lili

    2015-03-15

    We report here for the first time by using dry-reagent cotton thread-based point-of-care diagnosis devices for low-cost, sensitive and rapid detection of a lung cancer related biomarker, squamous cell carcinoma antigen (SCCA) and a human genetic disease, hereditary tyrosinemia type I related DNA sequences. A model system comprising SCCA as an analyte and a pair of monoclonal antibodies is used to demonstrate the proof-of-concept on the dry-reagent cotton thread based immunoassay device. An enhancement protocol was employed by using two kinds of gold nanoparticle labels for SCCA test which greatly improved the sensitivity of the device. The assay avoids the multiple incubation and washing steps performed in most conventional protein analyses, which is similar with the lateral flow strip technology. Under optimal conditions, the thread based immunoassay device was capable of measuring 1ng/mL SCCA in 20min which meet the requirement for clinical diagnosis. DNA detection was successfully realized by using a novel adenosine based molecular beacon probe as reporter probes in the cotton thread based device, the linear range is 75-3000fmol which is suitable for quantitative test.

  16. Fat accumulation, fatty acids and melting point changes in broiler chick abdominal fat as affected by time of dietary fat feeding and slaughter age.

    PubMed

    Carmona, J M; Lopez-Bote, C J; Daza, A; Rey, A I

    2017-03-23

    1. This work aims to quantify changes in fatty acid profile, melting point, abdominal fat accumulation and 2-thiobarbituric acid-reactive substances production depending on dietary fat source and age at slaughter, and to estimate the optimal date for the change from an unsaturated fat to a saturated fat diet or vice versa. 2. Treatments established were (1) birds fed 8% tallow from 21 to 49 d (TTT); (2) birds fed 8% tallow from 21 to 37 d and 8% sunflower oil from d 38 to 49 (TSS); (3) birds fed 8% sunflower oil from 21 to 37 d and 8% tallow from d 38 to 49 (STT); (4) birds fed 8% sunflower oil from 21 to 41 d and 8% tallow from d 42 to 49 (SST); (5) birds fed 8% sunflower oil from 21 to 49 d (SSS). Birds from each group were slaughtered on d 21, 29, 38, 40, 42, 44, 46 and 49. 3. The polyunsaturated fatty acids (PUFAs) proportion in the SSS group reached maximum values at d 40 and fitted a quadratic response. This group also showed a decrease in saturated fatty acids (SATs) and monounsaturated fatty acids (MUFAs) of lower intensity than the PUFA increase. The highest synthesis of SAT + MUFA was found in the SSS and TSS groups, whereas these had the lowest body-to-dietary PUFA ratio. 4. A high and quadratic increase in the MUFA proportion was observed during the first 10 d of feeding with the tallow-enriched diet at the expenses of the proportion of PUFA that quadratically decreased (minimum values at d 38). 5. Lipogenic and desaturation capacity decreased with age. 6. The TSS group increased tissue PUFA content faster that the SST group decreased PUFA content after the change in diet which indicates that the earlier feeding has to be taken into consideration for obtaining higher or lower changes in quality parameters. 7. The melting point of the SSS group showed a lower response to the dietary treatment in the initial period when compared to the TTT treatment. 8. The TTT, STT, SST and TSS groups showed similar fat accumulation, and changes in lipid

  17. Detection of intrathecal immunoglobulin G synthesis by capillary isoelectric focusing immunoassay in oligoclonal band negative multiple sclerosis.

    PubMed

    Halbgebauer, Steffen; Huss, André; Buttmann, Mathias; Steinacker, Petra; Oeckl, Patrick; Brecht, Isabel; Weishaupt, Andreas; Tumani, Hayrettin; Otto, Markus

    2016-05-01

    Oligoclonal immunoglobulin G bands (OCBs) restricted to the cerebrospinal fluid indicate intrathecal inflammation. Using isoelectric focusing and immunoblotting, they are detected in about 95 % of patients with clinically definite multiple sclerosis (MS). To elucidate whether in the remaining 5 % OCBs are truly absent or alternatively missed due to insufficient sensitivity of the routine measurement, we employed a new, highly sensitive nanoscale method for OCB detection. Capillary isoelectric focusing followed by immunological detection served to analyze OCBs in 33 well-characterized OCB-negative and 10 OCB-positive MS patients as well as in 100 OCB-negative control patients with non-inflammatory neurological diseases and 30 OCB-positive control patients with inflammatory neurological diseases. We detected intrathecal immunoglobulin G production in 10 out of 33 MS patients (30 %), initially diagnosed as being OCB-negative, and in all 10 OCB-positive MS patients, but in only 3 out of 100 non-inflammatory neurological controls (3 %) and in 29 of 30 inflammatory neurological controls (97 %). At least about one-third of MS patients without intrathecal immunoglobulin G synthesis according to standard methods are OCB-positive. Advanced methods for OCB detection may increase the analytical sensitivity for detecting OCB in patients with MS who are OCB-negative according to current routine methods.

  18. Seizure control by derivatives of medium chain fatty acids associated with the ketogenic diet show novel branching-point structure for enhanced potency.

    PubMed

    Chang, Pishan; Zuckermann, Alexandra M E; Williams, Sophie; Close, Adam J; Cano-Jaimez, Marife; McEvoy, James P; Spencer, John; Walker, Matthew C; Williams, Robin S B

    2015-01-01

    The medium chain triglyceride (MCT) ketogenic diet is a major treatment of drug-resistant epilepsy but is problematic, particularly in adults, because of poor tolerability. Branched derivatives of octanoic acid (OA), a medium chain fat provided in the diet have been suggested as potential new treatments for drug-resistant epilepsy, but the structural basis of this functionality has not been determined. Here we investigate structural variants of branched medium chain fatty acids as new seizure-control treatments. We initially employ a series of methyl-branched OA derivatives, and using the GABAA receptor antagonist pentylenetetrazol to induce seizure-like activity in rat hippocampal slices, we show a strong, branch-point-specific activity that improves upon the related epilepsy treatment valproic acid. Using low magnesium conditions to induce glutamate excitotoxicity in rat primary hippocampal neuronal cultures for the assessment of neuroprotection, we also show a structural dependence identical to that for seizure control, suggesting a related mechanism of action for these compounds in both seizure control and neuroprotection. In contrast, the effect of these compounds on histone deacetylase (HDAC) inhibition, associated with teratogenicity, shows no correlation with therapeutic efficacy. Furthermore, small structural modifications of the starting compounds provide active compounds without HDAC inhibitory effects. Finally, using multiple in vivo seizure models, we identify potent lead candidates for the treatment of epilepsy. This study therefore identifies a novel family of fatty acids, related to the MCT ketogenic diet, that show promise as new treatments for epilepsy control and possibly other MCT ketogenic diet-responding conditions, such as Alzheimer disease.

  19. Isolation and characterization of two forms of an acidic bromelain stem proteinase.

    PubMed

    Harrach, T; Eckert, K; Maurer, H R; Machleidt, I; Machleidt, W; Nuck, R

    1998-05-01

    Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

  20. Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

    PubMed

    Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

    2016-12-01

    A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (Km = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere.

  1. Molecular systems pharmacology: isoelectric focusing signature of protein kinase Cδ provides an integrated measure of its modulation in response to ligands.

    PubMed

    Kedei, Noemi; Chen, Jin-Qiu; Herrmann, Michelle A; Telek, Andrea; Goldsmith, Paul K; Petersen, Mark E; Keck, Gary E; Blumberg, Peter M

    2014-06-26

    Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure-activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.

  2. Isoelectric focusing purity criteria and /sup 1/H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata

    SciTech Connect

    Constantinidis, I.; Satterlee, J.D.

    1987-12-01

    Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7 position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here the authors demonstrate that, for all of the Glycera momomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparation. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displays two bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handling prior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin. The NMR results show that two types of spectroscopic heterogeneity are also present in component II, and these are unrelated to the protein purity.

  3. Preliminary crystallographic study of an acidic phospholipase A2 from Ophiophagus hannah (king cobra).

    PubMed

    Xu, Sujuan; Gu, Lichuan; Wang, Qiuyan; Shu, Yuyan; Lin, Zhengjiong

    2002-10-01

    An acidic phospholipase A(2) (OH APLA(2)-II) with an isoelectric point (pI) of 4.0 was recently isolated from Ophiophagus hannah (king cobra) from Guangxi province, China. Comparison of this enzyme to a previously reported homologous phospholipase A(2) from the same venom shows that it lacks toxicity and exhibits a greater phospholipase activity. OH APLA(2)-II has been crystallized by the hanging-drop vapour-diffusion method using 1,6-hexanediol and magnesium chloride as precipitants. The crystal belongs to space group P6(3), with unit-cell parameters a = b = 98.06, c = 132.39 A. The diffraction data were collected under cryoconditions (100 K) and reduced to 2.1 A resolution. A molecular-replacement solution has been determined and shows that there are six molecules in one asymmetric unit.

  4. Amino acid rejection behaviour as a function of concentration.

    PubMed

    Shirley, Jason; Mandale, Stephen; Williams, Paul M

    2011-05-11

    The solute rejection versus concentration behaviour of five different amino acids has been investigated using a Nitto Denko NTR7450 nanofiltration membrane. The experimental data for amino acid rejection was also compared against a combined steric and charge rejection model. At its isoelectric point, lysine was effectively neutral and its behaviour was well described by the model incorporating a steric function only. For phenylalanine, the combined model was found to fit the data well. In contrast there was poor agreement between the model and rejection data for glutamine, glutamic acid and glycine whose rejection values at first increased with concentration. This result implied that another governing process was in operation. Dimerisation as an explanation for the observed phenomena was also investigated. Size analysis of amino acid molecules as a function of the prevailing concentration using dynamic light scattering was limited but showed no evidence of dimerisation. This data was supported by osmotic pressure measurements which demonstrated no evidence of non-linearity in the relation between osmotic pressure and concentration.

  5. Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy.

    PubMed

    Goodacre, R; Shann, B; Gilbert, R J; Timmins, E M; McGovern, A C; Alsberg, B K; Kell, D B; Logan, N A

    2000-01-01

    Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong to Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (including Bacillus niger and Bacillus globigii), Bacillus sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier-transform infrared spectroscopy (FT-IR). Chemometric methods based on rule induction and genetic programming were used to determine the physiological state (vegetative cells or spores) correctly, and these methods produced mathematical rules which could be simply interpreted in biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in vegetative cells; this was confirmed using pyrolysis-gas chromatography/mass spectrometry. In addition, a pyridine ring vibration at 1447-1439 cm-1 from DPA was found to be highly characteristic of spores in FT-IR analysis. Thus, although the original data sets recorded hundreds of spectral variables from whole cells simultaneously, a simple biomarker can be used for the rapid and unequivocal detection of spores of these organisms.

  6. Transcriptome and proteome profiling of colon mucosa from quercetin fed F344 rats point to tumor preventive mechanisms, increased mitochondrial fatty acid degradation and decreased glycolysis.

    PubMed

    Dihal, Ashwin A; van der Woude, Hester; Hendriksen, Peter J M; Charif, Halima; Dekker, Lennard J; Ijsselstijn, Linda; de Boer, Vincent C J; Alink, Gerrit M; Burgers, Peter C; Rietjens, Ivonne M C M; Woutersen, Ruud A; Stierum, Rob H

    2008-01-01

    Quercetin has been shown to act as an anticarcinogen in experimental colorectal cancer (CRC). The aim of the present study was to characterize transcriptome and proteome changes occurring in the distal colon mucosa of rats supplemented with 10 g quercetin/kg diet for 11 wk. Transcriptome data analyzed with Gene Set Enrichment Analysis showed that quercetin significantly downregulated the potentially oncogenic mitogen-activated protein kinase (Mapk) pathway. In addition, quercetin enhanced expression of tumor suppressor genes, including Pten, Tp53, and Msh2, and of cell cycle inhibitors, including Mutyh. Furthermore, dietary quercetin enhanced genes involved in phase I and II metabolism, including Fmo5, Ephx1, Ephx2, and Gpx2. Quercetin increased PPARalpha target genes, and concomitantly enhanced expression of genes involved in mitochondrial fatty acid (FA) degradation. Proteomics performed in the same samples revealed 33 affected proteins, of which four glycolysis enzymes and three heat shock proteins were decreased. A proteome-transcriptome comparison showed a low correlation, but both pointed out toward altered energy metabolism. In conclusion, transcriptomics combined with proteomics showed that dietary quercetin evoked changes contrary to those found in colorectal carcinogenesis. These tumor-protective mechanisms were associated with a shift in energy production pathways, pointing at decreased cytoplasmic glycolysis and toward increased mitochondrial FA degradation.

  7. A novel acid-stable, acid-active beta-galactosidase potentially suited to the alleviation of lactose intolerance.

    PubMed

    O'Connell, Shane; Walsh, Gary

    2010-03-01

    Extracellular beta-galactosidase produced by a strain of Aspergillus niger van Tiegh was purified to homogeneity using a combination of gel filtration, ion-exchange, chromatofocusing, and hydrophobic interaction chromatographies. The enzyme displayed a temperature optimum of 65 degrees C and a low pH optimum of between 2.0 and 4.0. The monomeric glycosylated enzyme displayed a molecular mass of 129 kDa and an isoelectric point of 4.7. Protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme shares homology with a previously sequenced A. niger beta-galactosidase. Unlike currently commercialised products, the enzyme displayed a high level of stability when exposed to simulated gastric conditions in vitro, retaining 68+/-2% of original activity levels. This acid-stable, acid-active beta-galactosidase was formulated, along with a neutral beta-galactosidase from Kluyveromyces marxianus DSM5418, in a novel two-segment capsule system designed to ensure delivery of enzymes of appropriate physicochemical properties to both stomach and small intestine. When subjected to simulated full digestive tract conditions, the twin lactase-containing capsule hydrolyzed, per unit activity, some 3.5-fold more lactose than did the commercial supplemental enzyme. The acid-stable, acid-active enzyme, along with the novel two-segment delivery system, may prove beneficial in the more effective treatment of lactose intolerance.

  8. Plasma polymerized allylamine coated quartz particles for humic acid removal.

    PubMed

    Jarvis, Karyn L; Majewski, Peter

    2012-08-15

    Allylamine plasma polymerization has been used to modify the surface of quartz particles for humic acid removal via an inductively coupled rotating barrel plasma reactor. Plasma polymerized allylamine (ppAA) films were deposited at a power of 25 W, allylamine flow rate of 4.4 sccm and polymerization times of 5-60 min. The influence of polymerization time on surface chemistry was investigated via X-ray photoelectron spectroscopy (XPS), time of flight secondary ion mass spectrometry (ToF-SIMS) and electrokinetic analysis. Acid orange 7 adsorption/desorption quantified the number of surface amine groups. Humic acid removal via ppAA quartz particles was examined by varying pH, removal time, humic acid concentration, and particle mass. Increasing the polymerization time increased the concentration of amine groups on the ppAA quartz surface, thus also increasing the isoelectric point. ToF-SIMS demonstrated uniform distribution of amine groups across the particle surface. Greatest humic acid removal was observed at pH 5 due to electrostatic attraction. At higher pH values, for longer polymerization times, humic acid removal was also observed due to hydrogen bonding. Increasing the initial humic acid concentration increased the mass of humic acid removed, with longer polymerization times exhibiting the greatest increases. Plasma polymerization using a rotating plasma reactor has shown to be a successful method for modifying quartz particles for the removal of humic acid. Further development of the plasma polymerization process and investigation of additional contaminants will aid in the development of a low cost water treatment system.

  9. Biomimetic synthesis of highly biocompatible gold nanoparticles with amino acid-dithiocarbamate as a precursor for SERS imaging

    NASA Astrophysics Data System (ADS)

    Li, Li; Liu, Jianbo; Yang, Xiaohai; Huang, Jin; He, Dinggeng; Guo, Xi; Wan, Lan; He, Xiaoxiao; Wang, Kemin

    2016-03-01

    Amino acid-dithiocarbamate (amino acid-DTC) was developed as both the reductant and ligand stabilizer for biomimetic synthesis of gold nanoparticles (AuNPs), which served as an excellent surface-enhanced Raman scattering (SERS) contrast nanoprobe for cell imaging. Glycine (Gly), glutamic acid (Glu), and histidine (His) with different isoelectric points were chosen as representative amino acid candidates to synthesize corresponding amino acid-DTC compounds through mixing with carbon disulfide (CS2), respectively. The pyrogenic decomposition of amino acid-DTC initiated the reduction synthesis of AuNPs, and the strong coordinating dithiocarbamate group of amino acid-DTC served as a stabilizer that grafted onto the surface of the AuNPs, which rendered the as-prepared nanoparticles a negative surface charge and high colloidal stability. MTT cell viability assay demonstrated that the biomimetic AuNPs possessed neglectful toxicity to the human hepatoma cell, which guaranteed them good biocompatibility for biomedical application. Meanwhile, the biomimetic AuNPs showed a strong SERS effect with an enhancement factor of 9.8 × 105 for the sensing of Rhodamine 6G, and two distinct Raman peaks located at 1363 and 1509 cm-1 could be clearly observed in the cell-imaging experiments. Therefore, biomimetic AuNPs can be explored as an excellent SERS contrast nanoprobe for biomedical imaging, and the amino acid-DTC mediated synthesis of the AuNPs has a great potential in bio-engineering and biomedical imaging applications.

  10. Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients

    NASA Technical Reports Server (NTRS)

    Knisley, Keith A.; Rodkey, L. Scott

    1988-01-01

    Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific antitetanus or antimicrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

  11. Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

  12. Orosomucoid typing of apes (family Pongidae) by isoelectric focusing: Among primates do only humans have two functional orosomucoid loci

    SciTech Connect

    Yuasa, I. ); Umetsu, K. ); Udono, T.; Sasaoka, S. ); Suzuki, R.; Shotake, T.; Kawamoto, Y.; Takenaka, O.; Nozawa, K. )

    1991-12-01

    It has been demonstrated that human orosomucoid (ORM) is controlled by more than one functional loci, while Macaca ORM is controlled by one locus. To examine the time when the ORM gene was duplicated in the evolution of primates, plasma samples from 118 apes (family Pongidae) belonging to 4 genera and 12 species were investigated for ORM polymorphism using isoelectric focusing followed by immunoprinting. The band patterns of ORM in the subfamily Ponginae showed quantitatively different products as in humans. A pedigree study of common chimpanzees supported the two-locus model for ORM. Gibbons (subfamily Hylobatinae) displayed highly variable band patterns, but the number of loci was not determined unequivocally. Thus, this study shows that duplication of the ORM gene in primates occurred either before or after the divergence of hylobatinae and Ponginae, consistent with a previous prediction from the molecular evolutionary rate of ORM.

  13. Acousto-optical deflection-based whole channel scanning for microchip isoelectric focusing with laser-induced fluorescence detection.

    PubMed

    Sanders, J C; Huang, Z; Landers, J P

    2001-12-01

    This paper describes the development of a technique amenable to the separation of proteins on a microchip by isoelectric focusing (IEF) with entire channel scanning laser-induced fluorescence detection using acousto-optical deflection (AOD). The ability to use AOD to scan the portions of or the entire length of an IEF separation channel allows for high-speed analysis since the mobilization step is circumvented with this technique. Employing no moving parts eliminates mechanical noise and, not only is there no loss of resolution, AOD scanning can potentially increase resolution. The ability of AOD to provide ultra-fast scanning rates (kHz timescale) allows for real-time imaging of the focusing process. This is demonstrated with the separation of naturally fluorescent proteins using entire channel (total scanning range of 2.4 cm) AOD-mediated scanning laser-induced fluorescence detection.

  14. Amino acid composition of two masticatory nuts (Cola acuminata and Garcinia kola) and a snack nut (Anacardium occidentale).

    PubMed

    Adeyeye, E I; Asaolu, S S; Aluko, A O

    2007-06-01

    The amino acid compositions of Cola acuminata, Garcinia kola and Anacardium occidentale were evaluated by ion-exchange chromatography. Glutamic acid was the most concentrated acid in the samples. In all the amino acids determined, A. occidentale had the most concentrated acid on a pairwise basis. The total amino acids were 356.24 mg/g protein, 112.90 mg/g protein and 659.17 mg/g protein for C. acuminata, G. kola and A. occidentale, respectively. The percentage total essential amino acids were 38.39% (C. acuminata), 47.05% (G. kola) and 51.04% (A. occidentale). Also the percentage total acidic amino acids were 38.16% (C. acuminata), 30.61% (G. kola) and 30.35% (A. occidentale). The calculated isoelectric points were 2.0 (C. acuminata), 0.7 (G. kola) and 3.9 (A. occidentale), showing they can all be precipitated at acidic pH. While threonine was the limiting amino acid in A. occidentale, it was valine in both C. acuminata and G. kola. The percentage cystine (Cys) levels in the total sulphur amino acid were 44.27% (C. acuminata), 37.75% (G. kola) and 50.51% (A. occidentale). The aim of this work was to compare the amino acid profile of the samples. It is recommended that C. acuminata and G. kola consumption be avoided by ulcer patients because of their high levels of acidic amino acids. A. occidentale amino acid scores ranged from 42% to 127%, suggesting that it could be used to enhance the protein quality of cereals through food complementation.

  15. Adsorption of Aspartic Acid onto Rutile: Implications for Biochirality

    NASA Astrophysics Data System (ADS)

    Estrada, C. F.; Jonsson, C. M.; Jonsson, C. L.; Sverjensky, D. A.; Hazen, R. M.

    2008-12-01

    Mineral surfaces may have facilitated the concentration and polymerization of simple biomolecules into macromolecules while promoting the development of biochirality. In this study, rutile and aspartic acid (Asp) were investigated as a possible system in this scenario. Batch adsorption experiments were performed to examine the adsorption of Asp as a function of total concentration and pH. A constant background electrolyte of 0.1 M NaCl was applied to the system, and all solutions were purged with argon gas to eliminate carbon dioxide contamination. Asp adsorbs onto rutile to the highest extent over the pH range 3-5.5 suggesting that an acidic environment is required for the adsorption between Asp and rutile to occur in significant amounts. This pH range of maximum adsorption is constrained between the isoelectric point of Asp and the point of zero charge of rutile, which indicates that electrostatic effects are influencing Asp adsorption. Both the L- and D- enantiomers of Asp were individually adsorbed onto the rutile surface to determine the potential of the system for chiral selection. Preliminary results indicate that D-Asp may possibly adsorb in greater amounts than L-Asp at higher Asp total concentrations. This trend is unexpected as the growth planes dominating the rutile are achiral, and a more thorough study is required to validate this difference in adsorption. Nevertheless, this result may provide insight on the emergence of chiral selection in macromolecules within what might be a predominantly achiral prebiotic system.

  16. Western Blot Analysis of the Exotoxins Components from Bacillus anthracis Separated by Isoelectric Focusing Gel Electrophoresis

    DTIC Science & Technology

    2004-07-01

    Biochemical and Biophysical Research Communications 317 (2004) 294–300 BBRC www.elsevier.com/locate/ybbrcWestern blot analysis...pag iden- tified five different point mutations from 26 different S.F. Little / Biochemical and Biophysical Research Communications 317 (2004) 294...identified as Iowa, it has subsequently been identified as Texas. 296 S.F. Little / Biochemical and Biophysical Research Communications 317 (2004)

  17. Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

    PubMed

    Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

    2012-01-01

    We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

  18. Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides.

    PubMed

    Chen, Lei; Zhu, Shusheng; Lu, Xiaohong; Pang, Zhili; Cai, Meng; Liu, Xili

    2012-01-01

    The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

  19. Predicting the Effect of Amino Acid Single-Point Mutations on Protein Stability-Large-Scale Validation of MD-Based Relative Free Energy Calculations.

    PubMed

    Steinbrecher, Thomas; Zhu, Chongkai; Wang, Lingle; Abel, Robert; Negron, Christopher; Pearlman, David; Feyfant, Eric; Duan, Jianxin; Sherman, Woody

    2017-04-07

    The stability of folded proteins is critical to their biological function and for the efficacy of protein therapeutics. Predicting the energetic effects of protein mutations can improve our fundamental understanding of structural biology, the molecular basis of diseases, and possible routes to addressing those diseases with biological drugs. Identifying the effect of single amino acid point mutations on the thermodynamic equilibrium between the folded and unfolded states of a protein can pinpoint residues of critical importance that should be avoided in the process of improving other properties (affinity, solubility, viscosity, etc.) and suggest changes at other positions for increasing stability in protein engineering. Multiple computational tools have been developed for in silico predictions of protein stability in recent years, ranging from sequence-based empirical approaches to rigorous physics-based free energy methods. In this work, we show that FEP+, which is a free energy perturbation method based on all-atom molecular dynamics simulations, can provide accurate thermal stability predictions for a wide range of biologically relevant systems. Significantly, the FEP+ approach, while originally developed for relative binding free energies of small molecules to proteins and not specifically fitted for protein stability calculations, performs well compared to other methods that were fitted specifically to predict protein stability. Here, we present the broadest validation of a rigorous free energy-based approach applied to protein stability reported to date: 700+ single-point mutations spanning 10 different protein targets. Across the entire data set, we correctly classify the mutations as stabilizing or destabilizing in 84% of the cases, and obtain statistically significant predictions as compared with experiment [average error of ~1.6kcal/mol and coefficient of determination (R(2)) of 0.40]. This study demonstrates, for the first time in a large

  20. Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis.

    PubMed

    Németh, Krisztina; Domonkos, Celesztina; Sarnyai, Virág; Szemán, Julianna; Jicsinszky, László; Szente, Lajos; Visy, Júlia

    2014-10-01

    The resolution power of permethylated 6-monoamino-6-monodeoxy-βCD (PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-AAs by varying pH and selector concentrations were investigated and correlated with their structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Chiral discrimination ability of PMMABCD was demonstrated by the separation of an artificial mixture of 8 Dns-AA pairs.

  1. Comparative Study of Novel Ratio Spectra and Isoabsorptive Point Based Spectrophotometric Methods: Application on a Binary Mixture of Ascorbic Acid and Rutin

    PubMed Central

    Darwish, Hany W.; Bakheit, Ahmed H.; Naguib, Ibrahim A.

    2016-01-01

    This paper presents novel methods for spectrophotometric determination of ascorbic acid (AA) in presence of rutin (RU) (coformulated drug) in their combined pharmaceutical formulation. The seven methods are ratio difference (RD), isoabsorptive_RD (Iso_RD), amplitude summation (A_Sum), isoabsorptive point, first derivative of the ratio spectra (1DD), mean centering (MCN), and ratio subtraction (RS). On the other hand, RU was determined directly by measuring the absorbance at 358 nm in addition to the two novel Iso_RD and A_Sum methods. The work introduced in this paper aims to compare these different methods, showing the advantages for each and making a comparison of analysis results. The calibration curve is linear over the concentration range of 4–50 μg/mL for AA and RU. The results show the high performance of proposed methods for the analysis of the binary mixture. The optimum assay conditions were established and the proposed methods were successfully applied for the assay of the two drugs in laboratory prepared mixtures and combined pharmaceutical tablets with excellent recoveries. No interference was observed from common pharmaceutical additives. PMID:26885440

  2. Adsorption of dissolved aluminum on sapphire-c and kaolinite: implications for points of zero charge of clay minerals

    PubMed Central

    2014-01-01

    We have studied the impact of dissolved aluminum on interfacial properties of two aluminum bearing minerals, corundum and kaolinite. The effect of intentionally adding dissolved aluminum on electrokinetic potential of basal plane surfaces of sapphire was studied by streaming potential measurements as a function of pH and was complemented by a second harmonic generation (SHG) study at pH 6. The electrokinetic data show a similar trend as the SHG data, suggesting that the SHG electric field correlates to zeta-potential. A comparable study was carried out on kaolinite particles. In this case electrophoretic mobility was measured as a function of pH. In both systems the addition of dissolved aluminum caused significant changes in the charging behavior. The isoelectric point consistently shifted to higher pH values, the extent of the shift depending on the amount of aluminum present or added. The experimental results imply that published isoelectric points of clay minerals may have been affected by this phenomenon. The presence of dissolved aluminum in experimental studies may be caused by particular pre-treatment methods (such as washing in acids and subsequent adsorption of dissolved aluminum) or even simply by starting a series of measurements from extreme pH (causing dissolution), and subsequently varying the pH in the very same batch. This results in interactions of dissolved aluminum with the target surface. A possible interpretation of the experimental results could be that at low aluminum concentrations adatoms of aluminum (we will refer to adsorbed mineral constituents as adatoms) can form at the sapphire basal plane, which can be rather easily removed. Simultaneously, once the surface has been exposed to sufficiently high aluminum concentration, a visible change of the surface is seen by AFM which is attributed to a surface precipitate that cannot be removed under the conditions employed in the current study. In conclusion, whenever pre-treatment or the

  3. Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans.

    PubMed Central

    Hansen, M T

    1980-01-01

    Four proteins, alpha beta, gamma, and delta, preferentially synthesized in ultraviolet light-treated cells of Micrococcus radiodurans, were characterized in terms of their molecular weights and isoelectric points. Within the sublethal-dose range, the differential rate of synthesis for these proteins increased linearly with the inducing UV dose. The degree of induction reached 100-fold, and the most abundant protein beta, amounted to approximately 2% of the total newly synthesized protein after irradiation. Damage caused by ionizing radiation or by treatment with mitomycin C also provoked the synthesis of the four proteins. The proportions between the individual proteins, however, varied strikingly with the damaging agent. In contrast to treatments which introduced damage in the cellular deoxyribonucleic acid, the mere arrest of deoxyribonucleic acid replication, caused by nalidixic acid or by starvation for thymine, failed to elicit the synthesis of either protein. Repair of deoxyribonucleic acid damage requires that a number of versatile and efficient processes by employed. It is proposed that the induced proteins participate in deoxyribonucleic acid repair in M. radiodurans. Mechanisms are discussed which would allow a differentiated cellular response to damages of sufficiently distinctive nature. Images PMID:7354007

  4. A review of the different techniques for solid surface acid-base characterization.

    PubMed

    Sun, Chenhang; Berg, John C

    2003-09-18

    In this work, various techniques for solid surface acid-base (AB) characterization are reviewed. Different techniques employ different scales to rank acid-base properties. Based on the results from literature and the authors' own investigations for mineral oxides, these scales are compared. The comparison shows that Isoelectric Point (IEP), the most commonly used AB scale, is not a description of the absolute basicity or acidity of a surface, but a description of their relative strength. That is, a high IEP surface shows more basic functionality comparing with its acidic functionality, whereas a low IEP surface shows less basic functionality comparing with its acidic functionality. The choice of technique and scale for AB characterization depends on the specific application. For the cases in which the overall AB property is of interest, IEP (by electrokinetic titration) and H(0,max) (by indicator dye adsorption) are appropriate. For the cases in which the absolute AB property is of interest such as in the study of adhesion, it is more pertinent to use chemical shift (by XPS) and the heat of adsorption of probe gases (by calorimetry or IGC).

  5. Potential of capillary zone electrophoresis for estimation of humate acid-base properties.

    PubMed

    Vanifatova, Natalia G; Zavarzina, Anna G; Spivakov, Boris Ya

    2008-03-07

    Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and

  6. Mercury distribution and lipid oxidation in fish muscle: Effects of washing and isoelectric protein precipitation

    USGS Publications Warehouse

    Gong, Y.; Krabbenhoft, D.P.; Ren, L.; Egelandsdal, B.; Richards, M.P.

    2011-01-01

    Nearly all the mercury (Hg) in whole muscle from whitefish (Coregonus clupeaformis) and walleye (Sander vitreus) was present as methyl mercury (MeHg). The Hg content in whole muscle from whitefish and walleye was 0.04-0.09 and 0.14-0.81 ppm, respectively. The myofibril fraction contained approximately three-fourths of the Hg in whitefish and walleye whole muscle. The sarcoplasmic protein fraction (e.g., press juice) was the next most abundant source of Hg. Isolated myosin, triacylglycerols, and cellular membranes contained the least Hg. Protein isolates prepared by pH shifting in the presence of citric acid did not decrease Hg levels. Addition of cysteine during washing decreased the Hg content in washed muscle probably through the interaction of the sulfhydryl group in cysteine with MeHg. Primary and secondary lipid oxidation products were lower during 2 ??C storage in isolates prepared by pH shifting compared to those of washed or unwashed mince from whole muscle. This was attributed to removing some of the cellular membranes by pH shifting. Washing the mince accelerated lipid peroxide formation but decreased secondary lipid oxidation products compared to that of the unwashed mince. This suggested that there was a lipid hydroperoxide generating system that was active upon dilution of aqueous antioxidants and pro-oxidants. ?? 2011 American Chemical Society.

  7. Characterization of human papillomavirus by capillary isoelectric focusing with whole-column imaging detection.

    PubMed

    Zhou, Chao-Ming

    2013-11-01

    CIEF with whole-column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF-WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF-WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF-WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single-point detection. In the example, the five HPVs displayed pI values of 8.43 ± 0.06 (n = 10; HPV 6L1), 8.70 ± 0.04 (n = 10; HPV 11L1), 7.94 ± 0.05 (n = 18; HPV 16L1), 7.57 ± 0.04 (n = 18; HPV 18L1), and 8.45 ± 0.05 (n = 10; HPV 58L1). This CIEF-WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry.

  8. The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids.

    PubMed

    Guidon, P T; Hightower, L E

    1986-08-01

    A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.

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

    PubMed

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

    2014-10-01

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

  10. High-Resolution Capillary Isoelectric Focusing of Proteins Using Highly Hydrophilic-Substituted Cellulose-Coated Capillaries

    SciTech Connect

    Shen, Yufeng ); Smith, Richard D. )

    1999-12-01

    Highly hydrophilic group-substituted celluloses were immobilized on the fused silica capillary inner wall for capillary isoelectric focusing (CIEF) of proteins. The resultant capillaries were evaluated under typical CIEF conditions, and the results were compared with those obtained by using polyvinyl alcohol (PVA)- and linear polyacrylamide (LPA)-coated capillaries. A minimal resolved pI difference of 0.013 was obtained with a carrier ampholyte (pH 3-10) for hydroxyethyl cellulose- and hydroxypropyl cellulose-coated capillaries, while 0.019 was obtained on PVA- and LPA coated capillaries. Changing substituted groups from hydroxyl to hydroxylmethyl decreased the CIEF resolution from {approx}2.5 to {approx}1.0 for tested proteins at mobilization speed of {approx}0.9 cm min-1. Little loss in CIEF resolution was observed when increasing capillary length from 30 to 150 cm, however, flat peaks were observed when increasing column diameter up to >100 um. The present work indicates that mobilization speed of&l t;1 cm min-1 should be used to achieve high CIEF resolution.

  11. Steady-state protein focusing in carrier ampholyte-based isoelectric focusing: Part II-validation and case studies.

    PubMed

    Shim, Jaesool; Yoo, Kisoo; Dutta, Prashanta

    2017-03-01

    In this study, we systematically investigate the validity and applicability of an analytical model developed for carrier ampholyte-based isoelectric focusing (IEF). Three different IEF cases are considered in order to evaluate the efficacy of the approximate analytical results by comparison with high-resolution computer simulations. In the first case, three proteins are separated in a narrow pH range (6-9) by using 50 carrier ampholytes. In the second and third cases, the separation of proteins is studied in broad pH range (3-10) IEF by using 100 carrier ampholytes. Results obtained from the approximate analytical models are in very good agreement with the numerical results for IEF separation of cardiac troponin I, albumin, and hemoglobin in both narrow and broad pH ranges. The sensitivity of the analytical model is also tested for different initial mass ratios of proteins to ampholytes. No appreciable differences are observed between the approximate analytical and numerical results within the mass ratio range studied. The effect of a nominal electric field and/or a nominal pH gradient on protein focusing is also examined to demonstrate the effectiveness of the analytical model. Our results indicate that the use of both nominal electric field and pH gradient will result in erroneous peak concentrations for proteins. Finally, we describe the limitations of the approximate analytical solutions.

  12. Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.

    PubMed

    Wagner, Leona; Wermann, Michael; Rosche, Fred; Rahfeld, Jens-Ulrich; Hoffmann, Torsten; Demuth, Hans-Ulrich

    2011-07-01

    Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

  13. Detection of recombinant human erythropoietin in urine for doping analysis: interpretation of isoelectric profiles by discriminant analysis.

    PubMed

    Lasne, Françoise; Thioulouse, Jean; Martin, Laurent; de Ceaurriz, Jacques

    2007-06-01

    The detection in urine of recombinant human erythropoietin (rHuEPO), a hormone misused by endurance athletes as a doping agent, is based on the differentiation of its isoelectric pattern from that of the corresponding natural hormone. Different empirical criteria have been proposed for discriminating the images of the patterns but none of them have been elaborated from a rational statistical approach. Discriminant analysis was applied to a dataset of profiles defined as positive (116 profiles from 26 subjects) (presence of rHuEPO and possibly residual natural endogenous hormone) and negative (131 profiles from 131 subjects) (presence of natural endogenous hormone only). The different bands were numbered according to a template of 16 possible positions and their relative intensities constituted the 16 variables of the statistical analysis. This method was then tested with data from an administration trial of low doses (6.7-10 IU/kg) following high-dose (265 IU/kg) injections (71 profiles from one subject). The analysis of the dataset clearly separated the negative and positive profiles. A cross-validation procedure confirmed that the analysis was extremely stable: with ten-fold cross-validation, no false positives were observed even with 100,000 simulations. Furthermore, the detection of rHuEPO in the profiles from the low-dose trial was greatly improved in comparison with a previously validated empirical criterion.

  14. Analysis of hemoglobin derivatives by capillary isoelectric focusing and its application in the antidotal research of cyanide poisoning

    SciTech Connect

    Shih, M.L.; Korte, W.D.

    1996-07-01

    Cyanide toxicity can be reduced by the use of methemoglobin (MetHb) formers, and antidotal dosage is based on the extent of MetHb formation. Hemoglobin and ferrihemoglobin (MetHb, hemimethemoglobins {alpha}{sup 3+}{beta}{sup 2+} and {alpha}{sup 2-}{beta}{sup 3-}, tetracyanmethemoglobin, and dicyanmethemoglobin) concentrations in human, pig, and mouse blood were determined after separation by isoelectric focusing with an octyl-bonded capillary. The predominant species formed in blood when MetHb formers, such as potassium ferricyanide, hydroxylamine, sodium nitrite, and 4-dimethylaminophenol (DMAP), added at molar ratios ranging from 1:10 to 1:1 to hemoglobin, are the valency hybrid intermediates {alpha}{sup 3+}{beta}{sup 2+} and {alpha}{sup 2+}{beta}{sup 3+}. In the detoxication of cyanide with methemoglobin, an intermediate dicyanhemimethemoglobin was demonstrated to be the predominant species in the formation of tetracyanmethemoglobin. Complex mixtures of hemoglobin derivatives were observed with DMAP at 1:1 or greater molar ratio to hemoglobin. Comparison of the MetHb values obtained with a hemoxometer indicated that the valency hybrids were measured as MetHb and the values of oxidized hemoglobin were overestimated. In cyanide poisoning, incorrect dosages of MetHb formers could be calculated, and misinterpretation of MetHb data would results from methods that fail to discriminate among the various species of MetHb. 24 refs., 4 figs., 2 tabs.

  15. Combination of micropreparative solution isoelectric focusing and high-performance liquid chromatography for differentiation of biofilm-positive and biofilm-negative Candida parapsilosis group from vascular catheter.

    PubMed

    Vykydalová, Marie; Horká, Marie; Růžička, Filip; Duša, Filip; Moravcová, Dana; Kahle, Vladislav; Slais, Karel

    2014-02-17

    This study utilizes the high-performance liquid chromatography technique in combination with the new micropreparative solution isoelectric focusing fractionation on non-woven fabric strip for the characterization and differentiation of biofilm-positive and biofilm-negative forms of Candida parapsilosis sensu stricto on the basis of the changes in the composition of their cell-surface. Treatment of yeasts by boiling in distilled water relased surface substances from yeasts cells. Consequently, the optimized procedure has been used for fast identification of the highly pathogenic biofilm-positive Candida parapsilosis group in real clinical material - sonicate from vascular catheters. Moreover, the capillary isoelectric focusing was used as supporting and control technique. Obtained results suggest that this new method can be used to distinguish between biofilm-positive and negative forms of Candida parapsilosis sensu stricto.

  16. Nanoliter-volume protein enrichment, tryptic digestion, and partial separation based on isoelectric points by CE for MALDI mass spectral analysis.

    PubMed

    Nesbitt, Chandra A; Jurcic, Kristina; Yeung, Ken K-C

    2008-01-01

    Sequence-specific proteolysis is an important part of protein identification by MS. Digestion of protein is commonly performed in-solution, in sample vials with volumes ranging from milli- to microliters. When digestion is performed with a sample volume below 1 microL, handling of solution and potential sample loss via adsorption become significant issues. In this report, a proof of concept for the digestion of a small volume protein solution inside a capillary was demonstrated using a discontinuous buffer system previously studied (Nesbitt, C. A., et al. J. Chromatogr. A 2005, 1073, 175-180). Upon voltage application, a pH junction was created by the discontinuous buffer. Using myoglobin as an example, the protein molecules were enriched at the junction with an estimated volume of a few nanoliters. A protease, trypsin, was then introduced to myoglobin at the junction by coenrichment to induce in-capillary digestion. The voltage application was then suspended to provide the necessary time (2 h) for the proteolysis to proceed. When completed, voltage application was resumed, and the discontinuous buffer reconcentrated the peptides formed from digestion. Importantly, the refocused peptides appeared to roughly elute according to their pIs, resulting in a partial separation. Direct sample deposition from capillary was performed to facilitate mass spectral analysis by MALDI. The partial separation, according to pI, offered the potential benefits of MALDI MS signal enhancement and provided supplementary pI information for peptide identity assignment.

  17. Side-by-side comparison of disposable microchips with commercial capillary cartridges for application in capillary isoelectric focusing with whole column imaging detection.

    PubMed

    Liu, Zhen; Ou, Junjie; Samy, Razim; Glawdel, Tomasz; Huang, Tiemin; Ren, Carolyn L; Pawliszyn, Janusz

    2008-10-01

    Simple-structured, well-functioned disposable poly(dimethylsiloxane) (PDMS) microchips were developed for capillary isoelectric focusing with whole column imaging detection (CIEF-WCID). Side-by-side comparison of the developed microchips with well-established commercial capillary cartridges demonstrated that the disposable microchips have comparable performance as well as advantages such as absence of lens effect and possibility of high-aspect-ratio accompanied with a dramatic reduction in cost.

  18. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    PubMed

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  19. Fatty acid ethyl esters in hair as alcohol markers: estimating a reliable cut-off point by evaluation of 1,057 autopsy cases.

    PubMed

    Hastedt, Martin; Bossers, Lydia; Krumbiegel, Franziska; Herre, Sieglinde; Hartwig, Sven

    2013-06-01

    Alcohol abuse is a widespread problem, especially in Western countries. Therefore, it is important to have markers of alcohol consumption with validated cut-off points. For many years research has focused on analysis of hair for alcohol markers, but data on the performance and reliability of cut-off values are still lacking. Evaluating 1,057 cases from 2005 to 2011, included a large sample group for the estimation of an applicable cut-off value when compared to earlier studies on fatty acid ethyl esters (FAEEs) in hair. The FAEEs concentrations in hair, police investigation reports, medical history, and the macroscopic and microscopic alcohol-typical results from autopsy, such as liver, pancreas, and cardiac findings, were taken into account in this study. In 80.2 % of all 1,057 cases pathologic findings that may be related to alcohol abuse were reported. The cases were divided into social drinkers (n = 168), alcohol abusers (n = 502), and cases without information on alcohol use. The median FAEEs concentration in the group of social drinkers was 0.302 ng/mg (range 0.008-14.3 ng/mg). In the group of alcohol abusers a median of 1.346 ng/mg (range 0.010-83.7 ng/mg) was found. Before June 2009 the hair FAEEs test was routinely applied to a proximal hair segment of 0-6 cm, changing to a routinely investigated hair length of 3 cm after 2009, as proposed by the Society of Hair Testing (SoHT). The method showed significant differences between the groups of social drinkers and alcoholics, leading to an improvement in the postmortem detection of alcohol abuse. Nevertheless, the performance of the method was rather poor, with an area under the curve calculated from receiver operating characteristic (ROC curve AUC) of 0.745. The optimum cut-off value for differentiation between social and chronic excessive drinking calculated for hair FAEEs was 1.08 ng/mg, with a sensitivity of 56 % and a specificity of 80 %. In relation to the "Consensus on Alcohol Markers 2012

  20. Short communication: Diet-induced variations in milk fatty acid composition have minor effects on the estimated melting point of milk fat in cows, goats, and ewes: Insights from a meta-analysis.

    PubMed

    Toral, P G; Bernard, L; Chilliard, Y; Glasser, F

    2013-02-01

    In ruminants, the ability to maintain milk fat melting point within physiological values could play a role in the regulation of milk fat secretion when milk fatty acid (FA) composition varies, such as in response to feeding factors. However, the relationship between milk fat fluidity and changes in milk FA composition is difficult to study experimentally. A meta-analysis was therefore conducted to compare the magnitude of diet-induced variations in milk FA composition and the calculated melting point of milk FA (used as a proxy to estimate the variations in the melting point of milk fat) in 3 dairy ruminant species (cow, goat, and sheep). The coefficient of variation (CV), a scale-free measure of statistical dispersion, was used to compare the variability of criteria differing in their order of magnitude. The analysis of a database of milk FA profiles from cows, goats, and sheep fed different dietary treatments (unsupplemented diets and diets supplemented with lipids rich in oleic acid, linoleic acid, linolenic acid, or C20-22 polyunsaturated FA) revealed that the variability of the calculated melting point of milk FA was narrow (CV of 5%) compared with the variability of milk FA percentages (CV of 18 to 72%). The regulation of the melting point of milk fat is thus probably involved in the control of diet-induced variations in milk fat secretion. The calculated melting point of ewe milk FA was approximately 3°C lower than that of goats or cows across all types of diets, which might be linked to differences in milk fat content (higher in sheep) or the structure of milk triacylglycerides among these species. Lipid supplementation increased the calculated melting point of C18 FA in milk, whereas that of total FA was significantly reduced by supplements rich in oleic, linoleic, and linolenic acids but not C20-22 polyunsaturated FA. However, the slight effects of dietary treatments on the calculated melting point of milk FA did not differ between cows, goats, and ewes.

  1. Aristolochic Acids

    MedlinePlus

    ... Sciences NIH-HHS www.niehs.nih.gov Aristolochic Acids Key Points Report on Carcinogens Status Known to be human carcinogens Aristolochia Clematitis Aristolochic Acids n Known human carcinogens n Found in certain ...

  2. Sampling strategies for capillary isoelectric focusing with electroosmotic zone mobilization assessed by high-resolution dynamic computer simulation.

    PubMed

    Takácsi-Nagy, Anna; Kilár, Ferenc; Páger, Csilla; Mosher, Richard A; Thormann, Wolfgang

    2012-03-01

    The impact of initial sample distribution on separation and focusing of analytes in a pH 3–11 gradient formed by 101 biprotic carrier ampholytes under concomitant electroosmotic displacement was studied by dynamic high-resolution computer simulation. Data obtained with application of the analytes mixed with the carrier ampholytes (as is customarily done), as a short zone within the initial carrier ampholyte zone, sandwiched between zones of carrier ampholytes, or introduced before or after the initial carrier ampholyte zone were compared. With sampling as a short zone within or adjacent to the carrier ampholytes, separation and focusing of analytes is shown to proceed as a cationic, anionic, or mixed process and separation of the analytes is predicted to be much faster than the separation of the carrier components. Thus, after the initial separation, analytes continue to separate and eventually reach their focusing locations. This is different to the double-peak approach to equilibrium that takes place when analytes and carrier ampholytes are applied as a homogenous mixture. Simulation data reveal that sample application between two zones of carrier ampholytes results in the formation of a pH gradient disturbance as the concentration of the carrier ampholytes within the fluid element initially occupied by the sample will be lower compared to the other parts of the gradient. As a consequence thereof, the properties of this region are sample matrix dependent, the pH gradient is flatter, and the region is likely to represent a conductance gap (hot spot). Simulation data suggest that sample placed at the anodic side or at the anodic end of the initial carrier ampholyte zone are the favorable configurations for capillary isoelectric focusing with electroosmotic zone mobilization.

  3. Chemical composition and acid-base properties of the surface of GaAs-CdS solid solutions

    NASA Astrophysics Data System (ADS)

    Kirovskaya, I. A.; Zemtsov, A. E.

    2007-01-01

    Chemical composition and acid-base properties of the surfaces of GaAs-CdS solid solutions and the constituent binary compounds exposed to air, vacuum, CO, and NH3 were analyzed using mass spectrometry, IR spectrometry, isoelectric-point pH (pHiso) analysis, and mechanochemical methods. Powders and films were prepared from the solid solutions by isothermal diffusion and discrete vacuum evaporation, respectively. XRD and Raman analysis revealed that a solid solution has a sphalerite or wurtzite structure, depending on its composition. The initial surface of all the systems studied exhibited acidic properties: the pH value ranged from 4.9 to 6.45. The surfaces contained Lewis (electron accepting) and Brönsted (adsorbed H2O and CO2 molecules, OH- groups) sites. The acidity of the surface increased upon treatment with CO and decreased upon treatment with NH3. For all types of treatment, the concentration dependence of the acid-base properties of the surface exhibited an extremum. The strongest acidic sites were revealed on the surface of the (GaAs)0.95 (CdS)0.05 solid solution. The adsorption activity of this solution, a promising material for manufacturing gas sensors, was evaluated.

  4. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  5. Isoelectric leukocyte focusing

    NASA Technical Reports Server (NTRS)

    Leise, E. M.

    1977-01-01

    Modified electrophoretic separation procedure yields 70 to 80 percent visible cells for small-scale immunological and clinical profiling. All reagents and preparations used in separation and characterization procedures are commercially available.

  6. Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

    PubMed

    Albani, J R; Plancke, Y D

    1999-05-31

    Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.

  7. The selective conversion of glutamic acid in amino acid mixtures using glutamate decarboxylase--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Sanders, Johan P M

    2014-01-01

    Amino acids (AAs) derived from hydrolysis of protein rest streams are interesting feedstocks for the chemical industry due to their functionality. However, separation of AAs is required before they can be used for further applications. Electrodialysis may be applied to separate AAs, but its efficiency is limited when separating AAs with similar isoelectric points. To aid the separation, specific conversion of an AA to a useful product with different charge behavior to the remaining compounds is desired. Here the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) was studied. L-Glutamate α-decarboxylase (GAD, Type I, EC 4.1.1.15) was applied to specifically convert Glu into γ-aminobutyric acid (GABA). GABA has a different charge behavior from Asp therefore allowing a potential separation by electrodialysis. Competitive inhibition and reduced operational stability caused by Asp could be eliminated by maintaining a sufficiently high concentration of Glu. Immobilization of GAD does not reduce the enzyme's initial activity. However, the operational stability was slightly reduced. An initial study on the reaction operating in a continuous mode was performed using a column reactor packed with immobilized GAD. As the reaction mixture was only passed once through the reactor, the conversion of Glu was lower than expected. To complete the conversion of Glu, the stream containing Asp and unreacted Glu might be recirculated back to the reactor after GABA has been removed. Overall, the reaction by GAD is specific to Glu and can be applied to aid the electrodialysis separation of Asp and Glu.

  8. Sample prefractionation with liquid isoelectric focusing enables in depth microbial metaproteome analysis of mesophilic and thermophilic biogas plants.

    PubMed

    Kohrs, F; Heyer, R; Magnussen, A; Benndorf, D; Muth, T; Behne, A; Rapp, E; Kausmann, R; Heiermann, M; Klocke, M; Reichl, U

    2014-10-01

    Biogas production from energy crops and biodegradable waste is one of the major sources for renewable energies in Germany. Within a biogas plant (BGP) a complex microbial community converts biomass to biogas. Unfortunately, disturbances of the biogas process occur occasionally and cause economic losses of varying extent. Besides technical failures the microbial community itself is commonly assumed as a reason for process instability. To improve the performance and efficiency of BGP, a deeper knowledge of the composition and the metabolic state of the microbial community is required and biomarkers for monitoring of process deviations or even the prediction of process failures have to be identified. Previous work based on 2D-electrophoresis demonstrated that the analysis of the metaproteome is well suited to provide insights into the apparent metabolism of the microbial communities. Using SDS-PAGE with subsequent mass spectrometry, stable protein patterns were evaluated for a number of anaerobic digesters. Furthermore, it was shown that severe changes in process parameters such as acidification resulted in significant modifications of the metaproteome. Monitoring of changing protein patterns derived from anaerobic digesters, however, is still a challenge due to the high complexity of the metaproteome. In this study, different combinations of separation techniques to reduce the complexity of proteomic BGP samples were compared with respect to the subsequent identification of proteins by tandem mass spectrometry (MS/MS): (i) 1D: proteins were tryptically digested and the resulting peptides were separated by reversed phase chromatography prior to MS/MS. (ii) 2D: proteins were separated by GeLC-MS/MS according to proteins molecular weights before tryptic digestion, (iii) 3D: proteins were separated by gel-free fractionation using isoelectric focusing (IEF) conducted before GeLC-MS/MS. For this study, a comparison of two anaerobic digesters operated at mesophilic and at

  9. Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

    PubMed

    Rodriguez-Calvo, M S; Carracedo, A; Muñoz, I; Concheiro, L

    1992-03-01

    A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.

  10. Application of reduced order modeling techniques to problems in heat conduction, isoelectric focusing and differential algebraic equations

    NASA Astrophysics Data System (ADS)

    Mathai, Pramod P.

    This thesis focuses on applying and augmenting 'Reduced Order Modeling' (ROM) techniques to large scale problems. ROM refers to the set of mathematical techniques that are used to reduce the computational expense of conventional modeling techniques, like finite element and finite difference methods, while minimizing the loss of accuracy that typically accompanies such a reduction. The first problem that we address pertains to the prediction of the level of heat dissipation in electronic and MEMS devices. With the ever decreasing feature sizes in electronic devices, and the accompanied rise in Joule heating, the electronics industry has, since the 1990s, identified a clear need for computationally cheap heat transfer modeling techniques that can be incorporated along with the electronic design process. We demonstrate how one can create reduced order models for simulating heat conduction in individual components that constitute an idealized electronic device. The reduced order models are created using Krylov Subspace Techniques (KST). We introduce a novel 'plug and play' approach, based on the small gain theorem in control theory, to interconnect these component reduced order models (according to the device architecture) to reliably and cheaply replicate whole device behavior. The final aim is to have this technique available commercially as a computationally cheap and reliable option that enables a designer to optimize for heat dissipation among competing VLSI architectures. Another place where model reduction is crucial to better design is Isoelectric Focusing (IEF) - the second problem in this thesis - which is a popular technique that is used to separate minute amounts of proteins from the other constituents that are present in a typical biological tissue sample. Fundamental questions about how to design IEF experiments still remain because of the high dimensional and highly nonlinear nature of the differential equations that describe the IEF process as well as

  11. Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique--isoelectric focusing of proteins and a single-stranded DNA fragment.

    PubMed

    Walowski, Britta; Hüttner, Wilhelm; Wackerbarth, Hainer

    2011-11-01

    Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application.

  12. Biomimetic synthesis of highly biocompatible gold nanoparticles with amino acid-dithiocarbamate as a precursor for SERS imaging.

    PubMed

    Li, Li; Liu, Jianbo; Yang, Xiaohai; Huang, Jin; He, Dinggeng; Guo, Xi; Wan, Lan; He, Xiaoxiao; Wang, Kemin

    2016-03-11

    Amino acid-dithiocarbamate (amino acid-DTC) was developed as both the reductant and ligand stabilizer for biomimetic synthesis of gold nanoparticles (AuNPs), which served as an excellent surface-enhanced Raman scattering (SERS) contrast nanoprobe for cell imaging. Glycine (Gly), glutamic acid (Glu), and histidine (His) with different isoelectric points were chosen as representative amino acid candidates to synthesize corresponding amino acid-DTC compounds through mixing with carbon disulfide (CS2), respectively. The pyrogenic decomposition of amino acid-DTC initiated the reduction synthesis of AuNPs, and the strong coordinating dithiocarbamate group of amino acid-DTC served as a stabilizer that grafted onto the surface of the AuNPs, which rendered the as-prepared nanoparticles a negative surface charge and high colloidal stability. MTT cell viability assay demonstrated that the biomimetic AuNPs possessed neglectful toxicity to the human hepatoma cell, which guaranteed them good biocompatibility for biomedical application. Meanwhile, the biomimetic AuNPs showed a strong SERS effect with an enhancement factor of 9.8 × 10(5) for the sensing of Rhodamine 6G, and two distinct Raman peaks located at 1363 and 1509 cm(-1) could be clearly observed in the cell-imaging experiments. Therefore, biomimetic AuNPs can be explored as an excellent SERS contrast nanoprobe for biomedical imaging, and the amino acid-DTC mediated synthesis of the AuNPs has a great potential in bio-engineering and biomedical imaging applications.

  13. Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah).

    PubMed

    Ahn, M Y; Lee, B M; Kim, Y S

    1997-06-01

    The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

  14. Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato.

    PubMed

    Plant, A L; Cohen, A; Moses, M S; Bray, E A

    1991-11-01

    The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.

  15. Selective transport of amino acids into the gas phase: driving forces for amino acid solubilization in gas-phase reverse micelles.

    PubMed

    Fang, Yigang; Bennett, Andrew; Liu, Jianbo

    2011-01-28

    We report a study on encapsulation of various amino acids into gas-phase sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT) reverse micelles, using electrospray ionization guided-ion-beam tandem mass spectrometry. Collision-induced dissociation of mass-selected reverse micellar ions with Xe was performed to probe structures of gas-phase micellar assemblies, identify solute-surfactant interactions, and determine preferential incorporation sites of amino acids. Integration into gas-phase reverse micelles depends upon amino acid hydrophobicity and charge state. For examples, glycine and protonated amino acids (such as protonated tryptophan) are encapsulated within the micellar core via electrostatic interactions; while neutral tryptophan is adsorbed in the surfactant layer. As verified using model polar hydrophobic compounds, the hydrophobic effect and solute-interface hydrogen-bonding do not provide sufficient driving force needed for interfacial solubilization of neutral tryptophan. Neutral tryptophan, with a zwitterionic structure, is intercalated at the micellar interface between surfactant molecules through complementary effects of electrostatic interactions between tryptophan backbone and AOT polar heads, and hydrophobic interactions between tryptophan side chain and AOT alkyl tails. Protonation of tryptophan could significantly improve its incorporation capacity into gas-phase reverse micelles, and displace its incorporation site from the micellar interfacial zone to the core; protonation of glycine, on the other hand, has little effect on its encapsulation capacity. Another interesting observation is that amino acids of different isoelectric points could be selectively encapsulated into, and transported by, reverse micelles from solution to the gas phase, based upon their competition for protonation and subsequent encapsulation within the micellar core.

  16. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

    PubMed

    Dulermo, Thierry; Lazar, Zbigniew; Dulermo, Rémi; Rakicka, Magdalena; Haddouche, Ramedane; Nicaud, Jean-Marc

    2015-09-01

    The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

  17. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    PubMed

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis.

  18. Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

    PubMed

    Toley, Bhushan J; Covelli, Isabela; Belousov, Yevgeniy; Ramachandran, Sujatha; Kline, Enos; Scarr, Noah; Vermeulen, Nic; Mahoney, Walt; Lutz, Barry R; Yager, Paul

    2015-11-21

    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

  19. Functional Analysis of Arabidopsis Mutants Points to Novel Roles for Glutathione in Coupling H2O2 to Activation of Salicylic Acid Accumulation and Signaling

    PubMed Central

    Han, Yi; Chaouch, Sejir; Mhamdi, Amna; Queval, Guillaume; Zechmann, Bernd

    2013-01-01

    Abstract Aims: Through its interaction with H2O2, glutathione is a candidate for transmission of signals in plant responses to pathogens, but identification of signaling roles is complicated by its antioxidant function. Using a genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed at establishing whether GSH plays an important functional role in the transmission of signals downstream of H2O2. Results: Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into cat2 blocked H2O2-triggered GSH oxidation and accumulation. While no effects on NADP(H) or ascorbate were observed, and H2O2-induced decreases in growth were maintained, blocking GSH modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype, expression of marker genes, nontargeted metabolite profiling, accumulation of SA, and bacterial resistance. Most of the effects of the cad2 mutation on H2O2-triggered responses were distinct from those produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. Innovation: A novel genetic approach was used in which GSH content or antioxidative capacity was independently modified in an H2O2 signaling background. Analysis of new double and triple mutants allowed us to infer previously undescribed regulatory roles for GSH. Conclusion: In parallel to its antioxidant role, GSH acts independently of NPR1 to allow increased intracellular H2O2 to activate SA signaling, a key defense response in plants. Antioxid. Redox Signal. 18, 2106–2121. PMID:23148658

  20. Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

    PubMed Central

    Liu, S Y; Wiegel, J; Gherardini, F C

    1996-01-01

    An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed

  1. Separation of Alkaline Phosphatase Isoenzymes from Various Rat Tissues Using Flat-Bed Acrylamide Gel Isoelectric Focusing,

    DTIC Science & Technology

    1981-12-22

    Technical Bulletin No. 104. Alkaline phosphatase activity was expressed as 1 micromole of p- nitrophenol hydrolyzed per hour and specific alkaline... phosphatase activity was defined as the number of micromoles of p- nitrophenol hydrolyzed per hour per microgram of protein. The total protein was determined... phosphatase activity is known or suspected. Importantly, the procedure is easily adapted for acid phosphatase examination by merely changing the pH and

  2. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning

    SciTech Connect

    Takahashi, N.; Takahashi, Y.; Blumberg, B.S.; Putnam, F.W.

    1987-07-01

    The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO/sub 4//PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.

  3. Cloning and Sequence Analysis of cDNAs Encoding Two Acidic PLA(2) from venom of Ophiophagus hannah(King Cobra), Guangxi Species.

    PubMed

    Wang, Qiu-Yan; Shu, Yu-Yan; Zhuang, Mao-Xing; Lin, Zheng-Jiong

    2001-01-01

    Total RNA was extracted from venom glands of Ophiophagus hannah, Guangxi species. The cDNAs encoding PLA(2) were amplified by RT-PCR and cloned into the PUCm-T vector. The positive clones encoding two acidic PLA(2) (APLA(2)-1 and APLA(2)-2) were selected and bidirectionally sequenced. Their complete amino acid sequences were deduced and found to be identical to the known amino acid sequences. Their isoelectric points calculated by computer agreed with the values determined with their protein. Homology analysis indicated that the mature peptide of APLA(2)-1 had high homology with PLA(2) from venoms of Ophiophagus hannah, Fujian and Taiwan species, but APLA(2)-2 had lower homology. The most striking difference between APLA(2)-2 and other PLA(2) from Ophiophagus hannah venoms is the missing of a extra "pancreatic loop" at residues 62--66 in APLA(2)-2, and it may be related to their species evolution and biological activity.

  4. Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A₂ from Bothrops (Rhinocerophis) ammodytoides venom.

    PubMed

    Clement, Herlinda; Costa de Oliveira, Vanessa; Zamudio, Fernando Z; Lago, Néstor R; Valdez-Cruz, Norma A; Bérnard Valle, Melisa; Hajos, Silvia E; Alagón, Alejandro; Possani, Lourival D; de Roodt, Adolfo R

    2012-12-01

    A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.

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

    PubMed

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-27

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

  6. Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets

    PubMed Central

    Tan, Bie; Wang, Jing; Kong, Xiangfeng; Guan, Guiping; Li, Fengna; Yin, Yulong

    2015-01-01

    The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine. PMID:26107628

  7. Penicillanic acid sulfone: nature of irreversible inactivation of RTEM beta-lactamase from Escherichia coli.

    PubMed

    Brenner, D G; Knowles, J R

    1984-11-20

    When penicillanic acid sulfone in large molar excess is incubated with the RTEM beta-lactamase, the enzyme becomes inactivated irreversibly. From studies of the consequential spectroscopic changes, from the use of specifically tritiated penicillanic acid sulfone, and from comparison by isoelectric focusing of the enzyme after inactivation by the sulfone and by clavulanic acid, the inactivated enzyme appears to be cross-linked by a beta-aminoacrylate fragment deriving from C-5, C-6, and C-7 of the original beta-lactam. Model studies on the behavior of alcoholic solutions of penicillanic acid sulfone in the presence of amines are entirely consistent with this interpretation.

  8. Differential binding of thyroxine and triiodothyronine to acidic isoforms of thyroid hormone binding globulin in human serum

    SciTech Connect

    Terasaki, T.; Pardridge, W.M.

    1988-05-17

    The differential availability of thyroxine (T/sub 4/) and 3,5,3'-triiodothyronine (T/sub 3/) to liver from the circulating thyroid hormone binding globulin (TBG)-bound pool suggests that the two thyroid hormones may bind to different TBG isoforms in human serum. In the present study, the binding of (/sup 125/I)T/sub 4/ and (/sup 125/I)T/sub 3/ to human serum proteins was investigated by using slab gel isoelectric focusing and chromatofocusing. In normal human male serum, (/sup 125/I)T/sub 4/ was localized to four isoforms of TBG called TBG-I, -II, -III, and -IV, with isoelectric points (pI's) of 4.30, 4.35, 4.45, and 4.55, respectively. (/sup 125/I)T/sub 3/ was localized to only two isoforms of TBG, TBG-III, and -IV, with pI's that were identical with those for (/sup 125/I)T/sub 4/. In normal female serum, (/sup 125/I)T/sub 4/ was localized to the same four isoforms of TBG as those of normal male serum, while (/sup 125/I)T/sub 3/ was localized to TBG-II, -III, -IV, and -V (pI = 4.65). In pregnant female serum, (/sup 125/I)T/sub 4/ was localized to five isoforms, whereas (/sup 125/I)T/sub 3/ was localized to four. IEF was also performed with male serum loaded with various concentrations of unlabeled T/sub 3/. The K/sub i/ values of T/sub 3/ binding to TBG-I, -II, -III, and -IV were 5.0, 2.4, 0.86, and 0.46 nM, respectively. The TBG isoforms in normal male serum were also separated by sequential concanavalin A-Sepharose affinity chromatography and the chromatofocusing (pH range of 3.5-5.0). T/sub 4/ preferentially bound to the most acidic isoforms of TBG in the pI range of 3.8-4.0, whereas the less acidic fractions (pH 4.0-4.2) bound both T/sub 4/ and T/sub 3/. In conclusion, this study shows that T/sub 4/ and T/sub 3/ do not bind to a single competitive binding site on TBG. Instead, T/sub 4/ is preferentially bound by the most acidic TBG isoforms owing to a 10-fold lower affinity of T/sub 3/ for these proteins.

  9. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans.

    PubMed

    Hakamada, Yoshihiro; Endo, Keiji; Takizawa, Shuichi; Kobayashi, Tohru; Shirai, Tsuyoshi; Yamane, Takashi; Ito, Susumu

    2002-04-15

    A high-isoelectric-point (pI), alkaline endo-1,4-beta-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 degrees C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-beta-glucanase. However, this enzyme was not active on p-nitrophenyl beta-D-cellotrioside and p-nitrophenyl beta-D-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl(2) as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 A resolution. It belongs to the space group P2(1)2(1)2(1) with unit cell parameters of a=62.5 A, b=71.7 A, and c=88.6 A.

  10. Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes.

    PubMed

    Mikkonen, Saara; Thormann, Wolfgang; Emmer, Åsa

    2015-10-01

    In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.

  11. Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins.

    PubMed

    Altenhofer, Pia; Schierhorn, Angelika; Fricke, Beate

    2006-10-01

    2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.

  12. Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS.

    PubMed

    Guo, Tong; Rudnick, Paul A; Wang, Weijie; Lee, Cheng S; Devoe, Don L; Balgley, Brian M

    2006-06-01

    Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.

  13. Isoelectric Focusing of Serum Apolipoprotein C-III as a Sensitive Screening Method for the Detection of O-glycosylation Disturbances.

    PubMed

    Ondrušková, Nina; Honzík, Tomáš; Kytnarová, Jitka; Matoulek, Martin; Zeman, Jiří; Hansíková, Hana

    2015-01-01

    Apolipoprotein C-III (ApoC-III) is a glycoprotein carrying the most common O-linked glycan structure and is abundantly present in serum, what renders it a suitable marker for analysis of O-glycosylation abnormalities. Isoelectric focusing followed by a Western blot of ApoC-III, using PhastSystem™ Electrophoresis System (GE Healthcare), was introduced as a rather simple and rapid method for screening of certain subtypes of inherited glycosylation disorders. The study's aim was to establish this method in our laboratory, what included performing the analysis in a group of 170 healthy individuals to set the reference range of detected relative amounts of sialylated ApoC-III isoforms and to evaluate the gender- and age-dependent differences. A significant relative increase of asialo-ApoC-III with growing age was found. Secondly, we examined serum from patients with selected metabolic disorders and detected minor O-glycosylation changes in diseases such as Prader-Willi syndrome, PGM1 (phosphoglucomutase 1) or MAN1B (class 1B alpha-1,2-mannosidase) deficiency. Our results show that this method allows for a sensitive detection of ApoC-III O-glycosylation status, however this might be modulated by several factors (i.e. nutrition, medication) whose exact role remains to be determined.

  14. On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants.

    PubMed

    Przybylski, Cédric; Mokaddem, Meriem; Prull-Janssen, Mehdi; Saesen, Els; Lortat-Jacob, Hugues; Gonnet, Florence; Varenne, Anne; Daniel, Régis

    2015-01-21

    The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.

  15. [Comparison of one-step and two-step methods for pI determination of proteins and polypeptides by capillary isoelectric focusing].

    PubMed

    Gao, Peifeng; Zhao, Xinying; He, Muyi; Liu, Qingsheng; Qu, Feng

    2013-06-01

    One-step and two-step capillary isoelectric focusing (clEF) methods were employed the separation and pI determination of proteins and polypeptides. The parameters affecting the analysis efficiency, such as the sample solution, injection volume, focusing voltage, focusing time and driving conditions were optimized. The comparison of the two methods for separation of cytochrome C, hemoglobin, myoglobin, transferrin, bovine serum albumin and six polypeptides showed that the one-step cIEF was simple and fast, which could determine the pI of single component as well as it was rapid for protein and polypeptide separation, but it could not get good resolution or accurate pI of each component in a mixed sample. The two-step cIEF was more complex and needed longer time, however, which could separate and exactly determine the pI of each component in the mixture, and the pI value of each component determined was consistent with that determined using a single sample. The two methods are complementary, and can be widely used in rapid and accurate determination of the pI of amphiphilic biological particles.

  16. Stabilization of thin-layer agarose gels after isoelectric focusing with polyacrylamide enables reverse imidazole-zinc staining and facilitates two-dimensional gel electrophoresis.

    PubMed

    Hellman, Jukka

    2008-09-01

    Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated.

  17. Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

    PubMed

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tsai, Yun-Long; Tsai, Chuan-Fu; Shen, Yu-Han; Chang, Hsiao-Fen Grace; Skillman, Ashley; Wang, Hwa-Tang Thomas; Pronost, Stéphane; Zhang, Yan

    2017-03-01

    Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.

  18. Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

    PubMed

    Iliadi, Alexandra; Petropoulou, Margarita; Ioannou, Penelope C; Christopoulos, Theodore K; Anagnostopoulos, Nikolaos I; Kanavakis, Emmanuel; Traeger-Synodinos, Jan

    2011-09-01

    In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

  19. An ab initio potential energy surface for the formic acid dimer: zero-point energy, selected anharmonic fundamental energies, and ground-state tunneling splitting calculated in relaxed 1-4-mode subspaces.

    PubMed

    Qu, Chen; Bowman, Joel M

    2016-09-14

    We report a full-dimensional, permutationally invariant potential energy surface (PES) for the cyclic formic acid dimer. This PES is a least-squares fit to 13475 CCSD(T)-F12a/haTZ (VTZ for H and aVTZ for C and O) energies. The energy-weighted, root-mean-square fitting error is 11 cm(-1) and the barrier for the double-proton transfer on the PES is 2848 cm(-1), in good agreement with the directly-calculated ab initio value of 2853 cm(-1). The zero-point vibrational energy of 15 337 ± 7 cm(-1) is obtained from diffusion Monte Carlo calculations. Energies of fundamentals of fifteen modes are calculated using the vibrational self-consistent field and virtual-state configuration interaction method. The ground-state tunneling splitting is computed using a reduced-dimensional Hamiltonian with relaxed potentials. The highest-level, four-mode coupled calculation gives a tunneling splitting of 0.037 cm(-1), which is roughly twice the experimental value. The tunneling splittings of (DCOOH)2 and (DCOOD)2 from one to three mode calculations are, as expected, smaller than that for (HCOOH)2 and consistent with experiment.

  20. Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

    PubMed

    Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2011-01-01

    To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.

  1. Point set registration: coherent point drift.

    PubMed

    Myronenko, Andriy; Song, Xubo

    2010-12-01

    Point set registration is a key component in many computer vision tasks. The goal of point set registration is to assign correspondences between two sets of points and to recover the transformation that maps one point set to the other. Multiple factors, including an unknown nonrigid spatial transformation, large dimensionality of point set, noise, and outliers, make the point set registration a challenging problem. We introduce a probabilistic method, called the Coherent Point Drift (CPD) algorithm, for both rigid and nonrigid point set registration. We consider the alignment of two point sets as a probability density estimation problem. We fit the Gaussian mixture model (GMM) centroids (representing the first point set) to the data (the second point set) by maximizing the likelihood. We force the GMM centroids to move coherently as a group to preserve the topological structure of the point sets. In the rigid case, we impose the coherence constraint by reparameterization of GMM centroid locations with rigid parameters and derive a closed form solution of the maximization step of the EM algorithm in arbitrary dimensions. In the nonrigid case, we impose the coherence constraint by regularizing the displacement field and using the variational calculus to derive the optimal transformation. We also introduce a fast algorithm that reduces the method computation complexity to linear. We test the CPD algorithm for both rigid and nonrigid transformations in the presence of noise, outliers, and missing points, where CPD shows accurate results and outperforms current state-of-the-art methods.

  2. Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.

    PubMed

    Zhang, Z Y; Van Etten, R L

    1990-10-01

    A low-molecular-weight acid phosphatase that is representative of a group recently shown to be phosphotyrosyl protein phosphatases was purified to homogeneity from bovine heart. The enzyme was a monomer with a molecular mass of 18 kDa and had an isoelectric point of 7.0. The absorption coefficient, E1% 1cm was 9.65 at 280 nm. The enzyme had pH optima of 5.3 and 6.0 with the substrates p-nitrophenyl phosphate and tyrosine phosphate, respectively. When measured at pH 5 and 37 degrees C, the enzyme had specific activities of 114 and 86 mumol min-1 mg-1 for p-nitrophenyl phosphate and tyrosine O-phosphate, respectively, while the Km values were 0.38 and 14 mM. The enzyme was highly specific for aryl monophosphate esters and showed little or no activity toward aliphatic phosphate esters, with the remarkable exception of flavin mononucleotide (FMN) and certain of its structural analogs. As shown by 31P NMR data, the activity toward FMN was due to the hydrolysis of one of the eight components present in the (commercial) sample. Both molybdate and vanadate were potent inhibitors, with inhibition constants of 37 and 29 microM, respectively; tartrate and fluoride had little effect on enzymatic activity. A two-stage reversible denaturation of the enzyme by guanidine HCl was observed with midpoints of 0.25 and 1.75 M, respectively. The amino acid composition was homologous to the low-molecular-weight acid phosphatases from other tissue. The enzyme showed immunological cross-reactivity against low-molecular-weight human liver acid phosphatase. There were 7 or 8 accessible cysteines on the monomeric protein and at least one was essential for enzyme activity. The enzyme also had phosphotransferase activity, for example transferring phosphate from p-nitrophenyl phosphate to a wide variety of alcohol acceptors.

  3. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges

    PubMed Central

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Introduction Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Methods Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Results Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, p<0.001) and Gly (χ2 = 73.35, p<0.001) are highly and Val moderately (χ2 = 144.43, p<0.001) occurring in the 85% of thermophilic proteins. Phospho-regulated Tyr and redox-sensitive Cys are also moderately distributed (χ2~20.0, p<0.01) in a larger number of thermophilic proteins. A consistent lower distribution of thermophilicity and discretely higher distribution of hydrophobicity is noticed in a large number of thermophilic versus their mesophilic protein homolog. The mean differences of isoelectric points and charges are found to be significantly less (7.11 vs. 6.39, p<0.05 and 1 vs. -0.6, p<0.01, respectively) in thermophilic proteins compared to their mesophilic counterpart. The possible sites for Tyr phosphorylation are noticed to be 25% higher (p<0.05) in thermophilic proteins. The 60% thermophiles are found with higher number of salt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (p<0.05 and p

  4. Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson-Tiselius differential equation and moving reaction boundary.

    PubMed

    Guo, Chen-Gang; Li, Si; Wang, Hou-Yu; Zhang, Dong; Li, Guo-Qing; Zhang, Jie; Fan, Liu-Yin; Cao, Cheng-Xi

    2013-07-15

    An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson-Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG-IEF and non-IPG-IEF (opposite to IPG-IEF) were developed to unveil stability mechanism of IPG-IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG-IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG-IEF system, (ii) there existed high diffusional current in non-IPG-IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG-IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG-IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG-IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.

  5. Impact of glutathione-HbA1c on HbA1c measurement in diabetes diagnosis via array isoelectric focusing, liquid chromatography, mass spectrometry and ELISA.

    PubMed

    Li, Si; Guo, Chen-Gang; Chen, Lu; Yin, Xiao-Yang; Wu, Yi-Xin; Fan, Liu-Yin; Fan, Hui-Zhi; Cao, Cheng-Xi

    2013-10-15

    Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis.

  6. Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

    PubMed Central

    Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

    2016-01-01

    Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

  7. Combining high-throughput MALDI-TOF mass spectrometry and isoelectric focusing gel electrophoresis for virtual 2D gel-based proteomics.

    PubMed

    Lohnes, Karen; Quebbemann, Neil R; Liu, Kate; Kobzeff, Fred; Loo, Joseph A; Ogorzalek Loo, Rachel R

    2016-07-15

    The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry (MS). Intact proteins separated by isoelectric focusing (IEF) gel electrophoresis are imaged from immobilized pH gradient (IPG) polyacrylamide gels (the first dimension of classic 2D-PAGE) by matrix-assisted laser desorption/ionization (MALDI) MS. Obtaining accurate intact masses from sub-picomole-level proteins embedded in 2D-PAGE gels or in IPG strips is desirable to elucidate how the protein of one spot identified as protein 'A' on a 2D gel differs from the protein of another spot identified as the same protein, whenever tryptic peptide maps fail to resolve the issue. This task, however, has been extremely challenging. Virtual 2D gel/MS provides access to these intact masses. Modifications to our matrix deposition procedure improve the reliability with which IPG gels can be prepared; the new procedure is described. Development of this MALDI MS imaging (MSI) method for high-throughput MS with integrated 'top-down' MS to elucidate protein isoforms from complex biological samples is described and it is demonstrated that a 4-cm IPG gel segment can now be imaged in approximately 5min. Gel-wide chemical and enzymatic methods with further interrogation by MALDI MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MSI-based virtual 2D gel/MS platform may potentially link the benefits of 'top-down' and 'bottom-up' proteomics.

  8. Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats

    PubMed Central

    Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

    2016-01-01

    Objectives This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. Methods FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Results Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Conclusions Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to

  9. Purification and characterization of 3-dehydroshikimate dehydratase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

    PubMed

    Strøman, P; Reinert, W R; Giles, N H

    1978-07-10

    3-Dehydroshikimate dehydratase catalyzes the third reaction in the inducible quinic acid catabolic pathway of Neurospora crassa and is encoded in the qa-4 gene of the qa gene cluster. As part of continuing genetic and biochemical studies concerning the organization and regulation of this gene cluster, 3-dehydroshikimate dehydratase has been purified and characterized biochemically. The enzyme was purified 1650-fold using the following techniques: 1) (NH4)2SO4 fractionation; 2) ion exchange chromatography on DEAE-cellulose; 3) gel filtration on Sephadex G-100; 4) ion exchange chromatography on Cellex QAE (quaternary aminoethyl); and 5) hydroxylapatite chromatography. 3-Dehydroshikimate dehydratase is a monomer with a molecular weight of about 37,000 and a sedimentation coefficient of 3.27 S. It has a Km value of 5.9 X 10(-4) and an average isoelectric point of 4.92. The purified enzyme is extremely sensitive to thermal denaturation but can be significantly stabilized by Mg2+ ions. The purified enzyme also exhibits maximal catalytic activity only when assayed in the presence of certain divalent cations, e.g. magnesium. The NH2-terminal residue of 3-dehydroshikimate dehydratase is proline, and its alpha-amino group is unblocked.

  10. Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

    PubMed Central

    Chang, Ya-Min; Chen, Li-Chun; Wang, Hsin-Yi; Chiang, Chui-Liang; Chang, Chen-Tien; Chung, Yun-Chin

    2014-01-01

    Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters Km and kcat were 245 µM and 10.31 min−1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides. PMID:25437446

  11. Characterization of an acidic chitinase from seeds of black soybean (Glycine max (L) Merr Tainan No. 3).

    PubMed

    Chang, Ya-Min; Chen, Li-Chun; Wang, Hsin-Yi; Chiang, Chui-Liang; Chang, Chen-Tien; Chung, Yun-Chin

    2014-01-01

    Using 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters Km and kcat were 245 µM and 10.31 min-1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.

  12. Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes

    SciTech Connect

    Jeanny, J.C.; Fayein, N.; Courtois, Y. ); Moenner, M.; Chevallier, B.; Barritault, D. )

    1987-07-01

    The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, the authors performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.

  13. A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer

    PubMed Central

    Rajapakse, R. P. V. Jayanthe; Kularatne, Senanayake A. M.; Lee, Pei-Yu Alison; Ku, Keun Bon; Nam, Sangwoo; Chou, Pin-Hsing; Tsai, Yun-Long; Liu, Yu-Lun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2016-01-01

    Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3′ untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. PMID:27030492

  14. Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings.

    PubMed

    Kim, Donggiun; Lee, Gunsup; Chang, Man; Park, Jongbum; Chung, Youngjae; Lee, Sukchan; Lee, Taek-Kyun

    2011-10-26

    Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.

  15. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids

    PubMed Central

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S. V. Santhana; Buettner, Garry R.; Robertson, Larry W.; Luthe, Gregor

    2016-01-01

    Covalent bond formations of free radical metabolites with biomolecules like DNA and proteins are thought to constitute a major mechanism of toxicity and carcinogenesis. Glutathione (GSH) is generally accepted as a radical scavenger protecting the cell. In the present study, we investigated a semiquinone radical (SQ•-) metabolite of the semivolatile 4-chlorobiphenyl, using electron paramagnetic resonance spectroscopy, and oxygen consumption. Proton nuclear magnetic resonance (1H NMR) and liquid chromatography–mass spectrometry (LC-MS) were also employed to elucidate the radical interaction with DNA, amino acids, and GSH. We found that DNA and oligonucleotides stabilized SQ•- by electron delocalization in the π-stacking system, resulting in persistent radical intercalated, rather than forming a covalent bond with SQ•-. This finding was strongly supported by the semiempirical calculation of the semioccupied molecular orbital and the linear combination of the atomic orbitals, indicating 9.8 kcal mol−1 energy gain. The insertion of SQ•- into the DNA strand may result in DNA strand breaks and interruption of DNA replication process or even activate radical mediated secondary reactions. The presence of amino acids resulted in a decrease of the electron paramagnetic resonance (EPR) signal of SQ•- and correlated with their isoelectric points. The pH shifts the equilibrium of the dianions of hydroquinone and influenced indirectly the formation of SQ•-. Similar findings were observed with GSH and Cys. GSH and Cys functioned as indirect radical scavengers; their activities depend on their chemical equilibria with the corresponding quinones, and their further reaction via Michael addition. The generally accepted role of GSH as radical scavenger in biological systems should be reconsidered based upon these findings, questioning the generally accepted view of radical interaction of semiquinones with biologically active compounds, like DNA, amino acids, proteins

  16. SPS antenna pointing control

    NASA Technical Reports Server (NTRS)

    Hung, J. C.

    1980-01-01

    The pointing control of a microwave antenna of the Satellite Power System was investigated emphasizing: (1) the SPS antenna pointing error sensing method; (2) a rigid body pointing control design; and (3) approaches for modeling the flexible body characteristics of the solar collector. Accuracy requirements for the antenna pointing control consist of a mechanical pointing control accuracy of three arc-minutes and an electronic phased array pointing accuracy of three arc-seconds. Results based on the factors considered in current analysis, show that the three arc-minute overall pointing control accuracy can be achieved in practice.

  17. Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus.

    PubMed

    Kasai, Kosuke; Ishikawa, Takashi; Komata, Takafumi; Fukuchi, Kaori; Chiba, Mitsuru; Nozaka, Hiroyuki; Nakamura, Toshiya; Sato, Tatsusuke; Miura, Tomisato

    2010-01-01

    Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin-resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of L-amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT-PCR using a specific primer, and immunohistochemical analysis with anti-LAAO IgG, demonstrated tissue-specific expression and localization in the gill. Moreover, the anti-LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin-resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus-derived epidermal mucus, is a novel LAAO-like protein with antibacterial activity, similar to snake LAAOs.

  18. Volume 2 - Point Sources

    EPA Pesticide Factsheets

    Point source emission reference materials from the Emissions Inventory Improvement Program (EIIP). Provides point source guidance on planning, emissions estimation, data collection, inventory documentation and reporting, and quality assurance/quality contr

  19. Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy.

    PubMed Central

    Federwisch, M; Hassiepen, U; Bender, K; Dewor, M; Rajewsky, M F; Wollmer, A

    1997-01-01

    Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered. PMID:9164873

  20. Comparing Point Clouds

    DTIC Science & Technology

    2004-04-01

    Point clouds are one of the most primitive and fundamental surface representations. A popular source of point clouds are three dimensional shape...acquisition devices such as laser range scanners. Another important field where point clouds are found is in the representation of high-dimensional...framework for comparing manifolds given by point clouds is presented in this paper. The underlying theory is based on Gromov-Hausdorff distances, leading

  1. Point-to-Point Multicast Communications Protocol

    NASA Technical Reports Server (NTRS)

    Byrd, Gregory T.; Nakano, Russell; Delagi, Bruce A.

    1987-01-01

    This paper describes a protocol to support point-to-point interprocessor communications with multicast. Dynamic, cut-through routing with local flow control is used to provide a high-throughput, low-latency communications path between processors. In addition multicast transmissions are available, in which copies of a packet are sent to multiple destinations using common resources as much as possible. Special packet terminators and selective buffering are introduced to avoid a deadlock during multicasts. A simulated implementation of the protocol is also described.

  2. Poly(lactic acid) microparticles coated with insulin-containing layer-by-layer films and their pH-dependent insulin release.

    PubMed

    Hashide, Ryosuke; Yoshida, Kentaro; Hasebe, Yasushi; Seno, Masaru; Takahashi, Shigehiro; Sato, Katsuhiko; Anzai, Jun-Ichi

    2014-04-01

    Poly(lactic acid) (PLA) microparticles were coated with layer-by-layer (LbL) films containing insulin and the pH-dependent release of insulin was studied. The LbL films were prepared on the surface of PLA microparticles by the alternate deposition of insulin and poly(allylamine hydrochloride) (PAH) through the electrostatic attraction between insulin and PAH. The insulin loading on the PLA microparticles depended on the film thickness, which corresponded to the number of insulin layers, and on the pH of the solution used to deposit insulin. The insulin loading increased with the film thickness and when the film was prepared at pH 7.4. The LbL films decomposed upon exposure to acidic solutions because the electrostatic attraction between the insulin and the PAH in the films disappeared when the charge on insulin changed from negative to positive at an acidic pH, which resulted in the release of insulin. The temperature and salt concentration did not affect the pH stability of the LbL films. The pH threshold for insulin release was pH 5.0-6.0, which corresponds to isoelectric point of insulin, 5.4. The release of insulin from the microparticles was rapid, and was almost complete within a few minutes. The circular dichroism spectra showed that the released insulin retained its original secondary structure. Our insulin-loaded PLA microparticles may be useful for the controlled release of insulin.

  3. Robust Critical Point Detection

    SciTech Connect

    Bhatia, Harsh

    2016-07-28

    Robust Critical Point Detection is a software to compute critical points in a 2D or 3D vector field robustly. The software was developed as a part of the author's work at the lab as a Phd student under Livermore Scholar Program (now called Livermore Graduate Scholar Program).

  4. Nickel Curie Point Engine

    ERIC Educational Resources Information Center

    Chiaverina, Chris; Lisensky, George

    2014-01-01

    Ferromagnetic materials such as nickel, iron, or cobalt lose the electron alignment that makes them attracted to a magnet when sufficient thermal energy is added. The temperature at which this change occurs is called the "Curie temperature," or "Curie point." Nickel has a Curie point of 627 K, so a candle flame is a sufficient…

  5. Model Breaking Points Conceptualized

    ERIC Educational Resources Information Center

    Vig, Rozy; Murray, Eileen; Star, Jon R.

    2014-01-01

    Current curriculum initiatives (e.g., National Governors Association Center for Best Practices and Council of Chief State School Officers 2010) advocate that models be used in the mathematics classroom. However, despite their apparent promise, there comes a point when models break, a point in the mathematical problem space where the model cannot,…

  6. The Lagrangian points

    NASA Astrophysics Data System (ADS)

    Linton, J. Oliver

    2017-03-01

    There are five unique points in a star/planet system where a satellite can be placed whose orbital period is equal to that of the planet. Simple methods for calculating the positions of these points, or at least justifying their existence, are developed.

  7. Constructive points of powerlocales

    NASA Astrophysics Data System (ADS)

    Vickers, Steven

    1997-09-01

    Results of Bunge and Funk and of Johnstone, providing constructively sound descriptions of the global points of the lower and upper powerlocales, are extended here to describe the generalized points and proved in a way that displays in a symmetric fashion two complementary treatments of frames: as suplattices and as preframes. Also described here are the points of the Vietoris powerlocale.In each of two special cases, an exponential D ( being the Sierpinski locale) is shown to be homeomorphic to a powerlocale: to the lower powerlocale when D is discrete, and to the upper powerlocale when D is compact regular.

  8. Arctic climate tipping points.

    PubMed

    Lenton, Timothy M

    2012-02-01

    There is widespread concern that anthropogenic global warming will trigger Arctic climate tipping points. The Arctic has a long history of natural, abrupt climate changes, which together with current observations and model projections, can help us to identify which parts of the Arctic climate system might pass future tipping points. Here the climate tipping points are defined, noting that not all of them involve bifurcations leading to irreversible change. Past abrupt climate changes in the Arctic are briefly reviewed. Then, the current behaviour of a range of Arctic systems is summarised. Looking ahead, a range of potential tipping phenomena are described. This leads to a revised and expanded list of potential Arctic climate tipping elements, whose likelihood is assessed, in terms of how much warming will be required to tip them. Finally, the available responses are considered, especially the prospects for avoiding Arctic climate tipping points.

  9. Collinear Points Problem

    ERIC Educational Resources Information Center

    Shultz, Harris S.; Shiflett, Ray C.

    2008-01-01

    Students were asked to find all possible values for A so that the points (1, 2), (5, A), and (A, 7) lie on a straight line. This problem suggests a generalization: Given (x, y), find all values of A so that the points (x, y), (5, A), and (A, 7) lie on a straight line. We find that this question about linear equations must be resolved using the…

  10. Nickel Curie point engine

    NASA Astrophysics Data System (ADS)

    Chiaverina, Chris; Lisensky, George

    2014-04-01

    Ferromagnetic materials such as nickel, iron, or cobalt lose the electron alignment that makes them attracted to a magnet when sufficient thermal energy is added. The temperature at which this change occurs is called the "Curie temperature," or "Curie point." Nickel has a Curie point of 627 K, so a candle flame is a sufficient heat source. A simple but elegant device illustrates this phenomenon beautifully.

  11. Unpredictable points and chaos

    NASA Astrophysics Data System (ADS)

    Akhmet, Marat; Fen, Mehmet Onur

    2016-11-01

    It is revealed that a special kind of Poisson stable point, which we call an unpredictable point, gives rise to the existence of chaos in the quasi-minimal set. The existing definitions of chaos are formulated in sets of motions. This is the first time in the literature that description of chaos is initiated from a single motion. The theoretical results are exemplified by means of the symbolic dynamics.

  12. Analysis of Organic Acids.

    ERIC Educational Resources Information Center

    Griswold, John R.; Rauner, Richard A.

    1990-01-01

    Presented are the procedures and a discussion of the results for an experiment in which students select unknown carboxylic acids, determine their melting points, and investigate their solubility behavior in water and ethanol. A table of selected carboxylic acids is included. (CW)

  13. Bloch points are sticky

    NASA Astrophysics Data System (ADS)

    Tchernyshyov, Oleg; Kim, Se Kwon

    2014-03-01

    Bloch points are zero-dimensional topological defects in three-dimensional ferromagnets. A representative magnetic configuration is a hedgehog with magnetization pointing away from a center. The singular nature of a Bloch point's core leads to interesting and observable consequences. A simple argument based on dimensional analysis shows that a magnetic lattice creates a periodic potential that can pin a Bloch point even if the lattice has no defects. The pinning force is of the order of the micromagnetic exchange constant, a few piconewtons in a typical ferromagnet. A domain wall in a cylindrical ferromagnetic wire with the diameter of a few tens of nanometers may contain a Bloch point. Such a domain wall will have a sizable depinning field, tens of oersteds. A Bloch point moving through an atomic lattice should emit electromagnetic waves at the frequency of a few hundred gigahertz. Research supported in part by the U.S. National Science Foundation under Grants No. DMR-0520491 and No. DMR-1104753.

  14. Reference Point Heterogeneity

    PubMed Central

    Terzi, Ayse; Koedijk, Kees; Noussair, Charles N.; Pownall, Rachel

    2016-01-01

    It is well-established that, when confronted with a decision to be taken under risk, individuals use reference payoff levels as important inputs. The purpose of this paper is to study which reference points characterize decisions in a setting in which there are several plausible reference levels of payoff. We report an experiment, in which we investigate which of four potential reference points: (1) a population average payoff level, (2) the announced expected payoff of peers in a similar decision situation, (3) a historical average level of earnings that others have received in the same task, and (4) an announced anticipated individual payoff level, best describes decisions in a decontextualized risky decision making task. We find heterogeneity among individuals in the reference points they employ. The population average payoff level is the modal reference point, followed by experimenter's stated expectation of a participant's individual earnings, followed in turn by the average earnings of other participants in previous sessions of the same experiment. A sizeable share of individuals show multiple reference points simultaneously. The reference point that best fits the choices of the individual is not affected by a shock to her income. PMID:27672374

  15. Reference Point Heterogeneity.

    PubMed

    Terzi, Ayse; Koedijk, Kees; Noussair, Charles N; Pownall, Rachel

    2016-01-01

    It is well-established that, when confronted with a decision to be taken under risk, individuals use reference payoff levels as important inputs. The purpose of this paper is to study which reference points characterize decisions in a setting in which there are several plausible reference levels of payoff. We report an experiment, in which we investigate which of four potential reference points: (1) a population average payoff level, (2) the announced expected payoff of peers in a similar decision situation, (3) a historical average level of earnings that others have received in the same task, and (4) an announced anticipated individual payoff level, best describes decisions in a decontextualized risky decision making task. We find heterogeneity among individuals in the reference points they employ. The population average payoff level is the modal reference point, followed by experimenter's stated expectation of a participant's individual earnings, followed in turn by the average earnings of other participants in previous sessions of the same experiment. A sizeable share of individuals show multiple reference points simultaneously. The reference point that best fits the choices of the individual is not affected by a shock to her income.

  16. Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom.

    PubMed

    Resende, L M; Almeida, J R; Schezaro-Ramos, R; Collaço, R C O; Simioni, L R; Ramírez, D; González, W; Soares, A M; Calderon, L A; Marangoni, S; da Silva, S L

    2017-03-01

    Phospholipases A2 (PLA2s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA2s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA2s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic

  17. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection. PMID:22235204

  18. Chemoenzymatic synthesis of surfactants from carbohydrates, amino acids, and fatty acids.

    PubMed

    Bellahouel, S; Rolland, V; Roumestant, M L; Viallefont, P; Martinez, J

    2001-02-01

    The chemoenzymatic synthesis of new surfactants is reported; they were prepared from unprotected carbohydrates, amino acids, and fatty acids. This study pointed out the factors that govern the possibility to enzymatically bind the carbohydrate to the amino acid.

  19. A holographic critical point

    SciTech Connect

    DeWolfe, Oliver; Rosen, Christopher; Gubser, Steven S.

    2011-04-15

    We numerically construct a family of five-dimensional black holes exhibiting a line of first-order phase transitions terminating at a critical point at finite chemical potential and temperature. These black holes are constructed so that the equation of state and baryon susceptibilities approximately match QCD lattice data at vanishing chemical potential. The critical end point in the particular model we consider has temperature 143 MeV and chemical potential 783 MeV. Critical exponents are calculated, with results that are consistent with mean-field scaling relations.

  20. SAMPEX special pointing mode

    NASA Technical Reports Server (NTRS)

    Markley, F. Landis; Flatley, Thomas W.; Leoutsakos, Theodore

    1995-01-01

    A new pointing mode has been developed for the Solar, Anomalous, and Magnetospheric Particle Explorer (SAMPEX) spacecraft. This pointing mode orients the instrument boresights perpendicular to the field lines of the Earth's magnetic field in regions of low field strength and parallel to the field lines in regions of high field strength, to allow better characterization of heavy ions trapped by the field. The new mode uses magnetometer signals and is algorithmically simpler than the previous control mode, but it requires increased momentum wheel activity. It was conceived, designed, tested, coded, uplinked to the spacecraft, and activated in less than seven months.

  1. Point and Shoot Astronomy

    NASA Astrophysics Data System (ADS)

    Hoot, John E.

    2011-05-01

    A new generation of point and shoot digital cameras, when combined with open source firmware enhancements can operate as astrographs. This paper explores the research and astro-photographic opportunities and capabilities offered by this pairing of mass production optics and open source functional extensions that retail for as little as $200.

  2. ACCESS Pointing Control System

    NASA Technical Reports Server (NTRS)

    Brugarolas, Paul; Alexander, James; Trauger, John; Moody, Dwight; Egerman, Robert; Vallone, Phillip; Elias, Jason; Hejal, Reem; Camelo, Vanessa; Bronowicki, Allen; O'Connor, David; Partrick, Richard; Orzechowski, Pawel; Spitter, Connie; Lillie, Chuck

    2010-01-01

    ACCESS (Actively-Corrected Coronograph for Exoplanet System Studies) was one of four medium-class exoplanet concepts selected for the NASA Astrophysics Strategic Mission Concept Study (ASMCS) program in 2008/2009. The ACCESS study evaluated four major coronograph concepts under a common space observatory. This paper describes the high precision pointing control system (PCS) baselined for this observatory.

  3. EcoTipping Points

    ERIC Educational Resources Information Center

    Marten, Gerald G.; Matthews, Catherine E.

    2009-01-01

    Contrary to what we often hear and teach, there is good news to be found on the environmental front. Environmental success stories show us not only that sustainability is possible, but also how people have made it happen. We can make these stories and their lessons accessible to students with help from the EcoTipping Points Project, which has…

  4. The Lagrange Points

    ERIC Educational Resources Information Center

    Lovell, M.S.

    2007-01-01

    This paper presents a derivation of all five Lagrange points by methods accessible to sixth-form students, and provides a further opportunity to match Newtonian gravity with centripetal force. The predictive powers of good scientific theories are also discussed with regard to the philosophy of science. Methods for calculating the positions of the…

  5. Decision Points in Cataloging.

    ERIC Educational Resources Information Center

    Bright, Franklyn F.

    Libraries are frequently faced with policy decisions which can affect the quality and cost of library services for years to come. This point can be illustrated by citing examples of decisions made at the University of Wisconsin Library in the areas of: (1) conforming to national cataloging standards; (2) producing catalog cards in-house; and (3)…

  6. Optical Pointing Sensor

    NASA Technical Reports Server (NTRS)

    Shields, Joel F.; Metz, Brandon C.

    2010-01-01

    The optical pointing sensor provides a means of directly measuring the relative positions of JPL s Formation Control Testbed (FCT) vehicles without communication. This innovation is a steerable infrared (IR) rangefinder that gives measurements in terms of range and bearing to a passive retroreflector.

  7. Building Temperature Set Point

    SciTech Connect

    Meincke, Carol L.; Evans, Christopher A.

    2014-09-01

    This white paper provides information and recommendations for an actionable and enforceable corporate policy statement on temperature set points for office and related spaces at Sandia and presents a strategy that balances the need to achieve the energy goals with optimizing employee comfort and productivity.

  8. Points and Practices

    ERIC Educational Resources Information Center

    Ahmed, Syed Jamil; Heddon, Dee; Mackey, Sally

    2007-01-01

    This collection of three articles represents the "Points and Practices" section of this month's issue of "Research in Drama Education." The first article, "'Fitting the Bill' for 'Helping Them.' A Response to 'Integrated Popular Theatre Approach in Africa' and 'Commissioned Theatre Projects on Human Rights in…

  9. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking

    SciTech Connect

    Uchiumi, T.; Wahba, A.J.; Traut, R.R.

    1987-08-01

    The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by diagonal (two-dimensional nonreducing-reducing) NaDodSO/sub 4//polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO/sub 4/ gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, PO. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO/sub 4//polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)/sub 2/ and (P2)/sub 2/ dimers, each of which is anchored to PO. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.

  10. A new point mutation in the deoxyribonuclic acid-binding domain of the vitamine D receptor in a kindred with hereditary 1,25-dihydroxyvitamin d-resistant rickets

    SciTech Connect

    Yagi, Hideki; Miyake, Hiroshi; Nagashima, Kanji; Kuroume, Takayoshi ); Ozone, K.; Pike, J.W. )

    1993-02-01

    Hereditary 1,25-dihydroxyvitamin D [1,25-(OH)[sub 2]D]-resistant rickets (HVDRR) is a rare disorder characterized by rickets, alopecia, hypocalcemia, secondary hyperparathyroidism, and normal or elevated serum 1,25-dihydroxyvitamin D levels. The authors describe a patient with typical clinical characteristics of HVDRR, except that elevated levels of serum phosphorus were present coincident with increased levels of serum intact PTH. The patient was treated with high dose calcium infusion after an ineffective treatment with 1[alpha]-hydroxyvitamin D[sub 3]; serum calcium and phosphorus as well as intact PTH and alkaline phosphatase levels were normalized. Evaluation of phytohemagglutinin-activated lymphocytes derived from this patient revealed that 1,25-(OH)[sub 2]D[sub 3] was unable to inhibit thymidine incooperation, a result that contrast with the capacity of 1,25-(OH)[sub 2]D[sub 3] to inhibit uptake into normal activated lymphocytes. 1,25-(OH)[sub 2]D[sub 3] did not induce human osteocalcin promoter activity after transfection of this DNA linked to a reporter gene into patient cells. Cointroduction of a human vitamin D receptor (VDR) cDNA expression vector with the reporter plasmid, however, restored the hormone response. Evaluation of extracts from the patient cells for VDR DNA binding revealed a defect in DNA binding. Analysis of genomic DNA from the patient's cells by PCR confirmed the presence of a point mutation in exon 2 of the VDR. This exon directs synthesis of a portion of the DNA-binding domain of the receptor. We conclude that the genetic basis for 1,25-(OH)[sub 2]D[sub 3] resistance in this kindred with VDR-positive HVDRR is due to a single base mutation in the VDR that leads to production of a receptor unable to interact appropriately with DNA. 20 refs., 3 figs., 1 tab.

  11. Quantum Change Point

    NASA Astrophysics Data System (ADS)

    Sentís, Gael; Bagan, Emilio; Calsamiglia, John; Chiribella, Giulio; Muñoz-Tapia, Ramon

    2016-10-01

    Sudden changes are ubiquitous in nature. Identifying them is crucial for a number of applications in biology, medicine, and social sciences. Here we take the problem of detecting sudden changes to the quantum domain. We consider a source that emits quantum particles in a default state, until a point where a mutation occurs that causes the source to switch to another state. The problem is then to find out where the change occurred. We determine the maximum probability of correctly identifying the change point, allowing for collective measurements on the whole sequence of particles emitted by the source. Then, we devise online strategies where the particles are measured individually and an answer is provided as soon as a new particle is received. We show that these online strategies substantially underperform the optimal quantum measurement, indicating that quantum sudden changes, although happening locally, are better detected globally.

  12. Point clouds in BIM

    NASA Astrophysics Data System (ADS)

    Antova, Gergana; Kunchev, Ivan; Mickrenska-Cherneva, Christina

    2016-10-01

    The representation of physical buildings in Building Information Models (BIM) has been a subject of research since four decades in the fields of Construction Informatics and GeoInformatics. The early digital representations of buildings mainly appeared as 3D drawings constructed by CAD software, and the 3D representation of the buildings was only geometric, while semantics and topology were out of modelling focus. On the other hand, less detailed building representations, with often focus on ‘outside’ representations were also found in form of 2D /2,5D GeoInformation models. Point clouds from 3D laser scanning data give a full and exact representation of the building geometry. The article presents different aspects and the benefits of using point clouds in BIM in the different stages of a lifecycle of a building.

  13. Quantum Change Point.

    PubMed

    Sentís, Gael; Bagan, Emilio; Calsamiglia, John; Chiribella, Giulio; Muñoz-Tapia, Ramon

    2016-10-07

    Sudden changes are ubiquitous in nature. Identifying them is crucial for a number of applications in biology, medicine, and social sciences. Here we take the problem of detecting sudden changes to the quantum domain. We consider a source that emits quantum particles in a default state, until a point where a mutation occurs that causes the source to switch to another state. The problem is then to find out where the change occurred. We determine the maximum probability of correctly identifying the change point, allowing for collective measurements on the whole sequence of particles emitted by the source. Then, we devise online strategies where the particles are measured individually and an answer is provided as soon as a new particle is received. We show that these online strategies substantially underperform the optimal quantum measurement, indicating that quantum sudden changes, although happening locally, are better detected globally.

  14. The boiling point of stratospheric aerosols.

    NASA Technical Reports Server (NTRS)

    Rosen, J. M.

    1971-01-01

    A photoelectric particle counter was used for the measurement of aerosol boiling points. The operational principle involves raising the temperature of the aerosol by vigorously heating a portion of the intake tube. At or above the boiling point, the particles disintegrate rather quickly, and a noticeable effect on the size distribution and concentration is observed. Stratospheric aerosols appear to have the same volatility as a solution of 75% sulfuric acid. Chemical analysis of the aerosols indicates that there are other substances present, but that the sulfate radical is apparently the major constituent.

  15. Oscillating stagnation point flow

    NASA Technical Reports Server (NTRS)

    Grosch, C. E.; Salwen, H.

    1982-01-01

    A solution of the Navier-Stokes equations is given for an incompressible stagnation point flow whose magnitude oscillates in time about a constant, non-zero, value (an unsteady Hiemenz flow). Analytic approximations to the solution in the low and high frequency limits are given and compared with the results of numerical integrations. The application of these results to one aspect of the boundary layer receptivity problem is also discussed.

  16. Oscillating stagnation point flow

    NASA Astrophysics Data System (ADS)

    Grosch, C. E.; Salwen, H.

    1982-11-01

    A solution of the Navier-Stokes equations is given for an incompressible stagnation point flow whose magnitude oscillates in time about a constant, non-zero, value (an unsteady Hiemenz flow). Analytic approximations to the solution in the low and high frequency limits are given and compared with the results of numerical integrations. The application of these results to one aspect of the boundary layer receptivity problem is also discussed.

  17. The effect of an acidic, copper sulfate-based commercial sanitizer on indicator, pathogenic, and spoilage bacteria associated with broiler chicken carcasses when applied at various intervention points during poultry processing.

    PubMed

    Russell, S M

    2008-07-01

    Studies were conducted to evaluate 1) the effect of an acidic, copper sulfate-based commercial sanitizer on pathogenic, indicator, and spoilage bacteria in a model scalder system, 2) the effect of this sanitizer on total aerobic bacteria (APC) and Escherichia coli counts, and Salmonella prevalence on broiler chicken carcasses when applied during scalding or scalding and postpick dipping, and 3) the ability of sanitizer to extend the shelf-life of broiler chicken carcasses. Exposure of Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas fluorescens, or Shewanella putrefaciens to the sanitizer in scalder water at 54 degrees C for 2 min resulted in complete elimination of these bacterial species. Exposure of E. coli to the treated scald water resulted in a 4.9 log(10) reduction. These data suggest that this sanitizer would be effective for use in scalders. When applied during scalding in a commercial processing plant, APC and E. coli counts were significantly (P

  18. Challenges of Diagnosing Acute HIV-1 Subtype C Infection in African Women: Performance of a Clinical Algorithm and the Need for Point-of-Care Nucleic-Acid Based Testing

    PubMed Central

    Mlisana, Koleka; Sobieszczyk, Magdalena; Werner, Lise; Feinstein, Addi; van Loggerenberg, Francois; Naicker, Nivashnee; Williamson, Carolyn; Garrett, Nigel

    2013-01-01

    Background Prompt diagnosis of acute HIV infection (AHI) benefits the individual and provides opportunities for public health intervention. The aim of this study was to describe most common signs and symptoms of AHI, correlate these with early disease progression and develop a clinical algorithm to identify acute HIV cases in resource limited setting. Methods 245 South African women at high-risk of HIV-1 were assessed for AHI and received monthly HIV-1 antibody and RNA testing. Signs and symptoms at first HIV-positive visit were compared to HIV-negative visits. Logistic regression identified clinical predictors of AHI. A model-based score was assigned to each predictor to create a risk score for every woman. Results Twenty-eight women seroconverted after a total of 390 person-years of follow-up with an HIV incidence of 7.2/100 person-years (95%CI 4.5–9.8). Fifty-seven percent reported ≥1 sign or symptom at the AHI visit. Factors predictive of AHI included age <25 years (OR = 3.2; 1.4–7.1), rash (OR = 6.1; 2.4–15.4), sore throat (OR = 2.7; 1.0–7.6), weight loss (OR = 4.4; 1.5–13.4), genital ulcers (OR = 8.0; 1.6–39.5) and vaginal discharge (OR = 5.4; 1.6–18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p<0.001). Conclusions Accurate recognition of signs and symptoms of AHI is critical for early diagnosis of HIV infection. Our algorithm may assist in risk-stratifying individuals for AHI, especially in resource-limited settings where there is no routine testing for AHI. Independent validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care antigen or viral load technology is required, however, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission. PMID:23646162

  19. Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

    PubMed

    Charnas, R L; Fisher, J; Knowles, J R

    1978-05-30

    Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.

  20. Vernal Point and Anthropocene

    NASA Astrophysics Data System (ADS)

    Chavez-Campos, Teodosio; Chavez S, Nadia; Chavez-Sumarriva, Israel

    2014-05-01

    The time scale was based on the internationally recognized formal chronostratigraphical /geochronological subdivisions of time: The Phanerozoic Eonathem/Eon; the Cenozoic Erathem/Era; the Quaternary System/Period; the Pleistocene and Holocene Series/Epoch. The Quaternary was divided into: (1) The Pleistocene that was characterized by cycles of glaciations (intervals between 40,000 and 100,000 years). (2) The Holocene that was an interglacial period that began about 12,000 years ago. It was believed that the Milankovitch cycles (eccentricity, axial tilt and the precession of the equinoxes) were responsible for the glacial and interglacial Holocene periods. The magnetostratigraphic units have been widely used for global correlations valid for Quaternary. The gravitational influence of the sun and moon on the equatorial bulges of the mantle of the rotating earth causes the precession of the earth. The retrograde motion of the vernal point through the zodiacal band is 26,000 years. The Vernal point passes through each constellation in an average of 2000 years and this period of time was correlated to Bond events that were North Atlantic climate fluctuations occurring every ≡1,470 ± 500 years throughout the Holocene. The vernal point retrogrades one precessional degree approximately in 72 years (Gleissberg-cycle) and approximately enters into the Aquarius constellation on March 20, 1940. On earth this entry was verify through: a) stability of the magnetic equator in the south central zone of Peru and in the north zone of Bolivia, b) the greater intensity of equatorial electrojet (EEJ) in Peru and Bolivia since 1940. With the completion of the Holocene and the beginning of the Anthropocene (widely popularized by Paul Crutzen) it was proposed the date of March 20, 1940 as the beginning of the Anthropocene. The date proposed was correlated to the work presented in IUGG (Italy 2007) with the title "Cusco base meridian for the study of geophysical data"; Cusco was

  1. Prelinguistic Vocalizations Distinguish Pointing Acts

    ERIC Educational Resources Information Center

    Grunloh, Thomas; Liszkowski, Ulf

    2015-01-01

    The current study investigated whether point-accompanying characteristics, like vocalizations and hand shape, differentiate infants' underlying motives of prelinguistic pointing. We elicited imperative (requestive) and declarative (expressive and informative) pointing acts in experimentally controlled situations, and analyzed accompanying…

  2. Quantitation of estrogen receptor in seventy-five specimens of breast cancer: comparison between an immunoassay (Abbott ER-EIA monoclonal) and a (3H)estradiol binding assay based on isoelectric focusing in polyacrylamide gel

    SciTech Connect

    Pousette, A.; Gustafsson, S.A.; Thoernblad, A.M.N.; Nordgren, A.; Saellstroem, J.Li.; Lindgren, A.; Sundelin, P.; Gustafsson, J.A.

    1986-08-01

    Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of (/sup 3/H)estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.

  3. Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells.

    PubMed

    Elschenbroich, Sarah; Ignatchenko, Vladimir; Sharma, Parveen; Schmitt-Ulms, Gerold; Gramolini, Anthony O; Kislinger, Thomas

    2009-10-01

    High-resolution peptide separation is pivotal for successful shotgun proteomics. The need for capable techniques propels invention and improvement of ever more sophisticated approaches. Recently, Agilent Technologies has introduced the OFFGEL fractionator, which conducts peptide separation by isoelectric focusing in an off-gel setup. This platform has been shown to accomplish high resolution of peptides for diverse sample types, yielding valuable advantages over comparable separation techniques. In this study, we deliver the first comparison of the newly emerging OFFGEL approach to the well-established on-line MudPIT platform. Samples from a membrane-enriched fraction isolated from murine C2C12 cells were subjected to replicate analysis by OFFGEL (12 fractions, pH 3-10) followed by RP-LC-MS/MS or 12-step on-line MudPIT. OFFGEL analyses yielded 1398 proteins (identified by 10,269 peptides), while 1428 proteins (11,078 peptides) were detected with the MudPIT approach. Thus, our data shows that both platforms produce highly comparable results in terms of protein/peptide identifications and reproducibility for the sample type analyzed. We achieve more accurate peptide focusing after OFFGEL fractionation with 88% of all peptides binned to a single fraction, as compared to 61% of peptides detected in only one step in MudPIT analyses. Our study suggests that both platforms are equally capable of high quality peptide separation of a sample with medium complexity, rendering them comparably valuable for comprehensive proteomic analyses.

  4. Pointing control for LDR

    NASA Technical Reports Server (NTRS)

    Yam, Y.; Briggs, C.

    1988-01-01

    One important aspect of the LDR control problem is the possible excitations of structural modes due to random disturbances, mirror chopping, and slewing maneuvers. An analysis was performed to yield a first order estimate of the effects of such dynamic excitations. The analysis involved a study of slewing jitters, chopping jitters, disturbance responses, and pointing errors, making use of a simplified planar LDR model which describes the LDR dynamics on a plane perpendicular to the primary reflector. Briefly, the results indicate that the command slewing profile plays an important role in minimizing the resultant jitter, even to a level acceptable without any control action. An optimal profile should therefore be studied.

  5. Modeling of formation and prevention of a pure water zone in capillary isoelectric focusing with narrow pH range carrier ampholytes.

    PubMed

    Takácsi-Nagy, Anna; Kilár, Ferenc; Thormann, Wolfgang

    2017-03-01

    This paper comprises a continuation of computer simulation studies dealing with carrier ampholyte based CIEF in presence of narrow pH gradients. With this technique, amphoteric sample components with pI values outside the pH gradient are migrating isotachophoretically toward the cathode or anode whereas components with pI values within the gradient become focused. In order to understand the processes occurring in presence of the electric field, the behavior of both carrier ampholytes and amphoteric sample components is investigated by computer modeling. Characteristics of two pH unit gradients with end components having pI values at or around 7.00 and conditions that lead to the formation of a water zone at neutrality were investigated. Data obtained reveal that a zone of water is formed in focusing with carrier ampholytes when the applied pH range does not cover the neutral region, ends at pH 7.00 or begins at pH 7.00. The presence of additional amphoteric components that cover the neutrality region prevent water zone formation under current flow. This situation is met in experiments with narrow pH gradients that end or begin around neutrality. Simulation data reveal that no water zone evolves when atmospheric carbon dioxide dissolved in the catholyte causes the migration of carbonic acid (in the form of carbonate and/or hydrogen carbonate ions) from the catholyte through the focusing structure. An electrolyte change in the electrode solution does not have an impact on the focusing part but does change the isotachophoretic pattern migrating behind the leading ion.

  6. pH-driven solubilization and isoelectric precipitation of proteins from the brown seaweed Saccharina latissima-effects of osmotic shock, water volume and temperature.

    PubMed

    Vilg, Jenny Veide; Undeland, Ingrid

    2017-01-01

    In the light of the global search for novel and sustainable protein sources, macroalgal proteins are becoming an attractive target. To date, mainly red and green macroalgae have been investigated in this respect, whereas the brown species are less studied, possibly because of the lower content of protein. In a biorefinery context, however, the protein content of brown macroalgae can still be economically interesting due to fast growth and the possibility to co-extract other compounds, such as alginates. The aim of this study was to develop a simple, scalable pH shift-based protein isolation technique applicable on wet Saccharina latissima biomass. Factors investigated were extraction volume, temperature, protein solubilization pH, osmoshock pretreatment and protein precipitation pH. Maximum protein solubility was obtained at pH 12, where 34 % of the total protein content could be extracted with 5.56 volumes of extraction solution (20 volumes on dry weight (dw) basis). Osmoshocking significantly increased the yield, and 20, 40 and 60 volumes of water (dw basis) gave 45.1, 46.8 and 59.5 % yield, respectively. The temperature during osmoshocking did not significantly affect the extraction yield, and extended time (16 vs. 1 or 2 h) reduced protein yield. Precipitation of solubilized proteins was possible below pH 4; the highest precipitation yield, 34.5 %, was obtained at pH 2. After combined alkaline extraction and acid precipitation, 16.01 % of the Saccharina proteins were recovered, which can be considered acceptable in comparison to other studies on algae but leaves some room for improvement when compared to protein extraction from, for instance, soy.

  7. Relative Critical Points

    NASA Astrophysics Data System (ADS)

    Lewis, Debra

    2013-05-01

    Relative equilibria of Lagrangian and Hamiltonian systems with symmetry are critical points of appropriate scalar functions parametrized by the Lie algebra (or its dual) of the symmetry group. Setting aside the structures - symplectic, Poisson, or variational - generating dynamical systems from such functions highlights the common features of their construction and analysis, and supports the construction of analogous functions in non-Hamiltonian settings. If the symmetry group is nonabelian, the functions are invariant only with respect to the isotropy subgroup of the given parameter value. Replacing the parametrized family of functions with a single function on the product manifold and extending the action using the (co)adjoint action on the algebra or its dual yields a fully invariant function. An invariant map can be used to reverse the usual perspective: rather than selecting a parametrized family of functions and finding their critical points, conditions under which functions will be critical on specific orbits, typically distinguished by isotropy class, can be derived. This strategy is illustrated using several well-known mechanical systems - the Lagrange top, the double spherical pendulum, the free rigid body, and the Riemann ellipsoids - and generalizations of these systems.

  8. Deconfined Quantum Critical Points

    NASA Astrophysics Data System (ADS)

    Senthi, T.; Vishwanath, Ashvin; Balents, Leon; Sachdev, Subir; Fisher, Matthew P. A.

    The theory of second-order phase transitions is one of the foundations of modern statistical mechanics and condensed-matter theory. A central concept is the observable order parameter, whose nonzero average value characterizes one or more phases. At large distances and long times, fluctuations of the order parameter(s) are described by a continuum field theory, and these dominate the physics near such phase transitions. We show that near second-order quantum phase transitions, subtle quantum interference effects can invalidate this paradigm, and we present a theory of quantum critical points in a variety of experimentally relevant two-dimensional antiferromagnets. The critical points separate phases characterized by conventional "confining" order parameters. Nevertheless, the critical theory contains an emergent gauge field and "deconfined" degrees of freedom associated with fractionalization of the order parameters. We propose that this paradigm for quantum criticality may be the key to resolving a number of experimental puzzles in correlated electron systems and offer a new perspective on the properties of complex materials.

  9. HORSESHOE CURVE IN GLACIER POINT ROAD NEAR GLACIER POINT. HALF ...

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

    HORSESHOE CURVE IN GLACIER POINT ROAD NEAR GLACIER POINT. HALF DOME AT CENTER REAR. SAME VIEW AT CA-157-2. LOOKING NNE. GIS: N-37' 43 44.3 / W-119 34 14.1 - Glacier Point Road, Between Chinquapin Flat & Glacier Point, Yosemite Village, Mariposa County, CA

  10. 2. HORSESHOE CURVE IN GLACIER POINT ROAD NEAR GLACIER POINT. ...

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

    2. HORSESHOE CURVE IN GLACIER POINT ROAD NEAR GLACIER POINT. HALF DOME AT CENTER REAR. LOOKING NNE. GIS N-37 43 44.3 / W-119 34 14.1 - Glacier Point Road, Between Chinquapin Flat & Glacier Point, Yosemite Village, Mariposa County, CA

  11. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Control points and dispatch points. 80.41... SERVICES STATIONS IN THE MARITIME SERVICES Applications and Licenses § 80.41 Control points and dispatch... dispatch points may be installed and used without obtaining any authorization from the Commission....

  12. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Control points and dispatch points. 80.41... SERVICES STATIONS IN THE MARITIME SERVICES Applications and Licenses § 80.41 Control points and dispatch... dispatch points may be installed and used without obtaining any authorization from the Commission....

  13. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Control points and dispatch points. 80.41... SERVICES STATIONS IN THE MARITIME SERVICES Applications and Licenses § 80.41 Control points and dispatch... dispatch points may be installed and used without obtaining any authorization from the Commission....

  14. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Control points and dispatch points. 80.41... SERVICES STATIONS IN THE MARITIME SERVICES Applications and Licenses § 80.41 Control points and dispatch... dispatch points may be installed and used without obtaining any authorization from the Commission....

  15. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Control points and dispatch points. 80.41... SERVICES STATIONS IN THE MARITIME SERVICES Applications and Licenses § 80.41 Control points and dispatch... dispatch points may be installed and used without obtaining any authorization from the Commission....

  16. Thin wire pointing method

    NASA Technical Reports Server (NTRS)

    Green, G.; Mattauch, R. J. (Inventor)

    1983-01-01

    A method is described for forming sharp tips on thin wires, in particular phosphor bronze wires of diameters such as one-thousandth inch used to contact micron size Schottky barrier diodes, which enables close control of tip shape and which avoids the use of highly toxic solutions. The method includes dipping an end of a phosphor bronze wire into a dilute solution of sulfamic acid and applying a current through the wire to electrochemically etch it. The humidity in the room is controlled to a level of less than 50%, and the voltage applied between the wire and another electrode in the solutions is a half wave rectified voltage. The current through the wire is monitored, and the process is stopped when the current falls to a predetermined low level.

  17. Repelling Point Bosons

    NASA Astrophysics Data System (ADS)

    McGuire, J. B.

    2011-12-01

    There is a body of conventional wisdom that holds that a solvable quantum problem, by virtue of its solvability, is pathological and thus irrelevant. It has been difficult to refute this view owing to the paucity of theoretical constructs and experimental results. Recent experiments involving equivalent ions trapped in a spatial conformation of extreme anisotropic confinement (longitudinal extension tens, hundreds or even thousands of times transverse extension) have modified the view of relevancy, and it is now possible to consider systems previously thought pathological, in particular point Bosons that repel in one dimension. It has been difficult for the experimentalists to utilize existing theory, mainly due to long-standing theoretical misunderstanding of the relevance of the permutation group, in particular the non-commutativity of translations (periodicity) and transpositions (permutation). This misunderstanding is most easily rectified in the case of repelling Bosons.

  18. Isolation and Characterization of Chinese Standard Fulvic Acid Sub-fractions Separated from Forest Soil by Stepwise Elution with Pyrophosphate Buffer

    NASA Astrophysics Data System (ADS)

    Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P.

    2015-03-01

    XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.

  19. Isolation and characterization of Chinese standard fulvic acid sub-fractions separated from forest soil by stepwise elution with pyrophosphate buffer.

    PubMed

    Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P

    2015-03-04

    XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.

  20. Isolation and Characterization of Chinese Standard Fulvic Acid Sub-fractions Separated from Forest Soil by Stepwise Elution with Pyrophosphate Buffer

    PubMed Central

    Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P.

    2015-01-01

    XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants. PMID:25735451

  1. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification.

    PubMed

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle

    2015-01-01

    This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography.

  2. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  3. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium salt may be safely used in accordance...: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum 98 percent (dry basis). Sodium salt...

  4. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  5. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  6. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  7. Function Point Analysis Depot

    NASA Technical Reports Server (NTRS)

    Muniz, R.; Martinez, El; Szafran, J.; Dalton, A.

    2011-01-01

    The Function Point Analysis (FPA) Depot is a web application originally designed by one of the NE-C3 branch's engineers, Jamie Szafran, and created specifically for the Software Development team of the Launch Control Systems (LCS) project. The application consists of evaluating the work of each developer to be able to get a real estimate of the hours that is going to be assigned to a specific task of development. The Architect Team had made design change requests for the depot to change the schema of the application's information; that information, changed in the database, needed to be changed in the graphical user interface (GUI) (written in Ruby on Rails (RoR and the web service/server side in Java to match the database changes. These changes were made by two interns from NE-C, Ricardo Muniz from NE-C3, who made all the schema changes for the GUI in RoR and Edwin Martinez, from NE-C2, who made all the changes in the Java side.

  8. Pointing at 'Puffin'

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site] [figure removed for brevity, see original site] Figure 1Figure 2

    The intriguing dunes at the bottom of 'Endurance Crater' presented a tantalizing target for the science team for NASA's Mars Exploration Rover Opportunity. After analyzing the soil near and around the dunes, however, the rover engineering team decided that it was too risky to send Opportunity any closer. The terrain between the rover and the dune tendril did not present clear evidence of rocky plates to give the rover sufficient traction. A finger-like dune tendril pictured here (left) is, essentially, pointing to the rover's current area of investigation. Opportunity rolled over the foreground rock, called 'Puffin.' During the past several sols the rover has been examining its new neighborhood, an area that includes the rocks 'Ellesmere' and 'Escher' (not pictured) and the soil targets 'Shag' and 'Auk' (also not pictured). Experiencing significant slippage, the rover did some unintended trenching and left deep tracks in this area. This view is a mosaic of two images taken by the rover's navigation camera on Opportunity's 206th sol on Mars (August 22, 2004) and presented in a perspective projection. Figure 1 is a cylindrical-perspective projection and Figure 2 is a cylindrical perspective.

  9. Hidden isosbestic point(s) in ultraviolet spectra.

    PubMed

    Pouët, M-F; Baures, E; Vaillant, S; Thomas, O

    2004-04-01

    The existence of isosbestic point(s) in a set of UV-visible spectra is rarely exploited for quantitative or qualitative information despite its interest. Indeed, the presence of isosbestic points means a quality and quantity conservation in the global composition of samples with a given relation between the concentration of absorbing compounds or mixtures of compounds. However, in some cases, for example, when a dilution occurs, no isosbestic point appears. This work shows that a simple operation, called normalization, can reveal the existence of hidden isosbestic point(s) (IP*). This paper first summarizes the required conditions for the presence of isosbestic points. Second, it demonstrates the significance of IP* revealed by normalization. An example of the exploitation of direct or hidden isosbestic points in the environmental field illustrates the interest in and application of the technique.

  10. What's the Point? The Benefits of Grading without Points

    ERIC Educational Resources Information Center

    Brilleslyper, Mike; Ghrist, Michelle; Holcomb, Trae; Schaubroeck, Beth; Warner, Bradley; Williams, Scott

    2012-01-01

    Most instructors desire to create a learning environment conducive to deep learning. However, in courses where grades are based upon the accumulation of points, students may focus on collecting points rather than on learning and achieving the course objectives. We discuss an alternative points-free course design based on the development of clear…

  11. Aspartic acid

    MedlinePlus

    ... also called asparaginic acid. Aspartic acid helps every cell in the body work. It plays a role in: Hormone production and release Normal nervous system function Plant sources of aspartic acid include: Legumes such as ...

  12. Folic Acid

    MedlinePlus

    Folic acid is a B vitamin. It helps the body make healthy new cells. Everyone needs folic acid. For women who may get pregnant, it is really important. Getting enough folic acid before and during pregnancy can prevent major birth ...

  13. Folic Acid

    MedlinePlus

    Folic acid is used to treat or prevent folic acid deficiency. It is a B-complex vitamin needed by ... Folic acid comes in tablets. It usually is taken once a day. Follow the directions on your prescription label ...

  14. On Pfaffian Random Point Fields

    NASA Astrophysics Data System (ADS)

    Kargin, V.

    2014-02-01

    We study Pfaffian random point fields by using the Moore-Dyson quaternion determinants. First, we give sufficient conditions that ensure that a self-dual quaternion kernel defines a valid random point field, and then we prove a CLT for Pfaffian point fields. The proofs are based on a new quaternion extension of the Cauchy-Binet determinantal identity. In addition, we derive the Fredholm determinantal formulas for the Pfaffian point fields which use the quaternion determinant.

  15. Acid Rain

    USGS Publications Warehouse

    Bricker, Owen P.; Rice, Karen C.

    1995-01-01

    Although acid rain is fading as a political issue in the United States and funds for research in this area have largely disappeared, the acidity of rain in the Eastern United States has not changed significantly over the last decade, and it continues to be a serious environmental problem. Acid deposition (commonly called acid rain) is a term applied to all forms of atmospheric deposition of acidic substances - rain, snow, fog, acidic dry particulates, aerosols, and acid-forming gases. Water in the atmosphere reacts with certain atmospheric gases to become acidic. For example, water reacts with carbon dioxide in the atmosphere to produce a solution with a pH of about 5.6. Gases that produce acids in the presence of water in the atmosphere include carbon dioxide (which converts to carbonic acid), oxides of sulfur and nitrogen (which convert to sulfuric and nitric acids}, and hydrogen chloride (which converts to hydrochloric acid). These acid-producing gases are released to the atmosphere through natural processes, such as volcanic emissions, lightning, forest fires, and decay of organic matter. Accordingly, precipitation is slightly acidic, with a pH of 5.0 to 5.7 even in undeveloped areas. In industrialized areas, most of the acid-producing gases are released to the atmosphere from burning fossil fuels. Major emitters of acid-producing gases include power plants, industrial operations, and motor vehicles. Acid-producing gases can be transported through the atmosphere for hundreds of miles before being converted to acids and deposited as acid rain. Because acids tend to build up in the atmosphere between storms, the most acidic rain falls at the beginning of a storm, and as the rain continues, the acids "wash out" of the atmosphere.

  16. Ground point filtering of UAV-based photogrammetric point clouds

    NASA Astrophysics Data System (ADS)

    Anders, Niels; Seijmonsbergen, Arie; Masselink, Rens; Keesstra, Saskia

    2016-04-01

    Unmanned Aerial Vehicles (UAVs) have proved invaluable for generating high-resolution and multi-temporal imagery. Based on photographic surveys, 3D surface reconstructions can be derived photogrammetrically so producing point clouds, orthophotos and surface models. For geomorphological or ecological applications it may be necessary to separate ground points from vegetation points. Existing filtering methods are designed for point clouds derived using other methods, e.g. laser scanning. The purpose of this paper is to test three filtering algorithms for the extraction of ground points from point clouds derived from low-altitude aerial photography. Three subareas were selected from a single flight which represent different scenarios: 1) low relief, sparsely vegetated area, 2) low relief, moderately vegetated area, 3) medium relief and moderately vegetated area. The three filtering methods are used to classify ground points in different ways, based on 1) RGB color values from training samples, 2) TIN densification as implemented in LAStools, and 3) an iterative surface lowering algorithm. Ground points are then interpolated into a digital terrain model using inverse distance weighting. The results suggest that different landscapes require different filtering methods for optimal ground point extraction. While iterative surface lowering and TIN densification are fully automated, color-based classification require fine-tuning in order to optimize the filtering results. Finally, we conclude that filtering photogrammetric point clouds could provide a cheap alternative to laser scan surveys for creating digital terrain models in sparsely vegetated areas.

  17. Imaging study on acupuncture points

    NASA Astrophysics Data System (ADS)

    Yan, X. H.; Zhang, X. Y.; Liu, C. L.; Dang, R. S.; Ando, M.; Sugiyama, H.; Chen, H. S.; Ding, G. H.

    2009-09-01

    The topographic structures of acupuncture points were investigated by using the synchrotron radiation based Dark Field Image (DFI) method. Four following acupuncture points were studied: Sanyinjiao, Neiguan, Zusanli and Tianshu. We have found that at acupuncture point regions there exists the accumulation of micro-vessels. The images taken in the surrounding tissue out of the acupuncture points do not show such kind of structure. It is the first time to reveal directly the specific structure of acupuncture points by X-ray imaging.

  18. Hormone purification by isoelectric focusing in space

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1988-01-01

    The objective of the program was the definition and development of optimal methods for electrophoretic separations in microgravity. The approach is based on a triad consisting of ground based experiments, mathematical modeling and experiments in microgravity. Zone electrophoresis is a rate process, where separation is achieved in uniform buffers on the basis of differences in electrophoretic mobilities. Optimization and modeling of continuous flow electrophoresis mainly concern the hydrodynamics of the flow process, including gravity dependent fluid convection due to density gradients and gravity independent electroosmosis. Optimization of focusing requires a more complex model describing the molecular transport processes involved in electrophoresis of interacting systems. Three different focusing instruments were designed, embodying novel principles of fluid stabilization. Fluid stability was achieved by: (1) flow streamlining by means of membrane elements in combination with rapid fluid recycling; (2) apparatus rotation in combination with said membrane elements; and (3) shear stress induced by rapid recycling through a narrow gap channel.

  19. Efficient algorithm for simulation of isoelectric focusing.

    PubMed

    Yoo, Kisoo; Shim, Jaesool; Liu, Jin; Dutta, Prashanta

    2014-03-01

    IEF simulation is an effective tool to investigate the transport phenomena and separation performance as well as to design IEF microchip. However, multidimensional IEF simulations are computationally intensive as one has to solve a large number of mass conservation equations for ampholytes to simulate a realistic case. In this study, a parallel scheme for a 2D IEF simulation is developed to reduce the computational time. The calculation time for each equation is analyzed to identify which procedure is suitable for parallelization. As expected, simultaneous solution of mass conservation equations of ampholytes is identified as the computational hot spot, and the computational time can be significantly reduced by parallelizing the solution procedure for that. Moreover, to optimize the computing time, electric potential behavior during transient state is investigated. It is found that for a straight channel the transient variation of electric potential along the channel is negligible in a narrow pH range (5∼8) IEF. Thus the charge conservation equation is solved for the first time step only, and the electric potential obtain from that is used for subsequent calculations. IEF simulations are carried out using this algorithm for separation of cardiac troponin I from serum albumin in a pH range of 5-8 using 192 biprotic ampholytes. Significant reduction in simulation time is achieved using the parallel algorithm. We also study the effect of number of ampholytes to form the pH gradient and its effect in the focusing and separation behavior of cardiac troponin I and albumin. Our results show that, at the completion of separation phase, the pH profile is stepwise for lower number of ampholytes, but becomes smooth as the number of ampholytes increases. Numerical results also show that higher protein concentration can be obtained using higher number of ampholytes.

  20. Acid Rain.

    ERIC Educational Resources Information Center

    Openshaw, Peter

    1987-01-01

    Provides some background information on acid deposition. Includes a historical perspective, describes some effects of acid precipitation, and discusses acid rain in the United Kingdom. Contains several experiments that deal with the effects of acid rain on water quality and soil. (TW)

  1. Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

    PubMed

    Dada, Oluwatosin O; Jaya, Nomalie; Valliere-Douglass, John; Salas-Solano, Oscar

    2015-08-20

    Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

  2. The Point of No Return

    PubMed Central

    Logan, Gordon D.

    2015-01-01

    Bartlett (1958) described the point of no return as a point of irrevocable commitment to action, which was preceded by a period of gradually increasing commitment. As such, the point of no return reflects a fundamental limit on the ability to control thought and action. I review the literature on the point of no return, taking three perspectives. First, I consider the point of no return from the perspective of the controlled act, as a locus in the architecture and anatomy of the underlying processes. I review experiments from the stop-signal paradigm that suggest that the point of no return is located late in the response system. Then I consider the point of no return from the perspective of the act of control that tries to change the controlled act before it becomes irrevocable. From this perspective, the point of no return is a point in time that provides enough “lead time” for the act of control to take effect. I review experiments that measure the response time to the stop signal as the lead time required for response inhibition in the stop-signal paradigm. Finally, I consider the point of no return in hierarchically controlled tasks, in which there may be many points of no return at different levels of the hierarchy. I review experiments on skilled typing that suggest different points of no return for the commands that determine what is typed and the countermands that inhibit typing, with increasing commitment to action the lower the level in the hierarchy. I end by considering the point of no return in perception and thought as well as action. PMID:25633089

  3. Development of a Chip/Chip/SRM platform using digital chip isoelectric focusing and LC-Chip mass spectrometry for enrichment and quantitation of low abundance protein biomarkers in human plasma

    PubMed Central

    Rafalko, Agnes; Dai, Shujia; Hancock, William S.; Karger, Barry L.; Hincapie, Marina

    2013-01-01

    Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC™) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ~1–2.5 ng/mL with a CV of ~13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r2 = 0.9459) was observed between standard clinical ELISA tests and the SRM-based-assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples. PMID:22098410

  4. Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat.

    PubMed

    Zhao, X; Xing, T; Chen, X; Han, M-Y; Li, X; Xu, X-L; Zhou, G-H

    2016-11-11

    Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G(')) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G(') was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH.

  5. Modeling the acid-base properties of bacterial surfaces: A combined spectroscopic and potentiometric study of the gram-positive bacterium Bacillus subtilis.

    PubMed

    Leone, Laura; Ferri, Diego; Manfredi, Carla; Persson, Per; Shchukarev, Andrei; Sjöberg, Staffan; Loring, John

    2007-09-15

    In this study, macroscopic and spectroscopic data were combined to develop a surface complexation model that describes the acid-base properties of Bacillus subtilis. The bacteria were freeze-dried and then resuspended in 0.1 M NaCl ionic medium. Macroscopic measurements included potentiometric acid-base titrations and electrophoretic mobility measurements. In addition, ATR-FTIR spectra of wet pastes from suspensions of Bacillus subtilis at different pH values were collected. The least-squares program MAGPIE was used to generate a surface complexation model that takes into account the presence of three acid-base sites on the surface: tripple bond COOH, tripple bond NH+, and tripple bond PO-, which were identified previously by XPS measurements. Both potentiometric titration data and ATR-FTIR spectra were used quantitatively, and electrostatic effects at the charged bacterial surface were accounted for using the constant capacitance model. The model was calculated using two different approaches: in the first one XPS data were used to constrain the ratio of the total concentrations of all three surface sites. The capacitance of the double layer, the total buffer capacity, and the deprotonation constants of the tripple bond NH+, tripple bond POH, and tripple bond COOH species were determined in the fit. A second approach is presented in which the ratio determined by XPS of the total concentrations of tripple bond NH+ to tripple bond PO- sites is relaxed. The total concentration of tripple bond PO- sites was determined in the fit, while the deprotonation constant for tripple bond POH was manually varied until the minimization led to a model which predicted an isoelectric point that resulted in consistency with electrophoretic mobility data. The model explains well the buffering capacity of Bacillus subtilis suspensions in a wide pH range (between pH=3 and pH=9) which is of considerable environmental interest. In particular, a similar quantitative use of the IR data

  6. Lysozyme adsorption in pH-responsive hydrogel thin-films: the non-trivial role of acid-base equilibrium.

    PubMed

    Narambuena, Claudio F; Longo, Gabriel S; Szleifer, Igal

    2015-09-07

    We develop and apply a molecular theory to study the adsorption of lysozyme on weak polyacid hydrogel films. The theory explicitly accounts for the conformation of the network, the structure of the proteins, the size and shape of all the molecular species, their interactions as well as the chemical equilibrium of each titratable unit of both the protein and the polymer network. The driving forces for adsorption are the electrostatic attractions between the negatively charged network and the positively charged protein. The adsorption is a non-monotonic function of the solution pH, with a maximum in the region between pH 8 and 9 depending on the salt concentration of the solution. The non-monotonic adsorption is the result of increasing negative charge of the network with pH, while the positive charge of the protein decreases. At low pH the network is roughly electroneutral, while at sufficiently high pH the protein is negatively charged. Upon adsorption, the acid-base equilibrium of the different amino acids of the protein shifts in a nontrivial fashion that depends critically on the particular kind of residue and solution composition. Thus, the proteins regulate their charge and enhance adsorption under a wide range of conditions. In particular, adsorption is predicted above the protein isoelectric point where both the solution lysozyme and the polymer network are negatively charged. This behavior occurs because the pH in the interior of the gel is significantly lower than that in the bulk solution and it is also regulated by the adsorption of the protein in order to optimize protein-gel interactions. Under high pH conditions we predict that the protein changes its charge from negative in the solution to positive within the gel. The change occurs within a few nanometers at the interface of the hydrogel film. Our predictions show the non-trivial interplay between acid-base equilibrium, physical interactions and molecular organization under nanoconfined conditions

  7. Fermat's point from five perspectives

    NASA Astrophysics Data System (ADS)

    Park, Jungeun; Flores, Alfinio

    2015-04-01

    The Fermat point of a triangle is the point such that minimizes the sum of the distances from that point to the three vertices. Five approaches to study the Fermat point of a triangle are presented in this article. First, students use a mechanical device using masses, strings and pulleys to study the Fermat point as the one that minimizes the potential energy of the system. Second, students use soap films between parallel planes connecting three pegs. The tension on the film will be minimal when the sum of distances is minimal. Third, students use an empirical approach, measuring distances in an interactive GeoGebra page. Fourth, students use Euclidean geometry arguments for two proofs based on the Torricelli configuration, and one using Viviani's Theorem. And fifth, the kinematic method is used to gain additional insight on the size of the angles between the segments joining the Fermat point with the vertices.

  8. Point contact silicon solar cells

    NASA Technical Reports Server (NTRS)

    Swanson, Richard M.

    1987-01-01

    A new type of silicon solar cell has been developed. It is called the point-contact cell because the metal semiconductor contacts are restricted to an array of small points on the back of the cell. The point contact cell has recently demonstrated 22 percent conversion efficiency at one sun and 27.5 percent at 100 suns under an AM1.5 spectrum.

  9. Method of forming pointed structures

    NASA Technical Reports Server (NTRS)

    Pugel, Diane E. (Inventor)

    2011-01-01

    A method of forming an array of pointed structures comprises depositing a ferrofluid on a substrate, applying a magnetic field to the ferrofluid to generate an array of surface protrusions, and solidifying the surface protrusions to form the array of pointed structures. The pointed structures may have a tip radius ranging from approximately 10 nm to approximately 25 micron. Solidifying the surface protrusions may be carried out at a temperature ranging from approximately 10 degrees C. to approximately 30 degrees C.

  10. An Extremely Halophilic Proteobacterium Combines a Highly Acidic Proteome with a Low Cytoplasmic Potassium Content*

    PubMed Central

    Deole, Ratnakar; Challacombe, Jean; Raiford, Douglas W.; Hoff, Wouter D.

    2013-01-01

    Halophilic archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant and have evolved highly acidic proteomes that function only at high salinity. We examined osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila and Halorhodospira halochloris. Genome sequencing and isoelectric focusing gel electrophoresis showed that the proteome of H. halophila is acidic. In line with this finding, H. halophila accumulated molar concentrations of KCl when grown in high salt medium as detected by x-ray microanalysis and plasma emission spectrometry. This result extends the taxonomic range of organisms using KCl as a main osmoprotectant to the Proteobacteria. The closely related organism H. halochloris does not exhibit an acidic proteome, matching its inability to accumulate K+. This observation indicates recent evolutionary changes in the osmoprotection strategy of these organisms. Upon growth of H. halophila in low salt medium, its cytoplasmic K+ content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. First, we conclude that proteome acidity is not driven by stabilizing interactions between K+ ions and acidic side chains but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. Second, we propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K+-binding sites on an increasingly acidic protein surface. PMID:23144460

  11. 40 CFR 417.140 - Applicability; description of the neutralization of sulfuric acid esters and sulfonic acids...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... neutralization of sulfuric acid esters and sulfonic acids subcategory. 417.140 Section 417.140 Protection of... MANUFACTURING POINT SOURCE CATEGORY Neutralization of Sulfuric Acid Esters and Sulfonic Acids Subcategory § 417.140 Applicability; description of the neutralization of sulfuric acid esters and sulfonic...

  12. 40 CFR 417.140 - Applicability; description of the neutralization of sulfuric acid esters and sulfonic acids...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... neutralization of sulfuric acid esters and sulfonic acids subcategory. 417.140 Section 417.140 Protection of... MANUFACTURING POINT SOURCE CATEGORY Neutralization of Sulfuric Acid Esters and Sulfonic Acids Subcategory § 417.140 Applicability; description of the neutralization of sulfuric acid esters and sulfonic...

  13. 40 CFR 417.140 - Applicability; description of the neutralization of sulfuric acid esters and sulfonic acids...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... neutralization of sulfuric acid esters and sulfonic acids subcategory. 417.140 Section 417.140 Protection of... MANUFACTURING POINT SOURCE CATEGORY Neutralization of Sulfuric Acid Esters and Sulfonic Acids Subcategory § 417.140 Applicability; description of the neutralization of sulfuric acid esters and sulfonic...

  14. 40 CFR 417.140 - Applicability; description of the neutralization of sulfuric acid esters and sulfonic acids...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... neutralization of sulfuric acid esters and sulfonic acids subcategory. 417.140 Section 417.140 Protection of... MANUFACTURING POINT SOURCE CATEGORY Neutralization of Sulfuric Acid Esters and Sulfonic Acids Subcategory § 417.140 Applicability; description of the neutralization of sulfuric acid esters and sulfonic...

  15. 40 CFR 417.140 - Applicability; description of the neutralization of sulfuric acid esters and sulfonic acids...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... neutralization of sulfuric acid esters and sulfonic acids subcategory. 417.140 Section 417.140 Protection of... MANUFACTURING POINT SOURCE CATEGORY Neutralization of Sulfuric Acid Esters and Sulfonic Acids Subcategory § 417.140 Applicability; description of the neutralization of sulfuric acid esters and sulfonic...

  16. Inertial Pointing and Positioning System

    NASA Technical Reports Server (NTRS)

    Yee, Robert (Inventor); Robbins, Fred (Inventor)

    1998-01-01

    An inertial pointing and control system and method for pointing to a designated target with known coordinates from a platform to provide accurate position, steering, and command information. The system continuously receives GPS signals and corrects Inertial Navigation System (INS) dead reckoning or drift errors. An INS is mounted directly on a pointing instrument rather than in a remote location on the platform for-monitoring the terrestrial position and instrument attitude. and for pointing the instrument at designated celestial targets or ground based landmarks. As a result. the pointing instrument and die INS move independently in inertial space from the platform since the INS is decoupled from the platform. Another important characteristic of the present system is that selected INS measurements are combined with predefined coordinate transformation equations and control logic algorithms under computer control in order to generate inertial pointing commands to the pointing instrument. More specifically. the computer calculates the desired instrument angles (Phi, Theta. Psi). which are then compared to the Euler angles measured by the instrument- mounted INS. and forms the pointing command error angles as a result of the compared difference.

  17. BEST POSSIBLE FLOATING POINT ARITHMETIC.

    DTIC Science & Technology

    The report presents an algorithm for floating point arithmetic, using single-length arithmetic registers, which yields the most accurate...approximation which can be expressed in the given floating point format, the greatest lower bound, or the least upper bound for the result of the operation

  18. PowerPointing the Way.

    ERIC Educational Resources Information Center

    Stafford, Deborah J.

    1997-01-01

    Describes instructional uses of Microsoft PowerPoint software as demonstrated in a summer workshop for educators. Notes three uses: open house-type presentations, tutorials, and student-produced projects. Discusses how PowerPoint was used to present general information about the school and gives examples of uses in science and music history…

  19. Brocard Point and Euler Function

    ERIC Educational Resources Information Center

    Sastry, K. R. S.

    2007-01-01

    This paper takes a known point from Brocard geometry, a known result from the geometry of the equilateral triangle, and bring in Euler's [empty set] function. It then demonstrates how to obtain new Brocard Geometric number theory results from them. Furthermore, this paper aims to determine a [triangle]ABC whose Crelle-Brocard Point [omega]…

  20. NON-POINT SOURCE POLLUTION

    EPA Science Inventory

    Non-point source pollution is a diffuse source that is difficult to measure and is highly variable due to different rain patterns and other climatic conditions. In many areas, however, non-point source pollution is the greatest source of water quality degradation. Presently, stat...