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Sample records for acidic isoelectric points

  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. ELECTROKINETIC PHENOMENA : XIII. A COMPARISON OF THE ISOELECTRIC POINTS OF DISSOLVED AND CRYSTALLINE AMINO ACIDS.

    PubMed

    Abramson, H A; Moyer, L S

    1938-07-20

    1. Although the isoelectric points of dissolved cystine, tyrosine, and aspartic acid molecules lie at widely differing pH values, the isoelectric points of the surfaces of these substances in the crystalline state are all near pH 2.3. This was found to be true in solutions of hydrochloric acid and in acetate buffers of approximately constant ionic strength. 2. When suspended in gelatin, tyrosine and cystine crystals adsorb the protein and attain a surface identical in behavior with gelatin-coated quartz or collodion particles. 3. Aluminum ions at low concentrations reduce the electric mobilities of tyrosine crystals to zero in a manner analogous to their effect on other surfaces. 4. Alkyl benzene droplets also have their electric mobility reduced to zero at low pH values but, unlike the amino acids, a change in sign was never noticed. 5. The mobility of tyrosine crystals is independent of crystal length between 2-100micro. Below this size the mobilities are decreased. 6. These results are discussed in connection with the concept of the general definition of the isoelectric point and the behavior of certain insoluble proteins such as wool and silk fibroin. PMID:19873079

  4. Effect of proteins with different isoelectric points on the gene transfection efficiency mediated by stearic acid grafted chitosan oligosaccharide micelles.

    PubMed

    Yan, Jingjing; Du, Yong-Zhong; Chen, Feng-Ying; You, Jian; Yuan, Hong; Hu, Fu-Qiang

    2013-07-01

    A stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection. PMID:23679858

  5. Method for estimation of protein isoelectric point.

    PubMed

    Pihlasalo, Sari; Auranen, Laura; Hänninen, Pekka; Härmä, Harri

    2012-10-01

    Adsorption of sample protein to Eu(3+) chelate-labeled nanoparticles is the basis of the developed noncompetitive and homogeneous method for the estimation of the protein isoelectric point (pI). The lanthanide ion of the nanoparticle surface-conjugated Eu(3+) chelate is dissociated at a low pH, therefore decreasing the luminescence signal. A nanoparticle-adsorbed sample protein prevents the dissociation of the chelate, leading to a high luminescence signal. The adsorption efficiency of the sample protein is reduced above the isoelectric point due to the decreased electrostatic attraction between the negatively charged protein and the negatively charged particle. Four proteins with isoelectric points ranging from ~5 to 9 were tested to show the performance of the method. These pI values measured with the developed method were close to the theoretical and experimental literature values. The method is sensitive and requires a low analyte concentration of submilligrams per liter, which is nearly 10000 times lower than the concentration required for the traditional isoelectric focusing. Moreover, the method is significantly faster and simpler than the existing methods, as a ready-to-go assay was prepared for the microtiter plate format. This mix-and-measure concept is a highly attractive alternative for routine laboratory work. PMID:22946671

  6. 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)

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

    PubMed Central

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

    2012-01-01

    We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the E222G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF were employed to monitor dark (non-fluorescent) 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, 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. PMID:23017083

  8. Tuning the isoelectric point of graphene by electrochemical functionalization

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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.

  9. Tuning the isoelectric point of graphene by electrochemical functionalization.

    PubMed

    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

  10. 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

  11. 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

    Koetting, Michael C; Peppas, Nicholas A

    2014-08-25

    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 1.5mM 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

  12. Influence of deflocculant on the isoelectric point of refractory powders: Considerations on the action of deflocculant

    NASA Technical Reports Server (NTRS)

    Naruse, Y.; Semba, K.; Kiwaki, S.; Mishima, M.

    1983-01-01

    Isoelectric point changes in suspensions of refractory materials vis-a-vis the role of deflocculants used in monolithic refractories were investigated by considering the mineral compositions and adsorbed ions in four kinds of clay. Three types of curves represented the relation between the isoelectric point and the deflocculant. The surface charge of clay particles in the suspensions became negative as a result of the deflocculant, since the isoelectric point of suspensions decreased as the deflocculant was added. The isoelectric point changes of calcined alumina were also compared with those of the clays, and a similar phenomenon was observed, except that the deflocculant dispersed the calcined alumina better than it did the clays. A simple model was used to analyze the results.

  13. 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. PMID:20079385

  14. Genetically Controlled Variation of "Acid" β-Galactosidase Detected in Rattus norvegicus by Isoelectric Focusing

    PubMed Central

    Douglas, Tommy C.; Kimmel, Kathryn A.; Dawson, Patti E.

    1982-01-01

    Two genetically variant forms of rat "acid" β-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney β-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of β-galactosidases derived from different organs. The β-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of β-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same β-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" β-galactosidase. PMID:6811372

  15. Determination of the surface isoelectric point of oxide films on metals by contact angle titration

    SciTech Connect

    McCafferty, E.; Wightman, J.P.

    1997-10-15

    The surface isoelectric point for the native air-formed oxide films on aluminum, chromium, and tantalum has been determined by measurement of contact angles at the hexadecane/aqueous solution interface as a function of pH of the aqueous phase. Application of Young`s equation, the Gibbs equation, and surface equilibria conditions for hydroxylated oxide films leads to a mathematical expression which shows that the contact angle goes through a maximum at the isoelectric point of the oxide. The experimentally determined isoelectric point of oxide-covered chromium is 5.2 to 5.3, of oxide-covered aluminum is 9.5, and of oxide-covered tantalum is approximately {minus}0.7. These values for the oxide films are within one to three pH units of the reported isoelectric points for the corresponding bulk oxide powders. The oxide-covered metal surfaces were cleaned by argon plasma treatment prior to measurement of contact angles, in that XPS measurements showed this treatment to be effective in reducing the thickness of the carbon contamination layer. In addition, interfacial tensions were measured at the hexadecane/aqueous solution interface and were observed to have only a slight dependence on the pH of the aqueous phase.

  16. 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. PMID:24316526

  17. Impact of isoelectric points of nanopowders in electrolytes on electrochemical characteristics of dye sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Mohanty, Shyama Prasad; Bhargava, Parag

    2012-11-01

    Nanoparticle loaded quasi solid electrolytes are important from the view point of developing electrolytes for dye sensitized solar cells (DSSCs) having long term stability. The present work shows the influence of isoelectric point of nanopowders in electrolyte on the photoelectrochemical characteristics of DSSCs. Electrolytes with nanopowders of silica, alumina and magnesia which have widely differing isoelectric points are used in the study. Adsorption of ions from the electrolyte on the nanopowder surface, characterized by zeta potential measurement, show that cations get adsorbed on silica, alumina surface while anions get adsorbed on magnesia surface. The electrochemical characteristics of nanoparticulate loaded electrolytes are examined through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). DSSCs fabricated using liquid, silica or alumina loaded electrolytes exhibit almost similar performance. But interestingly, the magnesia loaded electrolyte-based cell show lower short circuit current density (JSC) and much higher open circuit voltage (VOC), which is attributed to adsorption of anions. Such anionic adsorption prevents the dark reaction in magnesia loaded electrolyte-based cell and thus, enhances the VOC by almost 100 mV as compared to liquid electrolyte based cell. Also, higher electron life time at the titania/electrolyte interface is observed in magnesia loaded electrolyte-based cell as compared to others.

  18. 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.

  19. 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. PMID:21819665

  20. 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

  1. 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. PMID:27454881

  2. Combining Isoelectric Point-Based Fractionation, Liquid Chromatography and Mass Spectrometry to Improve Peptide Detection and Protein Identification

    PubMed Central

    Cologna, Stephanie M.; Russell, William K.; Lim, Peniel J.; Vigh, Gyula; Russell, David H.

    2010-01-01

    The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry compatible, isoelectric trapping device that provides isoelectric point (pI) based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample pre-fractionation include: (i) small sample volumes (~200 μl), (ii) customized membranes over a large pH range, (iii) flexibility in the number of desired fractions, (iv) membrane compatibility with a variety of solvents systems and (v) resulting fractions do not require sample cleanup prior to MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides. PMID:20537905

  3. Measuring the isoelectric point of the edges of clay mineral particles: the case of montmorillonite.

    PubMed

    Pecini, Eliana M; Avena, Marcelo J

    2013-12-01

    The isoelectric point (IEP) of the edge surface of a montmorillonite sample was determined by using electrophoretic mobility measurements. This parameter, which is fundamental for the understanding of the charging behavior of clay mineral surfaces, was never measured so far because of the presence of permanent negative charges within the montmorillonite structure, charges that mask the electrokinetic behavior of the edges. The strategy was to block or neutralize the structural charges with two different cations, methylene blue (MB(+)) and tetraethylenepentaminecopper(II) ([Cu(tetren)](2+)), so that the charging behavior of the particles becomes that of the edge surfaces. Adsorption isotherms of MB(+) and [Cu(tetren)](2+) at different ionic strengths (NaCl) were performed to establish the uptakes that neutralize the cation exchange capacity (CEC, 0.96 meq g(-1)) of the sample. At high adsorptive concentrations, there was a superequivalent adsorption of MB(+) (adsorption exceeding the CEC) and an equivalent adsorption of [Cu(tetren)](2+) (adsorption reaching the CEC). In both cases, structural charges were neutralized at uptakes very close to the CEC. Zeta potential (ζ) vs pH data at different ionic strengths of montmorillonite with adsorbed MB(+) allowed to estimate an upper limit of the edge's IEP, 5.3 ± 0.2. The same kind of data obtained with adsorbed [Cu(tetren)](2+) provided a lower limit of the IEP, 4.0 ± 0.2. These values are in agreement with previously informed IEP and point of zero charge of pyrophyllite, which is structurally analogous to montmorillonite but carries no permanent charges. The importance of knowing the IEP of the edge surface of clay minerals is discussed. This value characterizes the intrinsic reactivity of edges, that is, the protonating capacity of edge groups in absence of any electric field generated by structural charges. It also allows us to correct relative edge charge vs pH curves obtained by potentiometric titrations and to

  4. Rapid quantitative enrichment of carnosic acid from rosemary (Rosmarinus officinalis L.) by isoelectric focused adsorptive bubble chromatography.

    PubMed

    Backleh, Marlène; Leupold, Günther; Parlar, Harun

    2003-02-26

    For the first time, the potent but unstable antioxidative diterpene carnosic acid could be enriched from an aqueous extract of rosemary (Rosmarinus officinalis L.) by isoelectric focused adsorptive bubble chromatography. Enrichment of carnosic acid in the foam was influenced by the pH value and the flow rate of the foam-forming gas. Efficiency was highest with diluted samples at pH 4. Under these conditions, the conversion of carnosic acid to carnosol was negligible. Transfer of carnosic acid to the foam from a standard solution in the presence of saponin as surfactive substance was similar to that from the aqueous rosemary extract. PMID:12590472

  5. Isoelectric points and surface hydrophobicity of Gram-positive cocci as determined by cross-partition and hydrophobic affinity partition in aqueous two-phase systems.

    PubMed Central

    Miörner, H; Albertsson, P A; Kronvall, G

    1982-01-01

    Thirty-nine streptococcal strains belonging to groups A, C, and G and 12 staphylococcal strains were investigated with respect to surface charge and hydrophobicity. Isoelectric points of the bacteria were determined by cross-partition experiments in dextran-polyethylene glycol two-phase systems containing charged polymers. The results obtained indicate that group A, C, and G streptococci have isoelectric points of pH 3.75 +/- 0.15 standard deviation. Staphylococci show an isoelectric point of around pH 2 and thereby differ markedly from the streptococci. Pretreatment of bacteria with human serum resulted in a significant change in the isoelectric points of streptococci. In a second series of experiments, an aqueous dextran-polyethylene glycol two-phase system containing polyethylene glycol palmitate or stearate was used to study the hydrophobic surface properties of the bacterial cells. The partition of the staphylococci was not influenced by the addition of up to 1% (wt/wt) polyethylene glycol palmitate or stearate, whereas the streptococci showed a large variation in affinity for polyethylene glycol-bound hydrophobic groups. The bacterial strains included in the study were also tested for uptake of human serum proteins. A positive correlation was found between the hydrophobic affinity of group A streptococci and the density of receptors for aggregated beta-2-microglobulin. PMID:7042571

  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. Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

    PubMed

    Onda, M; Nagata, S; Tsutsumi, Y; Vincent, J J; Wang, Q; Kreitman, R J; Lee, B; Pastan, I

    2001-07-01

    Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity. PMID:11431343

  8. 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. PMID:21913922

  9. 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.

  10. Interaction of cationic dodecyl-trimethyl-ammonium bromide with oxy-HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point.

    PubMed

    Alves, Fernanda Rosa; Carvalho, Francisco Adriano O; Carvalho, José Wilson P; Tabak, Marcel

    2016-04-01

    In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy-form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB-oxy-HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher Tm values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy-HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy-HbGp-DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy-HbGp-cationic surfactant interaction at neutral pH. PMID:26574155

  11. Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11.

    PubMed

    Florio, Walter; Batoni, Giovanna; Esin, Semih; Bottai, Daria; Maisetta, Giuseppantonio; Pardini, Manuela; Campa, Mario

    2003-05-01

    Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions. For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different. The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function. PMID:12748957

  12. Microheterogeneity of mammalian haptoglobins in isoelectric focusing.

    PubMed

    Dobryszycka, W; Krawczyk, E

    1979-01-01

    1. Human haptoglobin type 1-1, porcine haptoglobin, and equine haptoglobin were isolated and purified. 2. These haptoglobins were similar in polyacrylamide gel electrophoresis and in subunit structure but showed microheterogeneity in isoelectric focusing. 3. Isoelectric points of human haptoglobin as determined with photopolymerized gels were found to be 4.03-4.24, of porcine haptoglobin 4.0-4.30, and of horse haptoglobin 3.80-4.15, respectively. 4. Results obtained with chemically polymerized gels were 0.08-0.3 pH units higher. 5. Examined haptoglobins differed also in the ability of complex formation with hemoglobin, in sialic acid content and in antigenic specificity. PMID:45546

  13. 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.

  14. Dynamics of gel isoelectric focusing with ampholytic dyes monitored by camera in real-time.

    PubMed

    Stastná, Miroslava; Slais, Karel

    2003-08-01

    The dynamics of gel isoelectric focusing were studied by using amphoteric low-molecular-mass colored substances (isoelectric point markers). The polyacrylamide gel in slab format was in direct contact with the electrodes. In addition to isoelectric focusing with a pH gradient composed of synthetic carrier ampholytes, pH gradients created by simple buffers of acetic acid, 2-(N-morpholino)ethanesulfonic acid, histidine and N,N,N',N'-tetramethylethylenediamine were applied. The progress of the electrofocusing process was monitored by a charge-coupled device camera and video recording. The gradient profile and dynamics were approximated from the positions of isoelectric point markers, which were focused both on boundaries between individual zones of simple buffers and within the zones themselves. The obtained animated records enabled the observation of the entire real focusing run within fractions of a minute, which is useful both for the understanding and optimization of the focusing. PMID:12967184

  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. 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. PMID:20510014

  17. Modified interactions among globular proteins below isoelectric point in the presence of mono-, di- and tri-valent ions: A small angle neutron scattering study

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    Both short range attraction and long range electrostatic repulsion exist among globular protein Bovine Serum Albumin in solution below its isoelectric point (pI ≈ 4.8). At pD ≈ 4.0, below pI, protein has a net positive surface charge although local charge inhomogeneity presents. Small angle neutron scattering study reveals that in the presence of both mono-(Na+) and di-(Ni2+) valent ions attractive interaction increases and repulsive interaction decreases with the increase of salt concentration. However, for tri-valent (Fe3+) ions, both attractive and repulsive interaction increases with increasing salt concentration but the relative strength of repulsion is more than the attraction.

  18. 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…

  19. 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...

  20. 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. PMID:15490454

  1. The pH at the First Equivalence Point in the Titration of a Diprotic Acid

    NASA Astrophysics Data System (ADS)

    Ault, Addison

    2003-12-01

    Some readers will note a similarity between this approach and the one I took in a paper entitled “Do pH in Your Head” (2). In an example in that article the isoelectric pH of glycine (the pH at which the average charge of a glycine molecule is zero), has the value of 6.0, which is exactly half-way between 2.4, the pKa of the carboxyl group of glycine, and 9.6, the pKa of the ammonium group of glycine. This is what one would expect when realizing that a solution of neutral glycine right out of the bottle is equivalent to glycine obtained by titration of the conjugate acid of glycine to the first equivalence point. Those who are interested might want to consider why the isoelectric pH of an “acidic” amino acid, such as alanine, is exactly half-way between the pKa values of the two carboxyl groups, and why the isoelectric pH of a “basic” amino acid such as lysine is exactly half-way between the pKa values of the two ammonium groups.

  2. 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. PMID:26049243

  3. 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.

  4. 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.

  5. Stibiconite (Sb3O6OH), senarmontite (Sb2O3) and valentinite (Sb2O3): Dissolution rates at pH 2-11 and isoelectric points

    NASA Astrophysics Data System (ADS)

    Biver, M.; Shotyk, W.

    2013-05-01

    . The isoelectric point (i.e.p.) of the minerals was determined electrokinetically, as a proxy for the zero point of charge (pHzpc), which was experimentally inaccessible; the measured i.e.p. do not correspond to the dissolution rate minima. I.e.p. of antimony oxides have not yet been documented in the literature. The geochemical implications for the weathering of antimony oxide minerals and stibnite, with particular reference to the mobilization of antimony in the context of an abandoned antimony mine (Goesdorf, Luxembourg), are discussed.

  6. Isoelectric Focusing in a Drop

    PubMed Central

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

    2010-01-01

    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 non-invasive 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. PMID:21117663

  7. 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...

  8. THE AMPHOTERIC PROPERTIES OF SOME AMINO-ACIDS AND PEPTIDES.

    PubMed

    Eckweiler, H; Noyes, H M; Falk, K G

    1921-01-20

    The titration curves of solutions of glycine, alanine, alpha-ammo-butyric acid, leucine, glycyl-glycine, alanyl-glycine, alanyl-alanine, acetone, acetamide, urea, acetic acid, and aceturic acid were determined and some of the relations as dependent upon the chemical structures discussed. The isoelectric points of some of the amphoteric electrolytes were found experimentally. The definition of isoelectric point, its theoretical significance, and method of calculation were considered in some detail. PMID:19871865

  9. 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.

  10. 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.

  11. 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.

  12. Subsetting of acetylcholine receptor-reactive antibodies by preparative isoelectric focusing.

    PubMed

    Thompson, P A; Krolick, K A

    1991-01-01

    The antibodies produced against most foreign antigens are composed of a family of immunoglobulins, a family composed of members that are of a number that often reflects the size/complexity of the molecule that stimulates their production. In other words, such responses involve the activation of a "polyclonal" B lymphocyte population. The antibody products of the B cells, although all capable of binding the original antigen, bind at various immunogenic sites (epitopes) on that antigen. Such differences in antigen-binding fine specificity is determined by amino acid residues in the antibody variable region domains found associated with the antigen combining site and tend to have a complimentary biochemistry with the molecule for which they are intended to interact. Furthermore, in addition to amino acid differences that dictate the isotypes and allotypes of antibody molecules, differences in the amino acids that compose the variable regions can produce differences in net charge of particular antibody molecules; thus, families of polyclonal antibodies, all reactive with the same antigen but with different fine specificities, can be separated and, as shown below, purified based on their isoelectric points by preparative isoelectric focusing (pIEF). PMID:1780274

  13. Preparative-scale isoelectric trapping separations in methanol-water mixtures.

    PubMed

    Shave, Evan; Vigh, Gyula

    2004-05-14

    The typically low aqueous solubilities of small, hydrophobic organic ampholytic molecules limit the production rates that can be achieved in their isoelectric trapping (IET) separations and call for the use of hydro-organic mixtures as solvents. The compatibility of methanol-water mixtures and poly(ethylene terephthalate) substrate-supported isoelectric polyacrylamide hydrogels, developed for binary IET separations in a Gradiflow BF200IET unit, was investigated. The isoelectric polyacrylamide-based hydrogels retained their functional and mechanical integrities when the methanol concentration in the hydro-organic solvent mixture was kept at or below 25% (v/v). The utility of the hydro-organic media was demonstrated in the purification of a hydrophobic ampholytic compound, technical grade 4-hydroxy-3-(morpholinomethyl) benzoic acid. Production rates as high as 7 mg/h were achieved using small, 15 cm2 active surface area isoelectric membranes. PMID:15139407

  14. Capillary isoelectric focusing of native and inactivated microorganisms.

    PubMed

    Horká, M; Kubícek, O; Růzicka, F; Holá, V; Malinovská, I; Slais, K

    2007-07-01

    The research of microorganisms includes the development of methods for the inactivation of viruses and other microbes. It also means to efficiently eliminate the infectivity of microorganisms without damage of their integrity and structure. According to the results of the last 5 years the capillary electromigration techniques appear to be very perspective for the comparison of the methods applicable for inactivation in the diagnostics and study of the pathogens. In this paper we suggest the capillary isoelectric focusing of the model microorganisms, Escherichia coli, Staphylococcus epidermidis, Candida albicans and bacteriophage PhiX 174, native or inactivated by different procedures. UV detection and fluorometric detection for the dynamically modified microbes by pyrenebutanoate on the basis of the non-ionogenic tenside were used here. Isoelectric points of native and/or dynamically modified microorganisms and other properties were compared with those obtained after microorganisms inactivation. The segmental injection of the sample pulse enabled the reproducible and efficient capillary isoelectric focusing in different pH gradients. The low-molecular-weight pI markers were used for tracing of the pH gradient. PMID:17328903

  15. 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. PMID:15273996

  16. Ischemia detection using Isoelectric Energy Function.

    PubMed

    Kumar, Amit; Singh, Mandeep

    2016-01-01

    A novel method has been proposed for the detection of ischemia using an isoelectric energy function (IEEF) resulting from ST segment deviations in ECG signals. The method consists of five stages: pre-processing, delineation, measurement of isoelectric energy, a beat characterization algorithm and detection of ischemia. The isoelectric energy threshold is used to differentiate ischemic beats from normal beats for ischemic episode detection. Then, ischemic episodes are classified as transmural or subendocardial. The method is validated for recordings of the annotated European ST-T database (EDB). The results show 98.12% average sensitivity (SE) and 98.16% average specificity (SP). These results are significantly better than those of existing methods cited in the literature. The advantage of the proposed method includes simplicity, ruggedness and automatic discarding of noisy beats. PMID:26623944

  17. 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.

  18. 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. PMID:17157942

  19. 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

    2016-08-01

    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. PMID:26809616

  20. 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

  1. 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. PMID:26025812

  2. Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes.

    PubMed

    Glukhovskiy, P; Landers, T A; Vigh, G

    2000-03-01

    The IsoPrime multicompartment electrolyzer, equipped with a series of isoelectric membranes with closely spaced pI values, was used for the first time for the preparative-scale separation of the enantiomers of dansyl phenylalanine with hydroxypropyl beta-cyclodextrin as resolving agent. The final separation conditions could be established easily in three successive experiments by rationally narrowing the pH steps between the neighboring isoelectric membranes. The final separation yielded products with an enantiomeric excess greater than 99.9%, at production rates of about 0.1 mg/h. The greatest experimental difficulty was caused by the relatively high salt content of the hydroxypropyl beta-cyclodextrin used, which resulted in high conductivity and limited the maximum field strength one could use. PMID:10733219

  3. 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.

  4. On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing.

    PubMed

    Guillo, Christelle; Karlinsey, James M; Landers, James P

    2007-01-01

    Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids. PMID:17180213

  5. Continuous flow isoelectric focusing for purification of proteins.

    PubMed

    Soulet, N; Roux-de Balmann, H; Sanchez, V

    1998-06-01

    To estimate the feasibility of isoelectric focusing in a continuous mode, the conditions for obtaining stable and linear pH gradients were first investigated, using a mixture of carrier ampholytes. Then the operating parameters allowing the isoelectric focusing of bovine serum albumin as model protein were determined. A theoretical approach was used to find conditions minimizing the electrohydrodynamic effect, mainly responsible for protein dispersion, for the separation of bovine serum albumin and alpha-lactalbumin from bovine milk. PMID:9694268

  6. 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. PMID:17165837

  7. 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.

  8. Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing.

    PubMed

    Nagai, A; Komoriya, H; Bunai, Y; Yamada, S; Jiang, X Y; Ohya, I

    1991-06-01

    The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach. PMID:1889397

  9. 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.

  10. 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)

  11. Isoelectric focusing technology quantifies protein signaling in 25 cells

    PubMed Central

    O'Neill, Roger A.; Bhamidipati, Arunashree; Bi, Xiahui; Deb-Basu, Debabrita; Cahill, Linda; Ferrante, Jason; Gentalen, Erik; Glazer, Marc; Gossett, John; Hacker, Kevin; Kirby, Celeste; Knittle, James; Loder, Robert; Mastroieni, Catherine; MacLaren, Michael; Mills, Thomas; Nguyen, Uyen; Parker, Nineveh; Rice, Audie; Roach, David; Suich, Daniel; Voehringer, David; Voss, Karl; Yang, Jade; Yang, Tom; Vander Horn, Peter B.

    2006-01-01

    A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms. PMID:17053065

  12. 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. PMID:24058669

  13. Human Brain Activity Patterns beyond the Isoelectric Line of Extreme Deep Coma

    PubMed Central

    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. PMID:24058669

  14. Chicken lutropin acts like follitropin in rat ovarian follitropin receptor: an isoelectric focusing study.

    PubMed

    Iwasawa, A; Hattori, M; Fukuhara, Y; Kawashima, M; Wakabayashi, K; Kamiyoshi, M

    1998-07-01

    This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity. PMID:9827020

  15. 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. PMID:27396497

  16. The relative importance of transport phenomena in recycling isoelectric focusing.

    PubMed

    Burgaud, C; Clifton, M J; Sanchez, V

    1992-03-01

    The various transport phenomena involved in recycling isoelectric focusing are analyzed for their contributions to band spreading so as to find ways of improving the resolution of this liquid-phase protein purification method. A numerical model is proposed that takes into account diffusion, electroosmosis and electrophoretic migration as a function of pH. The electrohydrodynamic effects have so far been neglected in these calculations. The results of these calculations are compared with experimental measurements performed in different chamber geometries, with a variety of proteins and under different operating conditions, always chosen to avoid flow instabilities. This comparison shows that the resolution of this process is greatly impaired if the electroosmotic slip velocity at the wall is not suppressed. PMID:1592042

  17. 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...

  18. [Properties of a cephalosporinase produced by Proteus penneri inhibited by clavulanic acid].

    PubMed

    Miro, E; Barthelemy, M; Peduzzi, J; Reynaud, A; Morand, A; Prats, G; Labia, R

    1994-05-01

    P. penneri produces an inducible cephalosporinase, as many Enterobacteriaceae. Nevertheless this betalactamase is susceptible to clavulanic acid which is an exception also encountered for P. vulgaris. The authors studied the enzyme produced by P. penneri 14HBC resistant to cefotaxime (MIC 16 mg/l) isolated in Spain in 1992. This betalactamase of isoelectric point 6.65 hydrolyzes first generation cephalosporins, amoxycillin and poorly ticarcillin as it occurs for all cephalosporinases. However, this enzyme hydrolyzes strongly oxyimino-cephalosporins: cefuroxime, cefotaxime, cefepime, cefpirome as it occurs with extended-spectrum betalactamases. Cephamycins and imipenem are not substrates. Clavulanic acid has a very good affinity for this betalactamase which is inactivated progressively. These properties are similar to those of the enzyme of P. vulgaris Ro104 of isoelectric point 8.3 which, contrarily to other cephalosporinases, belongs to the structural Ambler's class A. PMID:7824319

  19. 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

  20. Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis.

    PubMed

    Mai, Junyu; Sommer, Gregory J; Hatch, Anson V

    2012-04-17

    Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing. PMID:22409593

  1. The molecular characteristics of a human pancreatic acidic phosphoprotein that inhibits calcium carbonate crystal growth.

    PubMed Central

    De Caro, A; Multigner, L; Lafont, H; Lombardo, D; Sarles, H

    1984-01-01

    A CaCO3-crystal-growth inhibitor was isolated from human pancreatic stones by using EDTA demineralization, followed by DEAE-Trisacryl chromatography. The isolated inhibitor was found to be a phosphoglycoprotein with Mr 14017 and having an unusual chemical composition. It is characterized by a high (42%) acidic amino acid content, but lacks methionine and gamma-carboxyglutamic acid. The protein contains 2.65 mol of P/mol of protein, as phosphoserine (2 mol) and phosphothreonine (0.5 mol). Isoelectric focusing of the protein yields one major band corresponding to an isoelectric point of 4.2. Immunochemical quantification of the crystal-growth inhibitor in pure pancreatic juice reveals that it constitutes 14% of the normal exocrine secretion. Our findings demonstrate that this is a novel secretory protein, which has no enzymic activity and which maintains pancreatic juice in a supersaturated state with respect to CaCO3. Images Fig. 3. Fig. 4. PMID:6487269

  2. 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).

  3. [Isoelectric spectra of liver tissue and blood serum albumin for rabbits of different age].

    PubMed

    Sopkina, D A; Ostolovskiĭ, E M; Ivlev, V N

    1978-01-01

    The isoelectric spectrum of albumin isolated from the liver and blood serum of 30-, 45- and 90-day rabbits was studied. By the method of isoelectric focusing in the boron-borate buffer--mannitol system. It is shown that the liver albumin displays heterogeneity ans is separated into four-five fractions, with pJ 4.8-6.0. Age peculiarities are found for the isoelectric spectrum of this protein. The serum albumin spectrum for rabbits of the studied age groups is characterized by the presence of a homogeneous peak with pJ 5.59, 5.57 and 5.47 corresponding to 30, 45 and 90-day age, respectively. Identity of serum albumin and protein of some liver albumin spectrum components is established by analyzing the pattern of the isoelectric spectrum for a mixture of preparations of 90-day rabbit proteins under comparison. PMID:34911

  4. 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.

  5. Acid hydrolysis of cellulose as the entry point into biorefinery schemes.

    PubMed

    Rinaldi, Roberto; Schüth, Ferdi

    2009-01-01

    Cellulose is a major source of glucose because it is readily available, renewable, and does not compete with the food supply. Hydrolysis of cellulose is experiencing a new research and development cycle in which this reaction is carried out over solid catalysts and coupled to other reactions for increased efficiency. Cellulose is typically not soluble in conventional solvents and very resistant to chemical and biological transformations. This Review focuses on aspects related to the hydrolysis of cellulose as this process is a significant entry point into the biorefinery scheme based on carbohydrates for the production of biofuels and biochemicals. Structural features of cellulose, conventional acid-catalyzed reactions, and the use of solid acid catalysts for hydrolysis are discussed. The longterm success of the biorefinery concept depends on the development of energetically efficient processes to convert cellulose directly or indirectly into biofuels and chemicals. PMID:19950346

  6. 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. PMID:25770625

  7. Photocatalytic decomposition of organic micropollutants using immobilized TiO2 having different isoelectric points.

    PubMed

    Arlos, Maricor J; Hatat-Fraile, Melisa M; Liang, Robert; Bragg, Leslie M; Zhou, Norman Y; Andrews, Susan A; Servos, Mark R

    2016-09-15

    Organic micropollutants found in the environment are a diverse group of compounds that includes pharmaceuticals, personal care products, and endocrine disruptors. Their presence in the aquatic environment continues to be a concern as the risk they pose towards both the environment and human health is still inconclusive. Removal of these compounds from water and wastewater is difficult to achieve and often incomplete, but UV-TiO2 is a promising treatment approach. In this study, the efficiency of titanium dioxide (TiO2) immobilized on porous supports were tested for treatment of target pharmaceuticals and their metabolites under UV-LED exposure, a potential low energy and cost effective alternative to conventional UV lamps. Immobilization was completed using two different methods: (1) dip coating of TiO2 onto quartz fiber filters (QFT) or (2) thermal-chemical oxidation of porous titanium sheets (PTT). Comparison against experimental controls (dark QFT, dark PTT, and photolysis using UV-LED only) showed that UV-LED/PTT and UV-LED/QFT treatments have the potential to reduce the concentrations of the target compounds. However, the treatments were found to be selective, such that individual pharmaceuticals were removed well using QFT and PTT but not both. The complementary treatment behavior is likely driven by electrostatic interactions of charged compounds with the membranes. QFT membranes are negatively charged at the experimental pH (4.5-5) while PTT membranes are positively charged. As a result, cationic compounds interact more with QFT while anionic compounds with PTT. Neutral compounds, however, were found to be recalcitrant under any treatment conditions suggesting that ionic interactions were important for reactions to occur. This behavior can be advantageous if specificity is required. The behavior of pharmaceutical metabolites is similar to the parent compounds. However, isomeric metabolites of atorvastatin with functional groups in para and ortho configurations behave differently, suggesting that the positioning of functional groups can have an impact in their interaction with the immobilized TiO2. It was also apparent that PTT can be reused after cleaning by heat treatment. Overall, these newly synthesized membrane materials have potential applications for treatment of trace organic contaminants in water. PMID:27286470

  8. Using isoelectric point to determine the pH for initial protein crystallization trials

    PubMed Central

    Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

    2015-01-01

    Motivation: The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination. The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallization trials required. However, the pH is usually recorded as that of the buffer solution, which can be highly inaccurate. Results: Here, we show that a better estimate of the true pH can be predicted by considering not only the buffer pH but also any other chemicals in the crystallization solution. We use these more accurate pH values to investigate the disputed relationship between the pI of a protein and the pH at which it crystallizes. Availability and implementation: Data used to generate models are available as Supplementary Material. Contact: julie.wilson@york.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573921

  9. 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...

  10. A new D-stereospecific amino acid amidase from Ochrobactrum anthropi.

    PubMed

    Asano, Y; Mori, T; Hanamoto, S; Kato, Y; Nakazawa, A

    1989-07-14

    A new D-stereospecific amino acid amidase has been partially purified from Ochrobactrum anthropi SCRC SV3, which had been isolated and selected from soil. The Mr of the enzyme was estimated to be about 38,000, and its isoelectric point was 5.3. The enzyme catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-amino acid and ammonia. The major substrates included D-phenylalanine amide, D-tyrosine amide, D-tryptophan amide, D-leucine amide, and D-alanine amide. PMID:2751665

  11. 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

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

    PubMed

    Casanova, Lisa M; Sobsey, Mark D

    2015-11-01

    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

  13. 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

  14. 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

  15. 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

  16. Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review.

    PubMed

    Ahmad, Farhan; Hashsham, Syed A

    2012-07-01

    Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10-20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics. PMID:22704369

  17. Comparison of the N-acetylhexosaminidase components in the ram testis and epididymis by isoelectric focusing

    PubMed Central

    Winchester, B. G.

    1971-01-01

    1. Several enzymic components, with both N-acetyl-β-glucosaminidase and N-acetyl-β-galactosaminidase activity, have been demonstrated in the ram testis and epididymis by isoelectric focusing. 2. The component (I), which predominates in the testis and caput of the epididymis, is isoelectric at pH6.2±0.1, whereas the predominant component (II) in the epididymal isthmus and cauda is isoelectric at pH7.0±0.1. 3. The total activity and the relative proportions of the enzymic components vary in the different sections of the epididymis. 4. Although their pH optima are slightly different, the appropriate Km values for components I and II for the hydrolysis of 4-methylumbelliferyl-N-acetyl-β-glucosaminide and N-acetyl-β-galactosaminide and the ratios of the maximal velocities towards the two substrates are very similar. PMID:5131014

  18. 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.

  19. 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.

  20. 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

  1. 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. PMID:25553199

  2. 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...

  3. 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

    1982-08-01

    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. PMID:7115772

  4. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

    PubMed Central

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development. PMID:26338058

  5. 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.

  6. A multi-point sensor based on optical fiber for the measurement of electrolyte density in lead-acid batteries.

    PubMed

    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

  7. 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

  8. On the accuracy of acid-base determinations from potentiometric titrations using only a few points from the titration curve.

    PubMed

    Olin, A; Wallén, B

    1977-05-01

    There are several procedures which use only a few points on the titration curve for the calculation of equivalence volumes in acid-base titrations. The accuracy of such determinations will depend on the positions of the points on the titration curve. The effects of errors in the stability constants and in the pH measurements on the accuracy of the analysis have been considered, and the results are used to establish the conditions under which these errors are minimized. PMID:18962087

  9. Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

    PubMed

    Antonioli, Paolo; Mendieta, Martha E; Sebastiano, Roberto; Citterio, Attilio; Peltre, Gabriel; Busnel, Jean-Marc; Descroix, Stephanie; Candiano, Giovanni; Righetti, Pier Giorgio

    2006-03-20

    Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall. PMID:16289957

  10. The effect of sire breed on the melting point and fatty acid composition of subcutaneous fat in steers.

    PubMed

    Perry, D; Nicholls, P J; Thompson, J M

    1998-01-01

    Fatty acid composition and the melting point of subcutaneous fat was determined in 18 Hereford, 25 Brahman x Hereford, 22 Simmental x Hereford, and 15 Friesian x Hereford steers that were grown out on pasture at two sites and slaughtered when the mean weight of the Herefords at each site was ca. 450 kg. Multivariate and univariate analyses tested the relations of fatty acid composition, degree of saturation, and melting point with sire breed, environment, age, and carcass characteristics. Hereford and Brahman steers were fatter than the Simmental and Friesian steers. Fat from Brahman-sired steers had a melting point 2.5 degrees C lower than fat from the Bos taurus-sired steers at the same age and had a higher proportion of unsaturated fatty acids, independent of variation in carcass weight and fatness. Melting point and degree of saturation decreased as age increased. Step-down discriminant analyses identified a set of three acids (14:0, 16:0, and 17:1) that differed among sire breeds, independent of differences in melting point: the acids 14:0 and 16:0 discriminated between Brahman and Bos taurus steers and 17:1 between Hereford and Simmental and Friesian steers. Increase in fatness was associated with an increase in 17:1, but, at the same fatness, no acids discriminated among the Bos taurus-sired steers. The use of Bos indicus cattle or their crossbreeds in situations in which hard-setting fat is likely may mitigate the problem. PMID:9464888

  11. Determination of Critical Point of pO2 Level in the Production of Lactic Acid by Lactobacillus rhamnosus

    NASA Astrophysics Data System (ADS)

    Mel, Maizirwan; Karim, Mohamed Ismail Abdul; Salleh, Mohamad Ramlan Mohamed; Abdullah, Rohane

    The study was conducted to determine the critical point of pO2 level in the production of lactic acid by Lactobacillus rhamnosus. The fermentation process was successfully carried out in laboratory scale fermenter/bioreactor using different pO2 level (the main parameter that significantly affects the growth of L. rhamnosus and lactic acid production) together with two other parameters; the agitation rate and pH. From the result, it was observed that the best production of lactic acid with the concentration of 16.85 g L-1 or 1.68% production yield has been obtained at the operating parameters of 5% pO2 level, agitation speed of 100 rpm and sample pH 6. The critical point of pO2 was found to be between 5 and 10%.

  12. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

    PubMed Central

    Krempl, C; Schultze, B; Laude, H; Herrler, G

    1997-01-01

    Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV. PMID:9060696

  13. Chemical properties of ω-3 fortified gels made of protein isolate recovered with isoelectric solubilisation/precipitation from whole fish.

    PubMed

    Tahergorabi, Reza; Beamer, Sarah K; Matak, Kristen E; Jaczynski, Jacek

    2013-08-15

    Protein isolate was recovered from whole gutted fish using isoelectric solubilisation/precipitation (ISP). The objective was to determine chemical properties of heat-set gels made of the ISP protein isolate fortified with ω-3 polyunsaturated fatty acids (PUFAs)-rich oils (flaxseed, fish, algae, krill, and blend). The extent of the PUFAs increase, ω-6/ω-3 FAs and unsaturated/saturated FAs ratios, and the indices of thrombogenicity and atherogenicity depended on specific ω-3 PUFAs-rich oil used to fortify protein isolate gels. Lipid oxidation in ω-3 PUFAs fortified gels was minimal, although greater (P<0.05) than control gels (without ω-3 PUFAs fortification). However, all gels were in the slightly rancid, but acceptable range. The commonly used thiobarbituric-acid-reactive-substances (TBARS) assay to determine lipid oxidation in seafood may be inaccurate for samples containing krill oil due to its red pigment, astaxanthin. Protein degradation (total-volatile-basic-nitrogen) was greater (P<0.05) in ω-3 PUFAs fortified gels than control gels. However, all gels were considerably below the acceptability threshold for protein degradation. The shear stress of ω-3 PUFAs fortified gels was generally greater than the control gels and the shear strain was generally unchanged. This study demonstrates that ω-3 PUFAs fortification of protein isolates recovered with ISP from fish processing by-products or whole fish has potential application in the development of functional foods. PMID:23561173

  14. 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.

  15. 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.

  16. 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). PMID:26289722

  17. 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. PMID:24293057

  18. The use of microwave tissue fixation to demonstrate the in vivo phosphorylation of an acidic 80,000 molecular weight protein in the rat neocortex following treatment with soman

    SciTech Connect

    Mobley, P.L.; Gonzalez, N.E. )

    1991-01-01

    Studies were conducted to determine if soman, a cholinesterase inhibitor, could activate the protein kinase C system in the rat neocortex. Using microwave radiation for rapid tissue fixation, it was demonstrated that treatment with soman increased {sup 32}P incorporation into an acidic 80,000 molecular weight, heat-stable protein in vivo. Based on relative molecular weight and isoelectric point this protein appears to be identical to a protein identified as a substrate for protein kinase C. Additionally, a protein of the same molecular weight and isoelectric point could be phosphorylated in tissue slices prepared from the neocortex by cholinergic dependent mechanisms. Also, treatment with soman decreased protein kinase C in the soluble fraction of this brain region; however, no corresponding increase was observed in the particulate fraction. These results suggest that soman can activate protein kinase C in vivo, and demonstrate the utility of using microwave tissue fixation to study protein phosphorylation events in vivo.

  19. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  20. Identification of Tuber borchii Vittad. mycelium proteins separated by two-dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging.

    PubMed

    Vallorani, L; Bernardini, F; Sacconi, C; Pierleoni, R; Pieretti, B; Piccoli, G; Buffalini, M; Stocchi, V

    2000-11-01

    This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty-three proteins were excised and analyzed by the combination of amino acid and N-terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS-PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases. PMID:11271490

  1. 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.

  2. Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts

    PubMed Central

    2009-01-01

    Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction−oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds. PMID:19438264

  3. 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. PMID:21116613

  4. Extraction of amino acids by reverse iontophoresis: simulation of therapeutic monitoring in vitro.

    PubMed

    Sieg, Anke; Jeanneret, Fabienne; Fathi, Marc; Hochstrasser, Denis; Rudaz, Serge; Veuthey, Jean-Luc; Guy, Richard H; Delgado-Charro, M Begoña

    2008-11-01

    Reverse iontophoresis across the skin has been investigated as alternative, non-invasive method for clinical and therapeutic drug monitoring. This research investigated the reverse iontophoretic extraction of 19 amino acids present at clinically relevant levels in the subdermal compartment of an in vitro diffusion cell. Over a simulated, systemic concentration range of 0-500 microM, the extraction of amino acids was linear. Charged amino acids were extracted towards the electrode of opposite polarity, while zwitterionic species were extracted to both anode and cathode with the latter predominating. The reverse iontophoretic extraction flux was a linear function of amino acid isoelectric point, reflecting the different contributions of electromigration and electroosmosis to electrotransport. Overall, the results confirm the feasibility of monitoring amino acids at clinically relevant levels and provide an incentive for in vivo research to further explore the clinical potential of reverse iontophoresis for the non-invasive monitoring of amino acids. PMID:18675906

  5. 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. PMID:27473481

  6. Occurrence and point source characterization of perfluoroalkyl acids in sewage sludge.

    PubMed

    Alder, Alfredo C; van der Voet, Juergen

    2015-06-01

    The occurrence and levels of perfluoroalkyl acids (PFAAs) emitted from specific pollution sources into the aquatic environment in Switzerland were studied using digested sewage sludges from 45 wastewater treatment plants in catchments containing a wide range of potential industrial emitters. Concentrations of individual PFAAs show a high spatial and temporal variability, which infers different contributions from industrial technologies and activities. Perfluorooctane sulfonic acid (PFOS) was generally the predominant PFAA with concentrations varying between 4 and 2440μgkg(-1) (median 75μgkg(-1)). Elevated emissions were especially observed in catchments capturing discharges from metal plating industries (median 82μgkg(-1)), aqueous firefighting foams (median 215μgkg(-1)) and landfill leachates (median 107μgkg(-1)). Some elevated perfluoroalkyl carboxylic acids (PFCAs) levels could be attributed to emissions from textile finishing industries with concentrations up to 233μgkg(-1) in sewage sludge. Assuming sorption to sludge for PFOS and PFCAs of 15% and 2%, respectively, concentrations in wastewater effluents up to the low μgL(-1) level were estimated. Even if wastewater may be expected to be diluted between 10 and 100 times by the receiving waters, elevated concentrations may be reached at specific locations. Although sewage sludge is a minor compartment for PFAAs in WWTPs, these investigations are helpful for the identification of hot-spots from industrial emitters as well as to estimate monthly average concentrations in wastewater. PMID:25176581

  7. 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

  8. 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.

  9. 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.

  10. Demonstration of murine pancreas elastase and its interstrain variation by isoelectric focusing.

    PubMed

    von Deimling, O H; Gaa, A; Hameister, H

    1991-06-01

    Isoelectric focusing between pH 9 and 11 was used for separation of murine pancreas proteases. One of these proteases is characterized by its preference for N-acetyl-L-alanine-alpha-naphthylester as substrate and by its genetic linkage to bt, a coat color marker of chromosome 15. This protease was identified as elastase, and is probably elastase-1 (ELA-1). Because of the simple procedure and the excellent reproducibility of the focusing pattern, ELA-1 is recommended as a useful marker for mouse chromosome 15. PMID:1889396

  11. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    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.

  13. 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.

  14. Freezing points and small-scale deicing tests for salts of levulinic acid made from grain sorghum.

    PubMed

    Ganjyal, G; Fang, Q; Hanna, M A

    2007-11-01

    Deicers from renewable resources are needed to overcome the disadvantages of using traditional deicers. Salts made from levulinic acid produced using grain sorghum as raw material were tested as road deicing agents. Freezing points of these salts viz., sodium levulinate, magnesium levulinate and calcium levulinate along with rock salt (sodium chloride) were determined according to American Society for Testing and Materials (ASTM) D 1177-94 standard at concentrations of 10, 20, 30 and 40 % w/w. There were significant differences among the freezing points of the salts. Freezing points for rock salt, sodium levulinate, calcium levulinate and magnesium levulinate, for different concentrations, were in the ranges of -6.6 to -20.5, -2.9 to -15.0, -2.1 to -7.8 and -1.5 to -6.5 degrees C, respectively. Deicing effectiveness of the salts of levulinic acid were investigated by conducting small-scale deicing tests with aqueous solutions of various salt concentrations (2%, 5% and 10%) in a laboratory freezer and by spraying the deicer on a graveled surface covered by ice and snow with the average temperature during the testing at -2.7 degrees C. Deicing capabilities of the three salts of levulinic acid differed. At -2.7 degrees C, all three salts caused melting of the ice. Among the different levulinates studied sodium levulinate was the most effective deicing agent. These salts of levulinates could be a viable replacement for traditional deicers and could help in reducing the disadvantages of traditional deicers. PMID:17416518

  15. A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

    PubMed Central

    Volles, Michael J.; Lansbury, Peter T.

    2005-01-01

    A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 109 sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an

  16. 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

  17. 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

    2016-02-01

    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. PMID:26759062

  18. [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. PMID:3078008

  19. Usefulness of two-point Dixon fat-water separation technique in gadoxetic acid-enhanced liver magnetic resonance imaging

    PubMed Central

    Ding, Ying; Rao, Sheng-Xiang; Chen, Cai-Zhong; Li, Ren-Chen; Zeng, Meng-Su

    2015-01-01

    AIM: To compare differences between volumetric interpolated breath-hold examination (VIBE) using two-point Dixon fat-water separation (Dixon-VIBE) and chemically selective fat saturation (FS-VIBE) with magnetic resonance imaging examination. METHODS: Forty-nine patients were included, who were scanned with two VIBE sequences (Dixon-VIBE and FS-VIBE) in hepatobiliary phase after gadoxetic acid administration. Subjective evaluations including sharpness of tumor, sharpness of vessels, strength and homogeneity of fat suppression, and artifacts that were scored using a 4-point scale. The liver-to-lesion contrast was also calculated and compared. RESULTS: Dixon-VIBE with water reconstruction had significantly higher subjective scores than FS-VIBE in strength and homogeneity of fat suppression (< 0.0001) but lower scores in sharpness of tumor (P < 0.0001), sharpness of vessels (P = 0.0001), and artifacts (P = 0.034). The liver-to-lesion contrast on Dixon-VIBE images was significantly lower than that on FS-VIBE (16.6% ± 9.4% vs 23.9% ± 12.1%, P = 0.0001). CONCLUSION: Dixon-VIBE provides stronger and more homogenous fat suppression than FS-VIBE, while has lower clarity of focal liver lesions in hepatobiliary phase after gadoxetic acid administration. PMID:25945017

  20. Reexamination of the Association Between Melting Point, Buoyant Density, and Chemical Base Composition of Deoxyribonucleic Acid

    PubMed Central

    De Ley, J.

    1970-01-01

    The equations currently used for the calculation of the chemical base composition of deoxyribonucleic acid (DNA), expressed as moles per cent guanine plus cytosine (% GC), from either buoyant density (ρ) or midpoint of thermal denaturation (Tm) were recalculated by using only sets of data on DNA determined with the same strains. All available information from the literature was screened and supplemented by unpublished data. The results were calculated by regression and correlation analysis and treated statistically. From the data on 96 strains of bacteria, it was calculated that% GC = 2.44 (Tm – 69.4). Tm appears to be unaffected by the substitution of cytosine by hydroxymethylcytosine. This equation is also valid for nonbacterial DNA. From the data on 84 strains of bacteria, the relation% GC = 1038.47 (–1.6616) was calculated. The constants in this equation are slightly modified when data on nonbacterial DNA are included. Both correlations differ only slightly from those currently used, but now they lean on a statistically sound basis. As a control, the relation between ρ and Tm was calculated from data of 197 strains; it agrees excellently with the above two equations. PMID:5438045

  1. 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). PMID:22035097

  2. Optimization of the 3-Point Bending Failure of Anodized Aluminum Formed in Tartaric/Sulphuric Acid Using Doehlert Design

    NASA Astrophysics Data System (ADS)

    Bensalah, W.; Feki, M.; De-Petris Wery, M.; Ayedi, H. F.

    2015-02-01

    The bending failure of anodized aluminum in tartaric/sulphuric acid bath was modeled using Doehlert design. Bath temperature, anodic current density, sulphuric acid, and tartaric acid concentrations were retained as variables. Thickness measurements and 3-point bending experiments were conducted. The deflection at failure ( D f) and the maximum load ( F m) of each sample were, then, deducted from the corresponding flexural responses. The treatment of experimental results has established mathematical models of second degree reflecting the relation of cause and effect between the factors and the studied properties. The optimum path study of thickness, deflection at failure, and maximum load, showed that the three optima were opposite. Multicriteria optimization using the desirability function was achieved in order to maximize simultaneously the three responses. The optimum conditions were: C tar = 18.2 g L-1, T = 17.3 °C, J = 2.37 A dm-2, C sul = 191 g L-1, while the estimated response values were e = 57.7 µm, D f = 5.6 mm, and F m = 835 N. Using the established models, a mathematical correlation was found between deflection at failure and thickness of the anodic oxide layer. Before bending tests, aluminum oxide layer was examined by scanning electron microscopy (SEM) and atomic force microscopy. After tests, the morphology and the composition of the anodic oxide layer were inspected by SEM, optical microscopy, and glow-discharge optical emission spectroscopy.

  3. 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

  4. 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

    2016-01-01

    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

  5. Common amino acid domain among endopolygalacturonases of ascomycete fungi.

    PubMed Central

    Keon, J P; Waksman, G

    1990-01-01

    The endopolygalacturonase (EC 3.2.1.15) enzymes produced in vitro by three ascomycete fungi, Aspergillus niger, Sclerotinia sclerotiorum, and Colletotrichum lindemuthianum were studied by using thin-layer isoelectric focusing and activity stain overlay techniques. The polygalacturonases from A. niger and S. sclerotiorum consisted of numerous isoforms, whereas the endopolygalacturonase from C. lindemuthianum consisted of a single protein species. The most abundant endopolygalacturonase isoform produced by each of these organisms was purified and characterized. Biochemical parameters, including molecular weight, isoelectric point, kinetic parameters, temperature and pH optima, and thermal stability, were determined. Considerable differences in physical and chemical properties were demonstrated among these fungal polygalacturonases. Antibodies raised against individual proteins exhibited little cross-reaction, suggesting that these enzymes differ structurally as well as biochemically. In contrast, the analysis of the N-terminal amino acid sequences of the three proteins showed extensive homology, particularly in a region labeled domain 1 in which 84% of the amino acids were conserved. Images PMID:2403258

  6. 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. PMID:24892189

  7. 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.

  8. Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries

    PubMed Central

    Koshel, Brooke M.; Wirth, Mary J.

    2013-01-01

    This review presents the need for replacing gels in 2D separations for proteomics, where speed, high-throughput, and the ability to characterize trace level proteins or small samples are the current desires. The theme of the review is isoelectric focusing, which is a valuable tool because it pre-concentrates proteins in addition to separating with high peak capacity. The review traces the technological progress from gel IEF to cIEF to packed capillaries with immobilized gradients for cIEF. Multiple capillary techniques are progressing toward meeting the current desires, providing extremely high sensitivity with regard to concentration and to small samples, integrated automation, and high peak capacity from multiple dimensions of separation. Capillaries with immobilized pH gradients for cIEF are emerging, which will alleviate interference from ampholytes and improve reproducibility in separation times when this valuable technique can be used as one of the dimensions. PMID:22930445

  9. Use of isoelectric focusing to discriminate transient oligoclonal bands from monoclonal protein in treated myeloma.

    PubMed

    Henry, Robyn; Glegg, Debra

    2016-06-01

    A 62-year-old diabetic man with prostate cancer first presented to our clinical laboratory in 2003 with a normal serum protein electrophoresis and immunofixation. In March 2009 he was diagnosed with an IgG κ myeloma. He underwent treatment and went into remission with the original paraprotein band being undetectable. Over the following 5 years, he developed oligoclonal bands and then eventually relapsed. Serum protein electrophoresis and immunofixation were inconclusive, however, isoelectrofocusing identified the oligoclonal pattern then the return of the original band, indicating relapse. This case illustrates the usefulness of an isoelectric focusing method to correctly determine clonality of small abnormal protein bands. It also highlights the need for appropriate commenting on reported results so that they are not confusing for clinicians. PMID:26641967

  10. Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase).

    PubMed

    Yoshida, N; Tsuruyama, S; Nagata, K; Hirayama, K; Noda, K; Makisumi, S

    1988-09-01

    A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA. PMID:3149277

  11. 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

  12. 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.

  13. 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.

  14. Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

    PubMed

    Khoo, K S; Beeley, J A

    1990-06-01

    Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing. PMID:1697536

  15. Two-dimensional electrophoresis of Arenicola marina extracellular hemoglobin: separation of chains with identical molecular mass but different isoelectric point.

    PubMed

    Slitine, F E; Toulmond, A

    1991-01-01

    1. On the basis of their molecular masses, four types of polypeptides (A, B, C, D) were obtained by SDS-PAGE of the extracellular hemoglobin of the polychaete annelid Arenicola marina. 2. On 2-dimensional polyacrylamide gel electrophoresis, the erythrocruorin dissociated into six different types of polypeptide chains; A1, A2, B1, B2, C and D. 3. A1 and B1 migrate in 2-dimensional electrophoresis at the same position as alpha and beta chains of human hemoglobin. PMID:1814687

  16. Simultaneous and selective decarboxylation of L-serine and deamination of L-phenylalanine in an amino acid mixture--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Witte-van Dijk, Susan C M; Sanders, Johan P M

    2016-01-25

    Amino acids (AAs) obtained from the hydrolysis of biomass-derived proteins are interesting feedstocks for the chemical industry. They can be prepared from the byproduct of biofuel production and agricultural wastes. They are rich in functionalities needed in petrochemicals, providing the opportunity to save energy, reagents, and process steps. However, their separation is required before they can be applied for further applications. Electrodialysis (ED) is a promising separation method, but its efficiency needs to be improved when separating AAs with similar isoelectric points. Thus, specific conversions are required to form product with different charges. Here we studied the enzymatic conversions which can be used as a means to aid the ED separation of neutral AAs. A model mixture containing L-serine, L-phenylalanine and L-methionine was used. The reactions of L-serine decarboxylase and L-phenylalanine ammonia-lyase were employed to specifically convert serine and phenylalanine into ethanolamine and trans-cinnamic acid. At the isoelectric point of methionine (pH 5.74), the charge of ethanolamine and trans-cinnamic acid are +1 and -1, therefore facilitating potential separation into three different streams by electrodialysis. Here the enzyme kinetics, specificity, inhibition and the operational stabilities were studied, showing that both enzymes can be applied simultaneously to aid the ED separation of neutral AAs. PMID:25976628

  17. Adsorption of oleic acid at sillimanite/water interface.

    PubMed

    Kumar, T V Vijaya; Prabhakar, S; Raju, G Bhaskar

    2002-03-15

    The interaction of oleic acid at sillimanite-water interface was studied by adsorption, FT-IR, and zeta potential measurements. The isoelectric point (IEP) of sillimanite obtained at pH 8.0 was found to shift in the presence of oleic acid. This shift in IEP was attributed to chemisorption of oleic acid on sillimanite. Adsorption experiments were conducted at pH 8.0, where the sillimanite surface is neutral. The adsorption isotherm exhibited a plateau around 5 micromol/m2 that correspond to a monolayer formation. Adsorption of oleic acid on sillimanite, alumina, and aluminum hydroxide was studied by FT-IR. Chemisorption of oleic acid on the above substrates was confirmed by FT-IR studies. Hydroxylation of mineral surface was found to be essential for the adsorption of oleic acid molecules. These surface hydroxyl sites were observed to facilitate deprotonation of oleic acid and its subsequent adsorption. Thus protons from oleic acid react with surface hydroxyl groups and form water molecules. Based on the experimental results, the mechanism of oleic acid adsorption on mineral substrate was proposed. Free energy of adsorption was estimated using the Stern-Graham equation for a sillimanite-oleate system. PMID:16290466

  18. 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

  19. Polydimethylsiloxane-Paper Hybrid Lateral Flow Assay for Highly Sensitive Point-of-Care Nucleic Acid Testing.

    PubMed

    Choi, Jane Ru; Liu, Zhi; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Yang, Hui; Qu, Zhiguo; Pingguan-Murphy, Belinda; Xu, Feng

    2016-06-21

    In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future. PMID:27012657

  20. [Microchip free flow isoelectric focusing with immobilized pH gradient on monolithic materials].

    PubMed

    Han, Bin; Wang, Pingli; Zhang, Lihua; Qu, Feng; Liang, Zhen; Deng, Yulin; Zhang, Yukui

    2009-07-01

    Microchip free flow electrophoresis (microFFE) is a significant microscale technique for the continuous pre-fractionation and the preparation of valuable biological samples. In our recent work, monolithic polyacylamide (PAM) materials were polymerized in microchamber by ultraviolet (UV) initiated polymerization. With the further immobilization of a stable pH gradient on the monolith, a novel microchip free flow isoelectric focusing (microFF-IEF) with monolithic immobilized pH gradient (M-IPG) materials was developed, by which fluorescein-5-isothiocyanate (FITC) labeled glycin, proline and lysine, with a minimum pI difference of 0.33 units, were well separated with a resolution higher than that performed by traditional microFF-IEF. Our experimental results demonstrate that by microFF-IEF with M-IPG, not only the interference of mobile carrier ampholytes in buffer, usually indispensable in traditional microFF-IEF, on the further separation by other techniques and the identification by mass spectrometry (MS) could be avoided, but also the improved resolution and detection sensitivity could be obtained compared with traditional microFF-IEF. Therefore, such a novel technique might be promising in microscale consecutive separation and preparation of samples. PMID:19938489

  1. Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries.

    PubMed

    Conti, M; Gelfi, C; Righetti, P G

    1995-08-01

    Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6-8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N-acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier ampholyte buffers were added to 3% short-chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier ampholytes in the pH 6-8 range and 3% soluble polyacrylamide, is made to contain 50 mM beta-alanine, a "separator" known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65-75%); Hb A, 20.2% (range 15-25%); and HbFac, 9.5% (range 7-11%). PMID:8529619

  2. Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products.

    PubMed

    Anderson, Carrie L; Wang, Yang; Rustandi, Richard R

    2012-06-01

    CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), 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. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste'' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products. PMID:22736354

  3. 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. PMID:16765111

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

    SciTech Connect

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

    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.

  5. Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

    PubMed Central

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

    2013-01-01

    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 2nd-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. PMID:23819755

  6. Fatty Acid-binding Protein 4, a Point of Convergence for Angiogenic and Metabolic Signaling Pathways in Endothelial Cells*

    PubMed Central

    Harjes, Ulrike; Bridges, Esther; McIntyre, Alan; Fielding, Barbara A.; Harris, Adrian L.

    2014-01-01

    Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment. PMID:24939870

  7. Isoelectric focusing of superoxide dismutase: report of the unique SOD A*2 allele in a US white population.

    PubMed

    DeCroo, S; Kamboh, M I; Leppert, M; Ferrell, R E

    1988-01-01

    An isoelectric focusing procedure in an ultranarrow pH range (5.0-5.5) polyacrylamide gel is described for the determination of superoxide dismutase (SOD) phenotypes. The occurrence of the rare SOD A*2 allele in the Caucasian population of Utah is also reported at a polymorphic frequency (0.011). The presence of the SOD A 2 unique allele in the Mormons of Utah is compatible with their historical affinity with Scandinavians. PMID:3350528

  8. The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida). Involvement of acidic polysaccharides from glycoproteins in nacre mineralization.

    PubMed

    Marie, Benjamin; Luquet, Gilles; Pais De Barros, Jean-Paul; Guichard, Nathalie; Morel, Sylvain; Alcaraz, Gérard; Bollache, Loïc; Marin, Frédéric

    2007-06-01

    Among molluscs, the shell biomineralization process is controlled by a set of extracellular macromolecular components secreted by the calcifying mantle. In spite of several studies, these components are mainly known in bivalves from only few members of pteriomorph groups. In the present case, we investigated the biochemical properties of the aragonitic shell of the freshwater bivalve Unio pictorum (Paleoheterodonta, Unionoida). Analysis of the amino acid composition reveals a high amount of glycine, aspartate and alanine in the acid-soluble extract, whereas the acid-insoluble one is rich in alanine and glycine. Monosaccharidic analysis indicates that the insoluble matrix comprises a high amount of glucosamine. Furthermore, a high ratio of the carbohydrates of the soluble matrix is sulfated. Electrophoretic analysis of the acid-soluble matrix revealed discrete bands. Stains-All, Alcian Blue, periodic acid/Schiff and autoradiography with (45)Ca after electrophoretic separation revealed three major polyanionic calcium-binding glycoproteins, which exhibit an apparent molecular mass of 95, 50 and 29 kDa, respectively. Two-dimensional gel electrophoresis shows that these bands, provisionally named P95, P50 and P29, are composed of numerous isoforms, the majority of which have acidic isoelectric points. Chemical deglycosylation of the matrix with trifluoromethanesulfonic acid induces a drastic shift of both the apparent molecular mass and the isoelectric point of these matrix components. This treatment induces also a modification of the shape of CaCO(3) crystals grown in vitro and a loss of the calcium-binding ability of two of the main matrix proteins (P95 and P50). Our findings strongly suggest that post-translational modifications display important functions in mollusc shell calcification. PMID:17488282

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

    PubMed

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

    2016-05-01

    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 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. PMID:26945062

  10. 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

  11. Simultaneous analysis of serum immunoglobulins in patients with M protein using cellulose acetate membrane isoelectric focusing.

    PubMed

    Iijima, S; Shiba, K; Kurihara, Y; Kamei, S; Kimura, S; Kimura, M; Fukumura, Y; Kobayashi, I

    1999-01-01

    We developed a method for the simultaneous analysis of microheterogeneity of human serum IgG, IgA, IgM, IgD, and IgE, and serum protein pattern using cellulose acetate membrane isoelectric focusing, and analyzed in 11 healthy subjects and 67 patients with M protein (17 cases of multiple myeloma [MM] and 50 cases of monoclonal gammopathy of undetermined significance [MGUS]). Using this method, bands indicating the microheterogeneity of each immunoglobulin could clearly be detected.Among healthy subjects, the detected IgG, IgA, and IgM bands did not vary, but the detected IgE and IgD bands did vary. Therefore, IgA, IgM, and IgG were selected for comparison of serum immunoglobulins in MM and in MGUS. In the IgA-type M protein group, normal IgM and IgG bands were decreased in MM patients compared to MGUS patients, while the M band and other bands were increased in MM patients compared to MGUS patients, but the differences between the two groups were not significant. In the IgG-type M protein group, normal IgM, IgA, and IgG were significantly decreased in MM patients compared to MGUS patients. We examined the changes in electrophoretic pattern in six MM patients and eight MGUS patients with IgA-type M protein after neuraminidase treatment. The width of the M band in MM patients with IgA-type M protein decreased with neuraminidase treatment. On the other hand, the width of the M band in MGUS patients with IgA-type M protein increased with neuraminidase treatment. We concluded that the decrease of the normal immunoglobulins in MM patients with IgG type M protein could be detected by this method, and IgA type of M protein binding sugar chain were different between MM and MGUS patients. PMID:10414593

  12. Microfluidic Preparative Free-Flow Isoelectric Focusing: System Optimization for Protein Complex Separation

    PubMed Central

    Wen, Jian; Wilker, Erik W.; Yaffe, Michael B.; Jensen, Klavs F.

    2010-01-01

    Isoelectric Focusing (IEF) is the first step for two-dimensional (2D) gel electrophoresis and plays an important role in sample purification for proteomics. However, biases in protein size and pI resolution, as well as limitations in sample volume, gel capacity, sample loss, and experimental time, remain challenges. In order to address some of the limitations of traditional IEF, we present a microfluidic free flow IEF (FF-IEF) device for continuous protein separation into 24 fractions. The device reproducibly establishes a nearly linear pH gradient from 4 to 10. Optimized dynamic coatings of 4% poly (vinyl) alcohol (PVA) minimize peak broadening by transverse electrokinetic flows. Even though the device operates at high electric fields (up to 370V/cm) efficient cooling maintains solution temperature inside the separation channel controllably in the range 2 – 25 °C. Protein samples with a dynamic concentration range between µg/mL to mg/mL can be loaded into the micro device at a flow rate of 1 mL/hr and residence time of ~12 min. By using a protein complex of 9 proteins and 13 isoforms, we demonstrate improved separation with the FF-IEF system over traditional 2D gel electrophoresis. Device to device reproducibility is also illustrated through the efficient depletion of the albumin and hemoglobin assays. Post-device sample concentrations result in a 10 to 20-fold increase, which allow for isolation and detection of low abundance proteins. The separation of specific proteins from a whole cell lysate is demonstrated as an example. The micro device has the further benefits of retaining high molecular weight proteins, providing higher yield of protein that has a broader range in pI, and reducing experimental time compared to conventional IEF IGP gel strip approaches. PMID:20092256

  13. 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. PMID:26460178

  14. A simple screening test for variant transthyretins associated with familial transthyretin amyloidosis using isoelectric focusing.

    PubMed

    Connors, L H; Ericsson, T; Skare, J; Jones, L A; Lewis, W D; Skinner, M

    1998-09-30

    Variant forms of the plasma protein transthyretin (TTR) are associated with the most frequently occurring type of familial systemic amyloidosis. Organ system involvement in transthyretin type amyloidosis (ATTR) is often similar to that which occurs in light chain amyloid disease (AL). The proper diagnosis of ATTR is important since treatment (liver transplantation) differs from that in AL (chemotherapy). We present a two-step test to screen sera for variant TTRs using non-denaturing gel electrophoresis performed in 7.5% acrylamide (PAGE) followed by isoelectric focusing (IEF) between pH 4.0 and 7.0 in 2.5 M urea. Serum samples from 110 patients with amyloidosis and their relatives were tested using this IEF technique and compared to genetic mutation results. Sera from patients with ATTR who underwent liver transplantation were also examined prior to and following surgery. IEF analysis showed the presence of both wild-type and variant TTR in 74 of the 110 serum samples tested. Genomic DNA from peripheral blood was used to identify TTR gene mutations in 77 of the 110 patients. Fifteen variants including Val122Ile, preponderant in the African-American population, could be demonstrated by IEF. The sensitivity of IEF was 96% (74/77) and the specificity was 100% (33/33). The predictive values for a positive or negative result were 100% (74/74) and 92% (33/36), respectively. There were no false-positive results and 4% (3/77) false-negative results. In sera from patients with ATTR who underwent liver transplantation, variant TTR was detected by IEF before, but not after, surgery. A simple, accurate, sensitive method is presented as a useful screening test for variant transthyretins associated with ATTR. PMID:9748569

  15. 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

  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 iTRAQ

    PubMed Central

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

    2013-01-01

    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 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-ESIMS/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-ESIMS/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. PMID:23822771

  17. Membrane-assisted capillary isoelectric focusing coupling with matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry for neuropeptide analysis.

    PubMed

    Zhang, Zichuan; Wang, Junhua; Hui, Limei; Li, Lingjun

    2011-08-01

    Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI-FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis. PMID:21696746

  18. 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.

  19. Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

    PubMed Central

    Wu, S; Kriz, A L; Widholm, J M

    1994-01-01

    The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that

  20. 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. PMID:26794762

  1. 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

  2. ERK and AKT phosphorylation status in lung cancer and emphysema using nanocapillary isoelectric focusing

    PubMed Central

    Crosbie, Philip A J; Crosbie, Emma J; Aspinall-O'Dea, Mark; Walker, Michael; Harrison, Rebecca; Pernemalm, Maria; Shah, Rajesh; Joseph, Leena; Booton, Richard; Pierce, Andrew; Whetton, Anthony D

    2016-01-01

    Background Emphysema is an independent risk factor for the development of lung cancer in smokers. Activation of oncogenic signalling proteins AKT and ERK by phosphorylation has an established role in the development of lung cancer and has also been implicated in the pathogenesis of emphysema. The aim of this study was to compare the protein level and phosphorylation status of AKT and ERK in paired lung cancer and emphysema tissue using a highly sensitive phosphoprotein analysis approach. Methods An antibody-based, nanocapillary isoelectric focusing (cIEF) assay was used to determine the relative quantities and phosphorylation status of AKT and ERK in tumour and matched lung tissue from patients, with or without evidence of emphysema, undergoing curative resection for non-small cell lung cancer. Results 20 patients with adenocarcinoma (n=9) or squamous cell carcinoma (n=11) of the lung were included (mean age 67.3 years (SD 7.5, range 47–80 years)), 12 were men and all were current (n=10) or former smokers (n=10). Paired macroscopically normal lung tissue was either histologically normal (n=7) or showed emphysema (n=13). Total and phosphorylated AKT levels were fourfold (p=0.0001) and fivefold (p=0.001) higher in tumour compared with matched lung, respectively. There was no correlation with tumour histology, stage or differentiation; however, total AKT signal in tumour was significantly correlated with fluorodeoxyglucose avidity on positron emission tomography-CT scan (r=0.53, p=0.035). Total ERK was not differentially expressed, but doubly phosphorylated (activated) ERK was threefold higher in emphysema (23.5%, SD 9.2) than either matched tumour (8.8%, SD 8.6) or normal lung tissue (8.3%, SD 9.0) and correlated with the histological severity of emphysema (p=0.005). Conclusions cIEF offers opportunities for quantifying subtle shifts in the phosphorylation status of oncoproteins in nanogram amounts of lung tissue. ERK activation is a feature of emphysema. PMID

  3. Research results from the Advanced Lead-Acid Battery Consortium point the way to longer life and higher specific energy for lead/acid electric-vehicle batteries

    NASA Astrophysics Data System (ADS)

    Moseley, P. T.

    Amidst the welter of publicity devoted to the newer battery chemistries, the remarkable progress made by lead/acid battery technologists in response to the needs of the emerging electric-vehicle market has tended to be overlooked. The flooded design of battery, launched by Gaston Planté around 1860, has given way to a valve-regulated variant which has a history dating only from the 1970s. The key parameters of this `maintenance free' battery have been improved markedly during the course of the development programme of the Advanced Lead-Acid Battery Consortium (ALABC), and it is likely that lead/acid will continue to feature strongly in motive-power applications as a result of its cost advantage and of its enhanced effectiveness.

  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. PMID:26774997

  5. 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.

  6. 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.

  7. 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. PMID:24691723

  8. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

    PubMed

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

    2014-07-17

    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 mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  9. 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. PMID:27038651

  10. 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). PMID:24209377

  11. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25402946

  13. 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.

  14. 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.

  15. 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.

  16. Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

    PubMed Central

    Modak, Sayli S.; Barber, Cheryl A.; Geva, Eran; Abrams, William R.; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

    2016-01-01

    Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation. PMID:26819557

  17. Adsorption dynamics of L-glutamic acid copolymers at a heptane/water interface.

    PubMed

    Beverung, C J; Radke, C J; Blanch, H W

    1998-02-16

    Random copolymers of glutamic acid (glu-ala, glu-leu, glu-phe, glu-tyr) were employed to investigate the relationship between side chain structure and peptide charge on adsorption behavior at an oil/water boundary. Adsorption of a series of glutamate copolymers at a heptane/water interface was examined by the dynamic pendant-drop method to determine interfacial tension. Incorporation of leucine or phenylalanine into a glutamate copolymer results in greater tension reduction than incorporation of alanine or tyrosine. These effects are amplified at pH values near the isoelectric point of glutamate, where macroscopic adsorbed films of glu-leu and glu-phe exhibit gel-like properties in response to interfacial area compression. Differences in interfacial tension behavior of glu-tyr and glu-phe indicate the importance of the tyrosine p-hydroxyl group on adsorption and aggregation at the oil/water interface. PMID:9540205

  18. 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. PMID:12351830

  19. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-05-15

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  20. 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.

  1. Polarized Raman spectra and intensities of aliphatic amino acids

    NASA Astrophysics Data System (ADS)

    Himmler, Hans J.; Eysel, Hans H.

    1989-01-01

    Raman spectra of aliphatic α- L-amino acids, glycine, alanine, and valine were re-investigated both in aqueous solution and deuterium oxide solution. The spectra were taken of the zwitterionic and of the completely deprotonated form of the amino acids. Spectra of leucine and isoleucine were studied in water at the isoelectric point. Spectra were recorded both with parallel and perpendicular polarization and the isotropic and anisotropic scattering components were isolated. The integrated intensities of CH stretch, CC stretch and carboxylate bend vibrations are discussed. Linear relations between the number of CC and CH bonds and the total scattered intensity in the appropriate spectral regions are observed. The sum over the carboxylate modes shows characteristic intensities for the first three members of the aliphatic amino acids. An increase of isotropic scattering of ϱ co 2 near 510 cm -1 with increasing chain length of the amino acid (or with increasing concentration) is interpreted as the result of micelle formation.

  2. Potentiometric investigation of the effect of the pH on the ionic transfer of some amino acids at the interface between two immiscible electrolyte solutions.

    PubMed

    Spătaru, Tanta; Spătaru, Nicolae; Bonciocat, Nicolae; Luca, Constantin

    2004-04-01

    The effect of the pH on the ionic transfer of glycine and beta-alanine at the interface between two immiscible electrolyte solutions (ITIES) was investigated by a simple potentiometric method. Upon addition of small amounts of solution containing the investigated amino acids, a variation of the potential drop across the interface was recorded, which was found to be pH-dependent. This behavior was explained in terms of a preferential orientation of the amino acid molecules at the ITIES, induced by the different lipoficility of the functional groups. The results enabled the measurement of this voltage variation to be used as the basis for a simple and rapid method for determining the isoelectric point of the investigated compounds. The agreement between the pH(i) values thus estimated and those reported in the literature suggests the possibility of using the method for the interpretation of processes occurring at the level of biological membranes. PMID:14990327

  3. 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.

  4. 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. PMID:17566886

  5. Release of brain amino acids during hyposmolar stress and energy deprivation.

    PubMed

    Haugstad, T S; Langmoen, I A

    1996-04-01

    The release of 10 amino acids from rat hippocampal slices during exposure to hyposmotic stress or energy deprivation was measured by high-performance liquid chromatography. Exposing the slices to hyposmotic stress by lowering extracellular NaCl caused a 10-fold release of taurine (p < 0.01) and over a twofold increase of gamma-aminobutyric acid (GABA) and glutamate (p < 0.01). These changes were reversed by mannitol. Exposure to combined glucose and oxygen deprivation (energy deprivation) caused a 50-fold increase in the release of GABA, a 40-fold increase in glutamate release (p < 0.01), and a twofold to sixfold increase in taurine, aspartate, glycine, asparagine, serine, and alanine release (p < 0.05) but no change in glutamine. Energy deprivation increased the water content by 21%. Mannitol blocked this increase and further enhanced the release of glutamate and aspartate (p < 0.01) but not of GABA. The permissivity of the amino acids was plotted against the pI (pH at isoelectric point) and hydropathy indexes. Energy deprivation increased the permissivity in the following order: acidic > neutral > basic. Among neutral amino acids, permissivity increased with increasing hydrophobicity. These results indicate that the mechanisms of amino acid release are different during cerebral ischemia and hyposmotic stress. PMID:8829565

  6. The properties of solid Zn(II)-amino acid complexes in the form of suspensions.

    PubMed

    Dolińska, B

    2001-10-01

    An investigation was made into the experimental conditions for the formation of poorly soluble complexes of the divalent Zinc(II) combined with the following selected amino acids: tyrosine, tryptophan, cysteine, histidine, and alanine, in the form of suspensions for parenteral administration. The number of Zn(II)-binding sites in the amino acid (n) as well as the amino acid affinity to Zn(II) (Ka), were determined. Cysteine was found to have the highest number of Zn(II)-binding sites--3, whereas alanine the lowest--1. In the conditions described herein, Zn(II) amino acid complexes of diverse stability (durability) were obtained. The analysis of the kinetics of the binding revealed that the most stable complexes were those formed by Zn(II) in combination with tryptophan (Ka = 405.78 microM(-1) +/- 12.17), and with tyrosine (Ka = 343.88 microM +/- 22.35); whereas the least stable complexes were those formed by Zn(II) in combination with histidine (Ka = 29.90 microM +/- 4.78), and with alanine (Ka = 13.0 microM(-1) +/- 1.04). Cysteine formed complexes of intermediate stability (Ka = 168.53 microM(-1) +/- 12.36). The stability ofthe Zn(II) amino acid complexes obtained was conditioned by both the molecular weight (P = 0.033) of the amino acid and its isoelectric point (P < 0.001). PMID:11718265

  7. Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor.

    PubMed

    Chen, Yuting; Cheng, Nan; Xu, Yuancong; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2016-07-15

    This study describes a simple and sensitive approach for visual and point-of-care detection of P. aeruginosa and its toxin genes based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow nucleic acid biosensor (LFNAB). Differentiation of the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa was determined using FITC-, hex-and digoxin-modified primers in the mLAMP process. In the presence of biotin-and FITC- (hex-, digoxin-) modified primers and Bst DNA polymerase large fragments, the mLAMP produced numerous biotin- and FITC- (hex-, digoxin-) attached duplex DNA products. The products were detected by LFNAB through dual immunoreactions (anti-biotin antibodies on the gold nanoparticle (Au-NP) and biotin on the duplex, anti-FITC (hex, digoxin) antibodies on the LFNAB test line and FITC (hex, digoxin) on the duplex). The accumulation of Au-NPs produced a characteristic red band, enabling visual detection of P. aeruginosa and its toxin genes without instrumentation. After systematic optimization of LFNAB preparation and detecting conditions, the current approach was capable of detecting concentrations as low as 20 CFU/mL P. aeruginosa or its toxin genes within 50min without complicated instrument, which is more sensitive than PCR. Therefore, this approach provides a simple, pollution free, sensitive, and low-cost point-of-care test for the detection of P. aeruginosa and its toxin genes. PMID:26985584

  8. 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. PMID:25044151

  9. 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.

  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. 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.

  12. 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. PMID:25096199

  13. Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo.

    PubMed

    Altwegg-Boussac, Tristan; Mahon, Séverine; Chavez, Mario; Charpier, Stéphane; Schramm, Adrien E

    2016-01-01

    The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons' integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent. PMID:27078163

  14. [Agarose gel isoelectric focusing: application to the study of abnormalities of immunoglobulin clonality in CSF and serum].

    PubMed

    Lebrun-Fourcy, C; Rondot, J; Revol, C; Renversez, J C

    1996-01-01

    Since it is quite difficult to commonly use isoelectric focusing (IEF) of proteins in polyacrylamide gel for biological diagnosis, we have developed a method based on IEF in agarose gel, to split proteins from sera and cerebrospinal fluid (CSF). A prefocalisation at low voltage (250 V) is made on a custom thin gel of agarose (0.5 mm) containing some carrier ampholytes (pH 5-9). After deposition of biological samples, the gel is run at 500 V, thereafter at 1200 V. After focusing, the gel is fixed before being coloured by a simplified silver staining technique. In order to demonstrate the good resolution of the immunoglobulines (Ig) in the pH gradient, a transfer on a nitrocellulose membrane followed by an immunofixation was carried out from unstained gels after IEF. This separation on agarose gel shows several advantages, ie its speed (3H total), its lack of toxicity, its sensibility and its reproductibility. It is specially well suited for the diagnosis of diseases characterised by oligoclonal or monoclonal Ig, particularly those found in the CSF during neurologic diseases like multiple sclerosis. Several examples of focused sera and CSF are reviewed in the paper. PMID:8952725

  15. 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

  16. Adsorption of dissolved aluminum on sapphire-c and kaolinite: implications for points of zero charge of clay minerals.

    PubMed

    Lützenkirchen, Johannes; Abdelmonem, Ahmed; Weerasooriya, Rohan; Heberling, Frank; Metz, Volker; Marsac, Remi

    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

  17. 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

  18. Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.

    PubMed

    Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-02-01

    A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4 ± 0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03 ± 0.23, 15.27 ± 0.36, and 177.31 ± 25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation. PMID:23665639

  19. Investigation of surfactant mediated acid-base charging of mineral oxide particles dispersed in apolar systems.

    PubMed

    Gacek, Matthew M; Berg, John C

    2012-12-21

    The current work examines the role of acid-base properties on particle charging in apolar media. Manipulating the polarity and magnitude of charge in such systems is of growing interest to a number of applications. A major hurdle to the implementation of this technology is that the mechanism(s) of particle charging remain a subject of debate. The authors previously conducted a study of the charging of a series of mineral oxide particles dispersed in apolar systems that contained the surfactant AOT. It was observed that there was a correlation between the particle electrophoretic mobility and the acid-base nature of the particle, as characterized by aqueous point of zero charge (PZC) or the isoelectric point (IEP). The current study investigates whether or not a similar correlation is observed with other surfactants, namely, the acidic Span 80 and the basic OLOA 11000. This is accomplished by measuring the electrophoretic mobility of a series of mineral oxides that are dispersed in Isopar-L containing various concentrations of either Span 80 or OLOA 11000. The mineral oxides used have PZC values that cover a wide range of pH, providing a systematic study of how particle and surfactant acid-base properties impact particle charge. It was found that the magnitude and polarity of particle surface charge varied linearly with the particle PZC for both surfactants used. In addition, the point at which the polarity of charge reversed for the basic surfactant OLOA 11000 was shifted to a pH of approximately 8.5, compared to the previous result of about 5 for AOT. This proves that both surfactant and particle acid-base properties are important, and provides support for the theory of acid-base charging in apolar media. PMID:23157688

  20. Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Croux, C; Paquet, V; Goma, G; Soucaille, P

    1990-01-01

    Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91%. The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3. Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents. It was shown to be a calcium- and zinc-containing protease. It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C. It was stable at low pH and heat labile above 50 degrees C. It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A. Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported. Images PMID:2082818

  1. Gamma-aminobutyric acid and glutamic acid levels in the auditory pathway of rats with chronic tinnitus: a direct determination using high resolution point-resolved proton magnetic resonance spectroscopy (1H-MRS)

    PubMed Central

    Brozoski, Thomas; Odintsov, Boris; Bauer, Carol

    2012-01-01

    Damage to the auditory system following high-level sound exposure reduces afferent input. Homeostatic mechanisms appear to compensate for the loss. Overcompensation may produce the sensation of sound without an objective physical correlate, i.e., tinnitus. Several potential compensatory neural processes have been identified, such as increased spontaneous activity. The cellular mechanisms enabling such compensatory processes may involve down-regulation of inhibitory neurotransmission mediated by γ-amino butyric acid (GABA), and/or up-regulation of excitatory neurotransmission, mediated by glutamic acid (Glu). Because central processing systems are integrated and well-regulated, compensatory changes in one system may produce reactive changes in others. Some or all may be relevant to tinnitus. To examine the roles of GABA and Glu in tinnitus, high resolution point-resolved proton magnetic resonance spectroscopy (1H-MRS) was used to quantify their levels in the dorsal cochlear nucleus (DCN), inferior colliculus (IC), medial geniculate body (MGB), and primary auditory cortex (A1) of rats. Chronic tinnitus was produced by a single high-level unilateral exposure to noise, and was measured using a psychophysical procedure sensitive to tinnitus. Decreased GABA levels were evident only in the MGB, with the greatest decrease, relative to unexposed controls, obtained in the contralateral MGB. Small GABA increases may have been present bilaterally in A1 and in the contralateral DCN. Although Glu levels showed considerable variation, Glu was moderately and bilaterally elevated both in the DCN and in A1. In the MGB Glu was increased ipsilaterally but decreased contralaterally. These bidirectional and region-specific alterations in GABA and Glu may reflect large-scale changes in inhibitory and excitatory equilibrium accompanying chronic tinnitus. The present results also suggest that targeting both neurotransmitter systems may be optimal in developing more effective therapeutics

  2. Molecular characterization by high-resolution isoelectric focusing of the products encoded by the class II region loci of the major histocompatibility complex in humans. I. DR and DQ gene variants.

    PubMed

    Rodriguez de Cordoba, S; Nunez-Roldan, A; Winchester, R; Marshall, P; Carrier, C; Mollen, N; Walker, M; Ginsberg-Fellner, F; Rubinstein, P

    1987-09-01

    We describe a new approach to the analysis of the structural polymorphism of the DR beta, DQ alpha, and DQ beta polypeptide chains of human histocompatibility class II antigens. In comparison to conventional two-dimensional gel studies, this method provides sharper definition of the protein bands and side-by-side comparisons within the same gel, thereby permitting the detection of minor differences in the isoelectric points of the protein chains. Using this methodology we have analyzed the IEF polymorphism and the variability in the number of the DR beta chains encoded by different DR haplotypes. Twenty DR beta chain variants, which include the products of no less than two separate DR beta loci, have been thus far identified. Alleles at one of these loci are assumed to code for DR beta chains carrying the DR alloespecificities DR1, DR2, DR3, DR4, DR5, DRw6, DR7, and DR8. Alleles at a second DR beta locus encode DR beta chains that may be shared by serologically DR-different haplotypes and carry supertypic serologic specificities (i.e., DRw52 and DRw53). We also demonstrate here that the structural polymorphisms of the DQ alpha and DQ beta chains are more extensive than previously thought, report the characterization of 14 DQ beta variants, and define their relationship to the previously described DQw serologic specificities. In addition, we describe the class II haplotype associations observed for the different DR and DQ variants characterized. PMID:3679903

  3. 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

  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. Modulation of a recombinant invertebrate γ-aminobutyric acid receptor-chloride channel complex by isoflurane: effects of a point mutation in the M2 domain

    PubMed Central

    Edwards, Michelle D; Lees, George

    1997-01-01

    Inhalational anaesthetics modulate ligand-gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for γ-aminobutyric acid (GABA) receptor modulation by isoflurane. Wild-type Drosophila melanogaster homo-oligomeric GABA receptors were characterized and compared with an ion-channel mutant (alanine substituted to a serine in M2) by means of two-electrode voltage-clamp in membrane-invariant Xenopus oocytes. Both channel receptor isoforms generated outwardly rectifying, bicuculline-insensitive currents with reversal potentials characteristic of a chloride current. As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 μM picrotoxinin (PTX): circa 7 fold reduction at the GABA EC20. Isoflurane, 195–389 μM, enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. Isoflurane concentration-response curves (on circa GABA EC25) demonstrated that enhancement was effected at around 100–195 μM for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 μM: wild-type receptors exhibited concentration-dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 μM). The above divergence was not attributable to differential desensitization: neither wild-type nor mutant conductance desensitized significantly (P>0.05) in the absence or presence of anaesthetic. This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. The non

  6. 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

  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. PMID:24616376

  8. Structure and expression of an unusually acidic matrix protein of pearl oyster shells.

    PubMed

    Tsukamoto, Daiki; Sarashina, Isao; Endo, Kazuyoshi

    2004-08-01

    We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp)(2-10) sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata. PMID:15249213

  9. Purification, characterization, and complete amino acid sequence of a trypsin inhibitor from amaranth (Amaranthus hypochondriacus) seeds.

    PubMed Central

    Valdes-Rodriguez, S; Segura-Nieto, M; Chagolla-Lopez, A; Verver y Vargas-Cortina, A; Martinez-Gallardo, N; Blanco-Labra, A

    1993-01-01

    A protein proteinase inhibitor was purified from a seed extract of amaranth (Amaranthus hypochondriacus) by precipitation with (NH4)2SO4, gel-filtration chromatography, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography. It is a 69-amino acid protein with a high content of valine, arginine, and glutamic acid, but lacking in methionine. The inhibitor has a relative molecular weight of 7400 and an isoelectric point of 7.5. It is a serine proteinase inhibitor that recognizes chymotrypsin, trypsin, and trypsin-like proteinase activities extracted from larvae of the insect Prostephanus truncatus. This inhibitor belongs to the potato-I inhibitor family, showing the closest homology (59.5%) with the Lycopersicum peruvianum trypsin inhibitor, and (51%) with the proteinase inhibitor 5 extracted from the seeds of Cucurbita maxima. The position of the lysine-aspartic acid residues present in the active site of the amaranth inhibitor are found in almost the same relative position as in the inhibitor from C. maxima. PMID:8290633

  10. Some Properties of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

    PubMed Central

    Hashimoto, Hikotaka; Iwaasa, Takashi; Yokotsuka, Tamotsu

    1973-01-01

    A purified acid protease from a true thermophilic fungus, Penicillium duponti K1014, was most active at pH 2.5 for milk casein and at pH 3.0 for hemoglobin. The enzyme was stable at a pH range of 2.5 to 6.0 at 30 C for 20 h. The acid protease retained full activity after 1 h at 60 C at a pH range between 3.5 and 5.5. At the most stable pH of 4.5, more than 65% of its activity remained after heat treatment for 1 h at 70 C. These thermal properties show the enzyme as a thermophilic protein. The enzyme activity was strongly inhibited by sodium lauryl sulfate and oxidizing reagents such as potassium permanganate and N-bromosuccinimide. No inhibition was caused by chelating reagents, potato inhibitor, and those reagents which convert sulfhydryl groups to mercaptides. Reducing reagents showed an activating effect. The enzyme showed the trypsinogen-activating property at an acidic pH range; optimal trypsinogen activation was obtained at a pH of approximately 3.0. The isoelectric point of the enzyme was estimated to be pH 3.89 by disk electrofocusing. By using gel filtration, an approximate value of 41,000 was estimated for the molecular weight. PMID:4699217

  11. 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

  12. Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 2.

    PubMed

    Lim, Peniel J; Vigh, Gyula

    2011-06-01

    A micropreparative-scale multicompartmental electrolyzer called ConFrac has been developed and tested for isoelectric trapping separations. ConFrac can be operated in pass-by-pass mode or recirculating mode, using either asymmetrical feeding (feed enters only the anodic or the cathodic flow-through compartment) or symmetrical feeding (feed enters both the anodic and the cathodic flow-through compartment). Symmetrical feeding results in higher processing rates and is the preferred operation mode. Residence time in the flow-through compartments is set as a compromise between processing rate and temperature rise in the effluent. Ampholytic components have been isolated from 5 to 50 mL volumes of micromolar feed solutions and hundredfold concentrated into 100-μL collection compartments. Samples containing ampholytic analytes in highly conducting salt solutions were readily desalted and fractionated in ConFrac in one operation. pH transients formerly observed in other isoelectric trapping devices were observed in ConFrac as well. The pH transients were caused by the unequal ion transmission rates of the anodic- and cathodic-side buffering membranes. PMID:21647926

  13. Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

    PubMed

    Uchida, K; Tsuchida, J; Tanaka, H; Koga, M; Nishina, Y; Nozaki, M; Yoshinaga, K; Toshimori, K; Matsumiya, K; Okuyama, A; Nishimune, Y

    2000-10-01

    We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation. PMID:10993819

  14. Isolation and immunological characterization of fatty acid binding protein isoforms from Fasciola hepatica.

    PubMed

    Espino, A M; Rodríguez Medina, J R; Hillyer, G V

    2001-10-01

    A combination of molecular sieving chromatography and 2-step preparative isoelectric focusing showed that native Fh12, a fatty acid-binding protein isolated from Fasciola hepatica adult worms, is a protein complex of at least 8 isoforms with identical molecular mass but different isoelectric points. Using enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA assays, immunological differences were observed between native (nFh12) and a recombinant molecule denoted rFh15 that was obtained after screening a cDNA library from F. hepatica adult worms with an anti-Fh12 monospecific polyclonal antibody. It was confirmed that in infected rabbits, antibodies to nFh12 appear by the second week postinfection, whereas antibodies to rFh15 appear much later, by 6 wk postinfection. Four acidic forms (Fh12(1-4)) showed more immunological identity with rFh15 than with nFh12, based on the observation that they inhibited ELISA activity by nearly 50% when they were added to the anti-rFh15 polyclonal antibody at 20 microg/ml of protein concentration. Moreover, the Fh12(1-4) isoforms were poorly reactive with sera from rabbits 2-4 wk postinfection. However, the 2 acidic forms, denoted Fh12(5) and Fh12(6), and the neutral/basic forms, denoted Fh12(7) and Fh12(8), showed more immunological identity with the native nFh12 molecule than with the recombinant rFh15 because they were highly reactive with sera of rabbits with early 2-wk F. hepatica infection and inhibited ELISA activity nearly 50% when they were quantitatively added to the anti-nFh12 polyclonal antibody. These results suggest that rFh15 could be one of the acidic forms of nFh12, and that it, in fact, may be one of the less immunogenic or immunoprotective members, or both, of the nFh12 protein complex. PMID:11695360

  15. 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. PMID:26867113

  16. Tipping Point

    MedlinePlus Videos and Cool Tools

    ... Tipping Point by CPSC Blogger September 22 appliance child Childproofing CPSC danger death electrical fall furniture head ... TV falls with about the same force as child falling from the third story of a building. ...

  17. Purification of an acidic phospholipase A2 from Bothrops lanceolatus (fer de lance) venom: molecular and enzymatic properties.

    PubMed

    de Araújo, A L; Radvanyi, F; Bon, C

    1994-09-01

    The acidic phospholipase A2 from Bothrops lanceolatus venom has been purified by gel filtration on Sephadex G-50 and ion exchange chromatography on CM-cellulose. Analysis by FPLC on Mono-Q column of the purified phospholipase A2 indicated that it is a mixture of several isoenzymes. The two major isoforms consist of a single polypeptide chain with mol. wts of 14,500 and 15,000, which slightly differ in their isoelectric point (4.9 and 5.3) and amino acid composition. However, enzymatic and pharmacological properties of the various isoenzymes are identical. The phospholipase from B. lanceolatus venom is characterized by a progressive increase in the rate of hydrolysis when enzymatic activity is determined with crude egg yolk as substrate in the absence of detergent. This phenomenon, which is not observed with mixed micelles of lecithin-detergent, is not due to the presence of a phospholipase A2 inhibitor in the venom, as previously suggested by several investigators in the case of other Bothrops and Cobra venoms. It is rather a catalytic characteristics of B. lanceolatus venom phospholipase, the enzymatic activity of which depends on the physical state of phospholipids. Bothrops lanceolatus acidic phospholipase A2 is non-toxic. PMID:7801343

  18. D-amino acid oxidase from the yeast Trigonopsis variabilis.

    PubMed

    Kubicek-Pranz, E M; Röhr, M

    1985-04-01

    D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme. PMID:2865242

  19. 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. PMID:21140022

  20. pH-Triggered release from surface-modified poly(lactic-co-glycolic acid) nanoparticles

    PubMed Central

    Häuser, Manuel; Langer, Klaus

    2015-01-01

    Summary Nanoparticles (NP) of poly(lactic-co-glycolic acid) (PLGA) represent a promising biodegradable drug delivery system. We suggest here a two-step release system of PLGA nanoparticles with a pH-tunable polymeric shell, providing an initial pH-triggered step, releasing a membrane-toxic cationic compound. PLGA nanoparticles are coated by polyelectrolytes using the layer-by-layer self-assembly technique, employing poly(acrylic acid) (PAA) as a pH-sensitive component and poly(diallyldimethylammonium chloride) (PDADMAC) as the releasable polycation. The pH during multilayer deposition plays a major role and influences the titration curve of the layer system. The pH-tunability of PAA is intensively investigated with regard to the pH region, in which the particle system becomes uncharged. The isoelectric point can be shifted by employing suitable deposition pH values. The release is investigated by quantitative 1H NMR, yielding a pH-dependent release curve. A release of PDADMAC is initiated by a decrease of the pH value. The released amount of polymer, as quantified by 1H NMR analysis, clearly depends on the pH value and thus on the state of deprotonation of the pH-sensitive PAA layer. Subsequent incubation of the nanoparticles with high concentrations of sodium chloride shows no further release and thus demonstrates the pH-driven release to be quantitative. PMID:26885463

  1. Mannose 6 Dephosphorylation of Lysosomal Proteins Mediated by Acid Phosphatases Acp2 and Acp5

    PubMed Central

    Makrypidi, Georgia; Damme, Markus; Müller-Loennies, Sven; Trusch, Maria; Schmidt, Bernhard; Schlüter, Hartmut; Heeren, Joerg; Lübke, Torben; Saftig, Paul

    2012-01-01

    Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5−/−) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5−/− mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. PMID:22158965

  2. Amino-acid substitution in alpha-spectrin commonly coinherited with nondominant hereditary spherocytosis.

    PubMed

    Tse, W T; Gallagher, P G; Jenkins, P B; Wang, Y; Benoit, L; Speicher, D; Winkelmann, J C; Agre, P; Forget, B G; Marchesi, S L

    1997-03-01

    Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS. PMID:9067503

  3. Induction of thymidine phosphorylase as a pharmacodynamic end-point in patients with advanced carcinoma treated with 5-fluorouracil, folinic acid and interferon alpha

    PubMed Central

    Braybrooke, J P; Propper, D J; O’Byrne, K J; Koukourakis, M I; Patterson, A V; Houlbrook, S; Love, S D; Varcoe, S; Taylor, M; Ganesan, T S; Talbot, D C; Harris, A L

    2000-01-01

    Thymidine phosphorylase (TP) is an essential enzyme for the biochemical activation of 5-fluorouracil (5-FU). Interferon upregulates TP in vivo, although the dose and schedule of interferon for optimal biomodulation of 5-FU is not known. In this study, TP activity was measured in peripheral blood lymphocytes (PBLs) from patients with advanced carcinoma receiving treatment with 5-FU and folinic acid. Cohorts of patients were treated with interferon alpha (IFNα), immediately prior to 5-FU/folinic acid, at doses of 3 MIU m–2, 9 MIU m–2and 18 MIUm–2. IFNα was administered on day 0 cycle two, day –1 and day 0 cycle three and day –2, day –1 and day 0 cycle four. A fourth cohort was treated with IFNα 9 MIU m–2three times per week from cycle 2 onwards. Twenty-one patients were entered into the study with 19 evaluable for response. Six patients (32%) had stable disease and 13 (68%) progressive disease. There were no grade-IV toxicities. TP activity was detected in PBLs from all patients with wide interpatient variability in constitutive TP activity prior to chemotherapy, and in response to IFNα. 5-FU/folinic acid alone did not induce TP activity but a single dose of IFNα led to upregulation of TP within 2 h of administration with a further increase by 24 h (signed rank test, P = 0.006). TP activity remained elevated for at least 13 days (signed rank test, P = 0.02). There were no significant differences in TP activity between schedules or with additional doses of IFNα. A single dose of IFNα as low as 3 MIU m–2can cause sustained elevation of PBL TP activity in vivo indicating that biochemical markers are important pharmacodynamic endpoints for developing optimal schedules of IFNα for biomodulation of 5-FU. © 2000 Cancer Research Campaign PMID:10901374

  4. Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

    PubMed

    Buhr, A; Baur, R; Malherbe, P; Sigel, E

    1996-06-01

    Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces. PMID:8649346

  5. 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

  6. 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. PMID:25959598

  7. Predicting points of departure for risk assessment based on in vitro cytotoxicity data and physiologically based kinetic (PBK) modeling: The case of kidney toxicity induced by aristolochic acid I.

    PubMed

    Abdullah, Rozaini; Alhusainy, Wasma; Woutersen, Jasper; Rietjens, Ivonne M C M; Punt, Ans

    2016-06-01

    Aristolochic acids are naturally occurring nephrotoxins. This study aims to investigate whether physiologically based kinetic (PBK) model-based reverse dosimetry could convert in vitro concentration-response curves of aristolochic acid I (AAI) to in vivo dose response-curves for nephrotoxicity in rat, mouse and human. To achieve this extrapolation, PBK models were developed for AAI in these different species. Subsequently, concentration-response curves obtained from in vitro cytotoxicity models were translated to in vivo dose-response curves using PBK model-based reverse dosimetry. From the predicted in vivo dose-response curves, points of departure (PODs) for risk assessment could be derived. The PBK models elucidated species differences in the kinetics of AAI with the overall catalytic efficiency for metabolic conversion of AAI to aristolochic acid Ia (AAIa) being 2-fold higher for rat and 64-fold higher for mouse than human. Results show that the predicted PODs generally fall within the range of PODs derived from the available in vivo studies. This study provides proof of principle for a new method to predict a POD for in vivo nephrotoxicity by integrating in vitro toxicity testing with in silico PBK model-based reverse dosimetry. PMID:27016491

  8. Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.

    PubMed

    Roberto, Patrícia G; Kashima, Simone; Soares, Andreimar M; Chioato, Lucimara; Faça, Victor M; Fuly, André L; Astolfi-Filho, Spartaco; Pereira, José O; França, Suzelei C

    2004-09-01

    The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities. PMID:15294287

  9. Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

    PubMed

    Rodrigues, Renata S; da Silva, Juliana F; Boldrini França, Johara; Fonseca, Fernando P P; Otaviano, Antônio R; Henrique Silva, Flávio; Hamaguchi, Amélia; Magro, Angelo J; Braz, Antônio Sérgio K; dos Santos, Juliana I; Homsi-Brandeburgo, Maria Inês; Fontes, Marcos R M; Fuly, André L; Soares, Andreimar M; Rodrigues, Veridiana M

    2009-04-01

    An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. PMID:19135502

  10. Separating nano graphene oxide from the residual strong-acid filtrate of the modified Hummers method with alkaline solution

    NASA Astrophysics Data System (ADS)

    Hu, Xuebing; Yu, Yun; Wang, Yongqing; Zhou, Jianer; Song, Lixin

    2015-02-01

    In the modified Hummers method for preparing graphene oxide, the yellow slurry can be obtained. After filtering through a quantitative filter paper, the strong-acid filtrate containing the unprecipitated nano graphene oxide was gained. The corresponding filtrate was added gradually with an alkaline (NaOH or KOH) solution at room temperature. The unprecipitated nano graphene oxide could undergo fast aggregation when the pH value of the filtrate was about 1.7 and formed the stable floccules. X-ray diffraction analysis shows the dominant peak of the floccules is about 11°, which accords to the peak of graphene oxide. Spectra of X-ray photoelectron spectroscopy confirm the presence in the floccules of an abundance of oxygen functional groups and the purified graphene oxide floccules can be obtained. Atomic force microscopy measurement shows the graphene oxide floccules consists of sheet-like objects, mostly containing only a few layers (about 5 layers). Zeta potential analysis demonstrates the surface charge of the graphene oxide is pH-sensitive and its isoelectric point is ∼1.7. The flocculation mechanism of graphene oxide ascribes to the acid-base interaction with the surface functional groups of the carbon layers.

  11. Amino acid side chain-like surface modification on magnetic nanoparticles for highly efficient separation of mixed proteins.

    PubMed

    Lee, Soo Youn; Ahn, Chi Young; Lee, Jiho; Chang, Jeong Ho

    2012-05-15

    This work reports on the realization of specific functionalized silica-coated magnetic nanoparticles (Si-MNPs) for effective protein separation through surface modification with various amino acid side chain-like functional groups such as thiol (-SH), disulfide (-S-S-), carbon chain (-C(n)), carboxyl (-COOH), amine (-NH(2)), and aldehyde (-CHO). This study also suggests an improved and convenient method for the synthesis of functionalized Si-MNPs by hydrolysis condensation with silan-coupling agents. The protein adsorption effects in a coexistent mixed state are explored using various proteins, which have different isoelectric point (pI) values and molecular weights, in order to elucidate the binding performance of different proteins one solution. The adsorption efficiency of bovine serum albumin (BSA; 66 kDa; pI=4.65) and lysozyme (LYZ; 14.3 kDa; pI=11) is 70-100% with various amino acid side chain-like functional groups. However, the adsorption efficiency of a mixed protein solution of BSA and LYZ was different. Although the relatively bulky BSA molecule displayed 50% and 20% adsorption corresponding to pH 4.65, and pH 11, respectively, the smaller LYZ provided almost 100% adsorption at both pH 4.65 and pH 11. PMID:22483893

  12. The effect of humic acid adsorption on pH-dependent surface charging and aggregation of magnetite nanoparticles

    SciTech Connect

    Illes, E.; Tombacz, E.

    2006-03-01

    The pH-dependent adsorption of humic acid (HA) on magnetite and its effect on the surface charging and the aggregation of oxide particles were investigated. HA was extracted from brown coal. Synthetic magnetite was prepared by alkaline hydrolysis of iron(II) and iron(III) salts. The pH-dependent particle charge and aggregation, and coagulation kinetics at pH around to 4 were measured by laser Doppler electrophoresis and dynamic light scattering. The charge of pure magnetite reverses from positive to negative at pH around 8, which may consider as isoelectric point (IEP). Near this pH, large aggregates form, while stable sols exist further from it. In the presence of increasing HA loading, the IEP shifts to lower pH, then at higher loading, magnetite becomes negatively charged even at low pHs, which indicate the neutralization and gradual recharging positive charges on surface. In acidic region, the trace HA amounts are adsorbed on magnetite surface as oppositely charged patches, systems become highly unstable due to heterocoagulation. Above the adsorption saturation, however, the nanoparticles are stabilized in a way of combined steric and electrostatic effects. The HA coated magnetite particles form stable colloidal dispersion, particle aggregation does not occur in a wide range of pH and salt tolerance is enhanced.

  13. 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. PMID:26036978

  14. Effect of dextran and dextran sulfate on the structural and rheological properties of model acid milk gels.

    PubMed

    Pachekrepapol, U; Horne, D S; Lucey, J A

    2015-05-01

    Various types of polysaccharides are widely used in cultured dairy products. However, the interaction mechanisms, between milk proteins and these polysaccharides, are not entirely clear. To explore the interactions between uncharged and charged polysaccharides and the caseins, we used a model acid-milk-gel system, which allowed acidification to occur separately from gelation. The effect of adding uncharged dextran (DX; molecular weight ~2.0×10(6) Da) and negatively charged dextran sulfate (DS; molecular weight ~1.4×10(6) Da) to model acid milk gels was studied. Two concentrations (0.075 and 0.5%, wt/wt) of DX or DS were added to cold milk (~0°C) that had been acidified to pH values 4.4, 4.6, 4.8, or 4.9. Acidified milks containing DX or DS were then quiescently heated at the rate of 0.5°C/min to 30°C, which induced gelation, and gels were then held at 30°C for 17 h to facilitate gel development. Dynamic small-amplitude-oscillation rheology and large-deformation (shear) tests were performed. Microstructure of gels was examined by fluorescence microscopy. Gels made with a high concentration of DX gelled at a lower temperature, but after 17 h at 30°C, these gels exhibited lower storage moduli and lower yield-stress values. At pH 4.8 or 4.9 (pH values greater than the isoelectric point of caseins), addition of 0.5% DS to acidified milk resulted in lower gelation temperature. At pH 4.4 (pH values less than the isoelectric point of caseins), addition of 0.5% DS to acidified milk resulted in gels with very high stiffness values. Gels made at pH 4.8 or 4.9 with both concentrations of DS had much lower stiffness and yield-stress values than control gels. Microstructural analysis indicated that gels made at pH 4.4 with the addition of 0.5% DX exhibited large protein strands and pores, whereas gels made with 0.075% DX or the control gels had a finer protein matrix. At higher pH values (>4.4), gels made with 0.5% DX had a finer structure. At all pH values, gels made

  15. Improved isoelectric focusing chromatography on strong anion exchange media via a new model that custom designs mobile phases using simple buffers.

    PubMed

    Choy, Derek Y C; Creagh, A Louise; Haynes, Charles

    2014-03-01

    Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge state with pH, is widely used at analytical scales and finding increasing interest in biologics manufacturing due to its exceptional resolving power. Here, a method is described for using simple monoprotic and diprotic buffers to create stable mobile phases for sample loading on a strong anion exchange column and for achieving an elution pH gradient of desired shape covering any pH range from pH 10.0 to 3. The buffers used are selected to satisfy cost constraints, and to permit facile detection of eluted biologics by UV spectroscopy and mass spectrometry. The method exploits a new model described here that combines multiple-chemical and adsorption-equilibria theory to enable in silico tailoring of elution pH profiles using mixtures of these simple buffers. It is shown to provide a versatile platform for optimizing and conducting ICF of protein mixtures on strong anion exchange media. PMID:24166014

  16. 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. PMID:26093664

  17. Effect of single doses of methoxypolyethylene glycol-epoetin beta (CERA, Mircera™) and epoetin delta (Dynepo™) on isoelectric erythropoietin profiles and haematological parameters.

    PubMed

    Dehnes, Yvette; Hemmersbach, Peter

    2011-05-01

    Erythropoietin (EPO) has been misused in sports for many years due to its performance-enhancing effect. In the last decade, detection of abuse has been possible with isoelectric focusing (IEF) based on the different isoform profiles of endogenous and recombinant EPO. The release of new EPOs on the market, such as the recombinant erythropoietin epoetin delta (Dynepo™) and the chemically modified EPO, CERA (Mircera™) potentially represents analytical challenges to the fight against doping. This study set out to investigate the possibility of and the time window for detecting the administration of a single dose of Dynepo™ and CERA. Our results are in agreement with earlier findings that detection of Dynepo™ is best achieved by combining IEF with SDS-PAGE. Haematological parameters were monitored for possible effects due to the long half-life (130 hours) of CERA in blood. Interestingly, although several haematological parameters were significantly changed after the injection of CERA, the endogenous EPO signal was still present in all collected samples. Due to the long half-life and the large size of the CERA molecule (about 60 kDa), it was uncertain whether CERA would be excreted into urine in detectable amounts unless urine collection was preceded by strenuous physical exercise. We find that CERA can be detected in urine without prior exercise in several, but not all, subjects. CERA is nevertheless best detected in serum with regard to both probability and length of detection, in addition to stability in matrix over time. PMID:21387570

  18. A simple purification procedure of D-amino-acid oxidase from Candida guilliermondii H(see symbol)-4.

    PubMed

    Gevorgyan, G K; Davtyan, M A; Hambardzumyan, A A

    2012-01-01

    D-amino-acid oxidase (EC 1.4.3.3) was purified about 1480-fold from the yeast Candida guilliermondii H(see symbol)-4 using chromatofocusing method. The purification procedure gave an enzyme preparation which is greater than 90% homogenous on SDS-polyacrylamide gels with a specific activity of 11.54 U/mg at 30 degrees C with D-proline as substrate with the yield of total activity 9.3%. The molecular weights of subunit and native enzyme were determined to be 38.4 and 78.6 kDa by SDS-polyacrylamide gel electrophoresis and gel-filtration, respectively, suggesting that the native enzyme exists as a homodimer. A single molecular form with an isoelectric point of 6.85 was detected in analytical isoelectrofocusing. The optimum pH and temperature were 8.0 and 33 degrees C. An enzyme shows stability in the pH range from 7.4 to 9.0 and at the temperature no higher than 38 degrees C. Activation energy for D-amino-acid oxidase reaction was calculated to be 60 kJ/mol at 30 degrees C. The strict D-isomer specificity of the enzyme is confirmed, since no reaction could be detected with L-amino acids, and a large number of D-amino acids could be substrates for this enzyme. K(m) and V(max) values were determined for D-proline and D-alanine, which, among 22 tested, were the best substrates of the enzyme. D-amino-acid oxidase from the yeast C. guilliermondii is a flavoprotein oxidase in which the prosthetic group is tightly, but not covalently, bound FAD. The enzyme is completely inhibited by sodium benzoate, SH-oxidizing agents, but not by sodium azide, toluene or chloroform. PMID:23156699

  19. A proteomics platform combining depletion, multi-lectin affinity chromatography (M-LAC), and isoelectric focusing to study the breast cancer proteome.

    PubMed

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M; Wang, Hong; Hancock, William S

    2011-06-15

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  20. 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. PMID:26826592

  1. A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC) and Isoelectric Focusing to Study the Breast Cancer Proteome

    PubMed Central

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J.; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M.; Wang, Hon; Hancock, William S.

    2011-01-01

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as simultaneously detecting glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation and LC-MS analysis has been applied to discover breast cancer associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  2. High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

    PubMed

    Kjellin, K G; Hallander, L B

    1982-01-01

    A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum

  3. Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas.

    PubMed

    Osatomi, K; Masuda, Y; Hara, K; Ishihara, T

    2001-04-01

    Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60 degrees C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD. PMID:11290457

  4. A farnesoic acid O-methyltransferase (FAMeT) from Exopalaemon carinicauda is responsive to Vibrio anguillarum and WSSV challenge.

    PubMed

    Duan, Yafei; Liu, Ping; Li, Jitao; Wang, Yun; Li, Jian; Chen, Ping

    2014-05-01

    Methyl farnesoate (MF), an analogue of the insect juvenile hormone III, is believed to play important roles in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) is the key enzyme in the juvenile hormone biosynthetic pathway, involved in the conversion of farnesoic acid (FA) to MF in the final step of MF synthesis. In this study, a FAMeT cDNA (named EcFAMeT) was cloned from the hemocytes of ridgetail white prawn Exopalaemon carinicauda by rapid amplification of cDNA ends (RACE) methods. The full-length cDNA of EcFAMeT was 1,620 bp, including contains a 5'-untranslated region (UTR) of 75 bp, 3'-UTR of 714 bp with a poly (A) tail, an open reading frame (ORF) of 831 bp, encoding a 276-amino-acid polypeptide with the predicted molecular weight of 31.57 kDa and estimated isoelectric point of 4.67. BLAST analysis revealed that amino acids of EcFAMeT shared high identity (75-90 %) with that of other crustaceans. Two conserved signatures domains of Methyltransf-FA superfamily were also identified in EcFAMeT. Real time quantitative RT-PCR analysis indicated that EcFAMeT could be detected in all the tested tissues and strongly expressed in hepatopancreas and ovary of E. carinicauda. After Vibrio anguillarum and WSSV challenge, EcFAMeT transcripts both in hemocytes and hepatopancreas increased significantly in the first 3 h, respectively. The results indicated that EcFAMeT might be associated with the immune defenses to V. anguillarum and WSSV in E. carinicauda. PMID:24136172

  5. 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

  6. 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. PMID:25437446

  7. Near point-of-care administration by the attending physician of the rapid influenza antigen detection immunochromatography test and the fully automated respiratory virus nucleic acid test: contribution to patient management.

    PubMed

    Boku, Soushin; Naito, Toshio; Murai, Kenji; Tanei, Mika; Inui, Akihiro; Nisimura, Hidekazu; Isonuma, Hiroshi; Takahashi, Hiroshi; Kikuchi, Ken

    2013-08-01

    Rapid influenza antigen detection tests (RIADTs) using immunochromatography are the most readily available tools for the diagnosis and management of influenza. This study was designed to assess whether near point-of-care administration by primary care physicians of the RIADT and a fully automated respiratory virus nucleic acid test (Verigene Respiratory Virus Plus®; RV+) would contribute to improved patient management. When viral culture and RT-PCR/bi-directional sequencing were used as the gold standard, sensitivities and specificities for RIADT and RV+ were 58.3% and 90.9%, and 97.2% and 100%, respectively. Within 12 hours from onset of fever, sensitivities were 44.4% and 94.4%, respectively, for RIADT and RV+. In clinical situations where a higher-sensitivity test is needed, such as during pre-admission evaluations, for testing of hospital employees during the prodromal phase of infection, during the therapeutic decision-making process, and during outbreaks, we suggest that patients testing negative by the RIADT can be reassessed with the RV+ test to achieve maximal diagnostic accuracy. PMID:23743175

  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. Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: purification, properties, and amino acid sequences.

    PubMed

    Haldar, U C; Saha, S K; Beavis, R C; Sinha, N K

    1996-02-01

    Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is at pH 4.55 for LA-1 and at pH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 A. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0 x 10(9) M-1 sec-1 for LA-1 and 0.8 x 10(9) M-1 sec-1 for LA-2 and that of K2HPO4 quenching is 1.6 x 10(11) M-1 sec-1 for LA-1 and 1.2 x 10(11) M-1 sec-1 for LA-2. Analysis of the circular dichroic spectra yields 40% alpha-helix and 60% beta-turn for La-1 and 45% alpha-helix and 55% beta-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzyme-inhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors. PMID:8924202

  10. Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I.

    PubMed

    Yan, Qi; Liu, Qin; Li, Meng-Meng; Li, Fang-Hui; Zhu, Bin; Wang, Jun-Zhong; Lu, Yin-Ping; Liu, Jia; Wu, Jun; Zheng, Xin; Lu, Meng-Ji; Wang, Bao-Ju; Yang, Dong-Liang

    2016-06-01

    Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection. PMID:27376800

  11. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids.

    PubMed

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S V Santhana; Buettner, Garry R; Robertson, Larry W; Luthe, Gregor

    2016-02-01

    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 ((1)H 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

  12. 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

    Go, Yun Young; 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; Balasuriya, Udeni B R

    2016-06-01

    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

  13. [Gastric Acid].

    PubMed

    Ruíz Chávez, R

    1996-01-01

    Gastric acid, a product of parietal cells secretion, full fills multiple biological roles which are absolutely necessary to keep corporal homeostasis. The production of the acid depends upon an effector cellular process represented in the first step by histamine, acetilcholine and gastrin, first messengers of the process. These interact with specific receptors than in sequence activate second messengers -cAMP and the calcium-calmodulin system- which afterwards activate a kinase. An specific protein is then phosphorilated by this enzyme, being the crucial factor that starts the production of acid. Finally, a proton bomb, extrudes the acid towards the gastric lumen. The secretion process mentioned above, is progressive lyactivated in three steps, two of which are stimulators -cephalic and gastric phases- and the other one inhibitor or intestinal phase. These stages are started by mental and neurological phenomena -thought, sight, smell or memory-; by food, drugs or other ingested substances; and by products of digestion. Changes in regulation of acid secretion, in the structure of gastro-duodenal mucosal barrier by a wide spectrum of factors and agents including food, drugs and H. pylori, are the basis of acid-peptic disease, entity in which gastric acid plays a fundamental role. From the therapeutic point of view, so at the theoretical as at the practical levels, t is possible to interfere with the secretion of acid by neutralization of some of the steps of the effector cellular process. An adequate knowledge of the basics related to gastric acid, allows to create strategies for the clinical handling of associated pathology, specifically in relation to peptic acid disease in all of the known clinical forms. PMID:12165790

  14. Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

    PubMed Central

    Eccleston, VS; Ohlrogge, JB

    1998-01-01

    Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids. PMID:9548986

  15. Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

    PubMed Central

    Green, L. S.; Faergestad, E. M.; Poole, A.; Chandler, P. M.

    1997-01-01

    Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression. PMID:12223700

  16. Translation initiation factor 5A in Picrorhiza is up-regulated during leaf senescence and in response to abscisic acid.

    PubMed

    Parkash, Jai; Vaidya, Tanmay; Kirti, Shruti; Dutt, Som

    2014-05-25

    Translation initiation, the first step of protein synthesis process is the principal regulatory step controlling translation and involves a pool of translation initiation factors. In plants, from recent studies it is becoming evident that these translation initiation factors impact various aspects of plant growth and development in addition to their role in protein synthesis. Eukaryotic translation initiation factor eIF5A is one such factor which functions in start site selection for the eIF2-GTP-tRNAi ternary complex within the ribosomal-bound preinitiation complex and also stabilizes the binding of GDP to eIF2. In the present study we have cloned and analysed a gene (eIF5a) encoding eIF5A from Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) a medicinal plant of the western Himalayan region. The full length eIF5a cDNA consisted of 838 bp with an open reading frame of 480 bp, 88 bp 5' untranslated region and 270 bp 3' untranslated region. The deduced eIF5A protein contained 159 amino acids with a molecular weight of 17.359 kDa and an isoelectric point of 5.59. Secondary structure analysis revealed eIF5A having 24.53% α-helices, 8.81% β-turns, 23.27% extended strands and 43.40% random coils. pk-eIF5a transcript was found to be expressing during the active growth phase as well as during leaf senescence stage, however, highest expression was observed during leaf senescence stage. Further, its expression was up-regulated in response to exogenous application of abscisic acid. Both high intensity as well as low intensity light decreased the expression of pk-eIF5a. The findings suggest eIF5a to be an important candidate to develop genetic engineering based strategies for delaying leaf senescence. PMID:24656625

  17. Floating Point Control Library

    2007-08-02

    Floating Point Control is a Library that allows for the manipulation of floating point unit exception masking funtions control exceptions in both the Streaming "Single Instruction, Multiple Data" Extension 2 (SSE2) unit and the floating point unit simultaneously. FPC also provides macros to set floating point rounding and precision control.

  18. Reversible Bacterial Adhesion on Mixed Poly(dimethylaminoethyl methacrylate)/Poly(acrylamidophenyl boronic acid) Brush Surfaces.

    PubMed

    Xiong, Xinhong; Wu, Zhaoqiang; Yu, Qian; Xue, Lulu; Du, Jun; Chen, Hong

    2015-11-10

    A simple and versatile method for the preparation of surfaces to control bacterial adhesion is described. Substrates were first treated with two catechol-based polymerization initiators, one for thermal initiation and one for visible-light photoinitiation. Graft polymerization in sequence of dimethylaminoethyl methacrylate (DMAEMA) and 3-acrylamidebenzene boronic acid (BA) from the surface-bound initiators to form mixed polymer brushes on the substrate was then carried out. The PDMAEMA grafts were thermally initiated and the PBA grafts were visible-light-photoinitiated. Gold, poly(vinyl chloride) (PVC), and poly(dimethylsiloxane) (PDMS) were used as model substrates. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), and ellipsometry analysis confirmed the successful grafting of PDMAEMA/PBA mixed brushes. We demonstrated that the resulting surfaces showed charge-reversal properties in response to change of pH. The transition in surface charge at a specific pH allowed the surface to be reversibly switched from bacteria-adhesive to bacteria-resistant. At pH 4.5, below the isoelectric points (IEP, pH 5.3) of the mixed brushes, the surfaces are positively charged and the negatively charged Gram-positive S. aureus adheres at high density (2.6 × 10(6) cells/cm(2)) due to attractive electrostatic interactions. Subsequently, upon increasing the pH to 9.0 to give negatively charged polymer brush surface, ∼90% of the adherent bacteria are released from the surface, presumably due to repulsive electrostatic interactions. This approach provides a simple method for the preparation of surfaces on which bacterial adhesion can be controlled and is applicable to a wide variety of substrates. PMID:26509287

  19. Myofascial trigger point pain.

    PubMed

    Jaeger, Bernadette

    2013-01-01

    Myofascial trigger point pain is an extremely prevalent cause of persistent pain disorders in all parts of the body, not just the head, neck, and face. Features include deep aching pain in any structure, referred from focally tender points in taut bands of skeletal muscle (the trigger points). Diagnosis depends on accurate palpation with 2-4 kg/cm2 of pressure for 10 to 20 seconds over the suspected trigger point to allow the referred pain pattern to develop. In the head and neck region, cervical muscle trigger points (key trigger points) often incite and perpetuate trigger points (satellite trigger points) and referred pain from masticatory muscles. Management requires identification and control of as many perpetuating factors as possible (posture, body mechanics, psychological stress or depression, poor sleep or nutrition). Trigger point therapies such as spray and stretch or trigger point injections are best used as adjunctive therapy. PMID:24864393

  20. Synthesis of a novel zwitterionic biodegradable poly (α,β-L-aspartic acid) derivative with some L-histidine side-residues and its resistance to non-specific protein adsorption.

    PubMed

    Wang, Xiaojuan; Wu, Guolin; Lu, Caicai; Wang, Yinong; Fan, Yunge; Gao, Hui; Ma, Jianbiao

    2011-08-01

    A novel zwitterionic polypeptide derivative, denoted as His-PAsp/PAsp, was successfully synthesized by amidation of Poly (α,β-L-aspartic acid) with L-histidine methyl ester. Turbidity, zeta potential and ¹H NMR measurements were used to study the aggregation behaviors of His-PAsp/PAsp under different pH values. The modified polypeptide derivative composed of electro-negatively carboxylic and electro-positively imidazole residues randomly so as to bear opposite charges at pH values above or below the isoelectric point. When the zwitterionic polypeptide was coated on silicon wafer as a model substrate material, the absorption resistance of fibrinogen, a blood protein resulting in the blood coagulation cascade, on the coated surface was measured. It was found that the adsorption amount of fibrinogen on the polypeptide-coated surface depended on the dose of the polypeptide on silicon wafer. Obvious resistance of the fibrinogen adsorption on the polypeptide-coated surface was observed. Since its good biodegradability and superior anti-protein-fouling property, this pH-responsive zwitterionic polypeptide is a promising candidate for surface modification in many biomedical applications, including medical implants, drug delivery carriers, and biosensors. PMID:21536417

  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. 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)

  3. Point by Point: Adding up Motivation

    ERIC Educational Resources Information Center

    Marchionda, Denise

    2010-01-01

    Students often view their course grades as a mysterious equation of teacher-given grades, teacher-given grace, and some other ethereal components based on luck. However, giving students the power to earn points based on numerous daily/weekly assignments and attendance makes the grading process objective and personal, freeing the instructor to…

  4. Splitting the Cartesian point

    SciTech Connect

    Blodwell, J.F.

    1987-10-01

    It is argued that the point structure of space and time must be constructed from the primitive extensional character of space and time. A procedure for doing this is laid down and applied to one-dimensional and two-dimensional systems of abstract extensions. Topological and metrical properties of the constructed point systems, which differ nontrivially from the usual R and R/sup 2/ models, are examined. Briefly, constructed points are associated with directions and the Cartesian point is split. In one-dimension each point splits into a point pair compatible with the linear ordering. An application to one-dimensional particle motion is given, with the result that natural topological assumptions force the number of left point, right point transitions to remain locally finite in a continuous motion. In general, Cartesian points are seen to correspond to certain filters on a suitable Boolean algebra. Constructed points correspond to ultrafilters. Thus, point construction gives a natural refinement of the Cartesian systems.

  5. Fatty acid binding protein 10 in the orange-spotted grouper (Epinephelus coioides): characterization and regulation under pH and temperature stress.

    PubMed

    Qi, Zeng-hua; Liu, Yu-feng; Wang, Wei-Na; Xin, Yu; Xie, Fu-xing; Wang, An-Li

    2012-04-01

    We have isolated and characterized a fatty acid binding protein from the liver of the orange-spotted grouper (Epinephelus coioides). Amino acid sequence similarity of the Ec-FABP (E. coioides-FABP) was highest to FABP10s isolated from the livers of catfish, chicken, salamander and iguana. The open-reading frame of the Ec-FABP codes for a protein of 14.0 kDa with a calculated isoelectric point of 8.5. We first expressed a FABP10 protein from orange-spotted grouper (E. coioides) in Pichia pastoris, and then characterized the antioxidative potential of our recombinant Ec-FABP by DCF fluorescence assay. RT-PCR assays showed that endogenous Ec-FABP mRNA is most strongly expressed in liver with the most abundance and intestine. Change in the groupers' blood cells respiratory burst activity was examined during and after exposure to the pH and temperature stress using flow cytometry. Further analysis of Ec-FABP gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-FABP transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its mRNA expression level peaked differed under these stresses. Western blot analyses confirmed that the Ec-FABP protein was strongly expressed in the liver after exposure to the pH and temperature stress. These results suggest that Ec-FABP expression is stimulated by pH and temperature stress and that it may play important roles in general adaptive and antioxidant responses. PMID:22182678

  6. Reduced immunogenicity of beta-lactoglobulin by conjugation with acidic oligosaccharides.

    PubMed

    Hattori, Makoto; Miyakawa, Shunpei; Ohama, Yukie; Kawamura, Hiroyuki; Yoshida, Tadashi; To-o, Kenji; Kuriki, Takashi; Takahashi, Koji

    2004-07-14

    Bovine beta-lactoglobulin (beta-LG) was conjugated with the acidic oligosaccharides, alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides (POs) by the Maillard reaction to reduce the immunogenicity of beta-LG. The molar ratios of beta-LG to ALGO and POs in the conjugates were 1:6 and 1:8. The carbohydrate-binding sites in the beta-LG-ALGO conjugate were partially identified to be (60)Lys, (77)Lys, (100)Lys, (138)Lys, and (141)Lys. The isoelectric point of each conjugate was lower than that of beta-LG. CD spectra indicated that the secondary structure of beta-LG was almost maintained after conjugation. The results of fluorescence studies indicated that the conformation around Trp had not changed in each conjugate and that the surface of each conjugate was covered with a saccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around (8)Lys-(19)Trp (beta-sheet, random coil, short helix) in the conjugates had changed, whereas the native structure was maintained around (15)Val-(29)Ile (beta-sheet) and (125)Thr-(135)Lys (alpha-helix). The beta-LG-ALGO and beta-LG-POs conjugates maintained 77 and 70% of the retinol binding activity of beta-LG. Conjugation with ALGO and POs substantially enhanced the thermal stability of beta-LG. The anti-beta-LG antibody response was markedly reduced after immunization with both conjugates in BALB/c, C57BL/6, and C3H/He mice. B cell epitopes of beta-LG and the conjugate recognized in these mice were determined with 15-mer multipin peptides, and the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, whereas the antibody response to each epitope was dramatically reduced. In particular, effective reduction of the antibody response was observed in the vicinity of the carbohydrate-binding sites. Conjugation of beta-LG with these acidic oligosaccharides was effective in reducing the immunogenicity of beta-LG. The conjugates obtained in this study are

  7. Purification and properties of two binding proteins for branched-chain amino acids in Salmonella typhimurium.

    PubMed

    Ohnishi, K; Kiritani, K

    1983-08-01

    Two leucine-binding proteins isolated from osmotic shock fluid of Salmonella typhimurium LT2 were purified by DEAE-cellulose and DEAE-Sephadex A-50 chromatography, and subsequent isoelectric focusing. These purified binding proteins could be crystallized by adding 2-methyl-2,4-pentanediol. One of the binding proteins, designated as LIVT-binding protein, binds L-leucine, L-isoleucine, L-valine, and L-threonine, while the other, L-binding protein, binds only L-leucine. The level of LIVT-binding protein in the shock fluid was about three-fold higher than that of L-binding protein. The molecular weight of the LIVT-binding protein was estimated to be 35,000 by gel filtration, and 39,000 by gel electrophoresis. The isoelectric point was pH 4.94. The dissociation constants of this protein for leucine, isoleucine, and valine were 0.43, 0.15, and 0.89 microM, respectively. For the L-binding protein, molecular weights of 34,000 (gel filtration), and 38,000 (gel electrophoresis) were obtained. The isoelectric point was pH 4.74. The dissociation constant of this protein for leucine was 0.54 microM. The LIVT-binding protein was more heat-stable than the L-binding protein. These two binding proteins showed an antigenic similarity, they could cross-react with each other's antiserum. This similarity was also found between the binding proteins of Salmonella typhimurium and Escherichia coli K-12. Both LIVT- and L-binding proteins in a regulatory mutant, KA2313, were found to be about three-fold the levels in the wild-type strain. PMID:6355077

  8. 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

  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. 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…

  11. Torsade de pointes.

    PubMed

    Munro, P T; Graham, C A

    2002-09-01

    A case is described of torsade de pointes in a 41 year old woman with pre-existing QTc prolongation, potentially exacerbated by treatment with sotalol. Previous cardiac investigations had been normal and after a second episode of ventricular fibrillation the patient was referred for electrophysiological studies. The authors review the physiology, causes, and treatment of QTc prolongation and torsade de pointes. PMID:12205024

  12. 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,…

  13. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

    PubMed Central

    Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

    2010-01-01

    The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct

  14. Multispectral Image Feature Points

    PubMed Central

    Aguilera, Cristhian; Barrera, Fernando; Lumbreras, Felipe; Sappa, Angel D.; Toledo, Ricardo

    2012-01-01

    This paper presents a novel feature point descriptor for the multispectral image case Far-Infrared and Visible Spectrum images. It allows matching interest points on images of the same scene but acquired in different spectral bands. Initially, points of interest are detected on both images through a SIFT-like based scale space representation. Then, these points are characterized using an Edge Oriented Histogram (EOH) descriptor. Finally, points of interest from multispectral images are matched by finding nearest couples using the information from the descriptor. The provided experimental results and comparisons with similar methods show both the validity of the proposed approach as well as the improvements it offers with respect to the current state-of-the-art.

  15. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    PubMed Central

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  16. Proteomic profiling combining solution-phase isoelectric fractionation with two-dimensional gel electrophoresis using narrow-pH-range immobilized pH gradient gels with slightly overlapping pH ranges.

    PubMed

    Lee, KiBeom; Pi, KyungBae

    2010-01-01

    This paper describes a simple new approach toward improving resolution of two-dimensional (2-D) protein gels used to explore the mammalian proteome. The method employs sample prefractionation using solution-phase isoelectric focusing (IEF) to split the mammalian proteome into well-resolved pools. As crude samples are thus prefractionated by pI range, very-narrow-pH-range 2-D gels can be subsequently employed for protein separation. Using custom pH partition membranes and commercially available immobilized pH gradient (IPG) strips, we maximized the total separation distance and throughput of seven samples obtained by prefractionation. Both protein loading capacity and separation quality were higher than the values obtained by separation of fractionated samples on narrow-pH-range 2-D gels; the total effective IEF separation distance was ~82 cm over the pH range pH 3-10. This improved method for analyzing prefractionated samples on narrow-pH-range 2-D gels allows high protein resolution without the use of large gels, resulting in decreased costs and run times. PMID:19813004

  17. Amino acids

    MedlinePlus

    Amino acids are organic compounds that combine to form proteins . Amino acids and proteins are the building blocks of life. When proteins are digested or broken down, amino acids are left. The human body uses amino acids ...

  18. 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. PMID:22270703

  19. Point tenderness - abdomen

    MedlinePlus

    ... medlineplus.gov/ency/article/003273.htm Point tenderness - abdomen To use the sharing features on this page, ... over a certain part of the belly area (abdomen). Considerations The abdomen is an area of the ...

  20. Triple Point Topological Metals

    NASA Astrophysics Data System (ADS)

    Zhu, Ziming; Winkler, Georg W.; Wu, QuanSheng; Li, Ju; Soluyanov, Alexey A.

    2016-07-01

    Topologically protected fermionic quasiparticles appear in metals, where band degeneracies occur at the Fermi level, dictated by the band structure topology. While in some metals these quasiparticles are direct analogues of elementary fermionic particles of the relativistic quantum field theory, other metals can have symmetries that give rise to quasiparticles, fundamentally different from those known in high-energy physics. Here, we report on a new type of topological quasiparticles—triple point fermions—realized in metals with symmorphic crystal structure, which host crossings of three bands in the vicinity of the Fermi level protected by point group symmetries. We find two topologically different types of triple point fermions, both distinct from any other topological quasiparticles reported to date. We provide examples of existing materials that host triple point fermions of both types and discuss a variety of physical phenomena associated with these quasiparticles, such as the occurrence of topological surface Fermi arcs, transport anomalies, and topological Lifshitz transitions.

  1. 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.

  2. 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.

  3. ATLAS solar pointing operations

    NASA Technical Reports Server (NTRS)

    Tyler, C. A.; Zimmerman, C. J.

    1994-01-01

    The ATLAS-series of Spacelab missions are comprised of a diverse group of scientific instruments including instruments for studying the sun and how the sun's energy changes across an eleven-year solar cycle. The ATLAS solar instruments are located on one or more pallets in the Orbiter payload bay and use the Orbiter as a pointing platform for their examinations of the sun. One of the ATLAS instruments contained a sun sensor which allowed scientists and engineers on the ground to see the pointing error of the sun with respect to the instrument and correct for the error based upon the information coming from the ATLAS 1 and ATLAS 2 missions with particular attention given to identifying the sources of pointing discrepancies of the solar instruments and to describe the crew and ground controller procedures that were developed to correct for these discrepancies. The Orbiter pointing behavior from the ATLAS 1 and ATLAS 2 flights presented in this paper can be applied to future flights which use the Orbiter as a pointing platform.

  4. SOFIA pointing history

    NASA Astrophysics Data System (ADS)

    Kärcher, Hans J.; Kunz, Nans; Temi, Pasquale; Krabbe, Alfred; Wagner, Jörg; Süß, Martin

    2014-07-01

    The original pointing accuracy requirement of the Stratospheric Observatory for Infrared Astronomy SOFIA was defined at the beginning of the program in the late 1980s as very challenging 0.2 arcsec rms. The early science flights of the observatory started in December 2010 and the observatory has reached in the mean time nearly 0.7 arcsec rms, which is sufficient for most of the SOFIA science instruments. NASA and DLR, the owners of SOFIA, are planning now a future 4 year program to bring the pointing down to the ultimate 0.2 arcsec rms. This may be the right time to recall the history of the pointing requirement and its verification and the possibility of its achievement via early computer models and wind tunnel tests, later computer aided end-to-end simulations up to the first commissioning flights some years ago. The paper recollects the tools used in the different project phases for the verification of the pointing performance, explains the achievements and may give hints for the planning of the upcoming final pointing improvement phase.

  5. 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.

  6. Precision Pointing System Development

    SciTech Connect

    BUGOS, ROBERT M.

    2003-03-01

    The development of precision pointing systems has been underway in Sandia's Electronic Systems Center for over thirty years. Important areas of emphasis are synthetic aperture radars and optical reconnaissance systems. Most applications are in the aerospace arena, with host vehicles including rockets, satellites, and manned and unmanned aircraft. Systems have been used on defense-related missions throughout the world. Presently in development are pointing systems with accuracy goals in the nanoradian regime. Future activity will include efforts to dramatically reduce system size and weight through measures such as the incorporation of advanced materials and MEMS inertial sensors.

  7. 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 Central

    Elschenbroich, Sarah; Ignatchenko, Vladimir; Sharma, Parveen; Schmitt-Ulms, Gerold; Gramolini, Anthony O.; Kislinger, Thomas

    2013-01-01

    High resolution peptide separation is pivotal for successful shot-gun 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. PMID:19670906

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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

    2015-01-01

    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

  14. 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.

  15. 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

  16. MountPointAttributes

    2001-06-16

    MountPointAttributes is a software component that provides client code with a technique to raise the local namespace of a file to a global namespace. Its abstractions and mechanisms allow the client code to gather global properties of a file and to use them in devising an effective storage access strategy on this file.

  17. GLAS Spacecraft Pointing Study

    NASA Technical Reports Server (NTRS)

    Born, George H.; Gold, Kenn; Ondrey, Michael; Kubitschek, Dan; Axelrad, Penina; Komjathy, Attila

    1998-01-01

    Science requirements for the GLAS mission demand that the laser altimeter be pointed to within 50 m of the location of the previous repeat ground track. The satellite will be flown in a repeat orbit of 182 days. Operationally, the required pointing information will be determined on the ground using the nominal ground track, to which pointing is desired, and the current propagated orbit of the satellite as inputs to the roll computation algorithm developed by CCAR. The roll profile will be used to generate a set of fit coefficients which can be uploaded on a daily basis and used by the on-board attitude control system. In addition, an algorithm has been developed for computation of the associated command quaternions which will be necessary when pointing at targets of opportunity. It may be desirable in the future to perform the roll calculation in an autonomous real-time mode on-board the spacecraft. GPS can provide near real-time tracking of the satellite, and the nominal ground track can be stored in the on-board computer. It will be necessary to choose the spacing of this nominal ground track to meet storage requirements in the on-board environment. Several methods for generating the roll profile from a sparse reference ground track are presented.

  18. 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.

  19. EndPoints 2000

    2009-08-13

    The application leads the user through a logical framework to determine the minimum effort and cost necessary to reach the desired end state for each space, system, and facility. Endpoints are used to plan the project work, track and manage the determination, management, verification, and closure of D&D endpoints, consistent with DOE End Point guidance documents.

  20. 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…

  1. 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.

  2. 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…

  3. Noncommutative Point Sources

    SciTech Connect

    Stern, A.

    2008-02-15

    We construct a perturbative solution to classical noncommutative gauge theory on R{sup 3} minus the origin using the Groenewald-Moyal star product. The result describes a noncommutative point charge. Applying it to the quantum mechanics of the noncommutative hydrogen atom gives shifts in the 1S hyperfine splitting which are first order in the noncommutativity parameter.

  4. 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 Pakistan,'" by Syed Jamil Ahmed,…

  5. 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.

  6. 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.

  7. 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 ...

  8. 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 ...

  9. 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 ...

  10. 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.

  11. Proximal Point Methods Revisited

    NASA Astrophysics Data System (ADS)

    Boikanyo, Oganeditse A.; Moroşanu, Gheorghe

    2011-09-01

    The proximal point methods have been widely used in the last decades to approximate the solutions of nonlinear equations associated with monotone operators. Inspired by the iterative procedure defined by B. Martinet (1970), R.T. Rockafellar introduced in 1976 the so-called proximal point algorithm (PPA) for a general maximal monotone operator. The sequence generated by this iterative method is weakly convergent under appropriate conditions, but not necessarily strongly convergent, as proved by O. Güler (1991). This fact explains the introduction of different modified versions of the PPA which generate strongly convergent sequences under appropriate conditions, including the contraction-PPA defined by H.K. Xu in 2002. Here we discuss Xu's modified PPA as well as some of its generalizations. Special attention is paid to the computational errors, in particular the original Rockafellar summability assumption is replaced by the condition that the error sequence converges to zero strongly.

  12. Purification and amino acid sequence of aminopeptidase P from pig kidney.

    PubMed

    Vergas Romero, C; Neudorfer, I; Mann, K; Schäfer, W

    1995-04-01

    Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. PMID:7744038

  13. At the Tipping Point

    SciTech Connect

    Wiley, H. S.

    2011-02-28

    There comes a time in every field of science when things suddenly change. While it might not be immediately apparent that things are different, a tipping point has occurred. Biology is now at such a point. The reason is the introduction of high-throughput genomics-based technologies. I am not talking about the consequences of the sequencing of the human genome (and every other genome within reach). The change is due to new technologies that generate an enormous amount of data about the molecular composition of cells. These include proteomics, transcriptional profiling by sequencing, and the ability to globally measure microRNAs and post-translational modifications of proteins. These mountains of digital data can be mapped to a common frame of reference: the organism’s genome. With the new high-throughput technologies, we can generate tens of thousands of data points from each sample. Data are now measured in terabytes and the time necessary to analyze data can now require years. Obviously, we can’t wait to interpret the data fully before the next experiment. In fact, we might never be able to even look at all of it, much less understand it. This volume of data requires sophisticated computational and statistical methods for its analysis and is forcing biologists to approach data interpretation as a collaborative venture.

  14. A point matching algorithm based on reference point pair

    NASA Astrophysics Data System (ADS)

    Zou, Huanxin; Zhu, Youqing; Zhou, Shilin; Lei, Lin

    2016-03-01

    Outliers and occlusions are important degradation in the real application of point matching. In this paper, a novel point matching algorithm based on the reference point pairs is proposed. In each iteration, it firstly eliminates the dubious matches to obtain the relatively accurate matching points (reference point pairs), and then calculates the shape contexts of the removed points with reference to them. After re-matching the removed points, the reference point pairs are combined to achieve better correspondences. Experiments on synthetic data validate the advantages of our method in comparison with some classical methods.

  15. 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)

  16. 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…

  17. 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

  18. Tipping point leadership.

    PubMed

    Kim, W Chan; Mauborgne, Renée

    2003-04-01

    When William Bratton was appointed police commissioner of New York City in 1994, turf wars over jurisdiction and funding were rife and crime was out of control. Yet in less than two years, and without an increase in his budget, Bratton turned New York into the safest large city in the nation. And the NYPD was only the latest of five law-enforcement agencies Bratton had turned around. In each case, he succeeded in record time despite limited resources, a demotivated staff, opposition from powerful vested interests, and an organization wedded to the status quo. Bratton's turnarounds demonstrate what the authors call tipping point leadership. The theory of tipping points hinges on the insight that in any organization, fundamental changes can occur quickly when the beliefs and energies of a critical mass of people create an epidemic movement toward an idea. Bratton begins by overcoming the cognitive hurdles that block organizations from recognizing the need for change. He does this by putting managers face-to-face with operational problems. Next, he manages around limitations on funds, staff, or equipment by concentrating resources on the areas that are most in need of change and that have the biggest payoffs. He meanwhile solves the motivation problem by singling out key influencers--people with disproportionate power due to their connections or persuasive abilities. Finally, he closes off resistance from powerful opponents. Not every CEO has the personality to be a Bill Bratton, but his successes are due to much more than his personality. He relies on a remarkably consistent method that any manager looking to turn around an organization can use to overcome the forces of inertia and reach the tipping point. PMID:12687920

  19. 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.

  20. 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

    Rafalko, Agnes; Dai, Shujia; Hancock, William S; Karger, Barry L; Hincapie, Marina

    2012-02-01

    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 (r(2) = 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Measurement of Carbon Dioxide Compensation Points of Freshwater Algae 1

    PubMed Central

    Birmingham, Brendan C.; Colman, Brian

    1979-01-01

    A technique is described for the measurement of total dissolved inorganic carbon by acid release as CO2 followed by its conversion to methane and detection by flame ionization in a modified gas chromatograph. This method was used to determine the dissolved inorganic carbon concentration reached at compensation point when algae were allowed to photosynthesize in a closed system in a buffer at known pH, and the CO2 compensation point was calculated from this concentration. The CO2 compensation points of 16 freshwater algae were measured at acid and alkaline pH in air-saturated medium: at acid pH the CO2 compensation points ranged from 4.8 to 41.5 microliters per liter while at alkaline pH they ranged from 0.2 to 7.2 microliters per liter. Removal of O2 from the medium caused a slight lowering of compensation point at acid pH but had little effect at alkaline pH. These low, O2-insensitive compensation points are characteristic of C4 plants. It is suggested that these low CO2 compensation points are maintained by an active bicarbonate uptake by algae especially at alkaline pH. PMID:16661077

  7. Point Source All Sky

    NASA Technical Reports Server (NTRS)

    2003-01-01

    This panoramic view encompasses the entire sky as seen by Two Micron All-Sky Survey. The measured brightnesses of half a billion stars (points) have been combined into colors representing three distinct wavelengths of infrared light: blue at 1.2 microns, green at 1.6 microns, and red at 2.2 microns. This image is centered on the core of our own Milky Way galaxy, toward the constellation of Sagittarius. The reddish stars seemingly hovering in the middle of the Milky Way's disc -- many of them never observed before -- trace the densest dust clouds in our galaxy. The two faint smudges seen in the lower right quadrant are our neighboring galaxies, the Small and Large Magellanic Clouds.

  8. ViewPoint

    PubMed Central

    Hallam, Jeffrey S.; Nagy, M. Christine

    2014-01-01

    ViewPoint features candid conversations with prominent health behavior scholars. The column focuses on the human or interpersonal side of research and addresses such topics as how renowned researchers were initiated into the world of research, why they continue to do it, personal challenges encountered over the years, ingredients necessary to be a success as an investigator, key characteristics and qualities of inspirational mentors, sources of consternation, challenges that lie ahead, hot topics worthy of pursuit, and books and materials that are must reading. The intention is to share the viewpoints and thinking processes of our leading health behavior scholars. We hope that the column will be an inspiration to young and seasoned scholars alike. PMID:19063654

  9. 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

    2015-09-01

    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

  10. pH-Triggered reversible morphological inversion of orthogonally-addressable poly(3-acrylamidophenylboronic acid)-block-poly(acrylamidoethylamine) micelles and their shell crosslinked nanoparticles

    PubMed Central

    Zou, Jiong; Zhang, Shiyi; Shrestha, Ritu; Seetho, Kellie; Donley, Carrie L.

    2012-01-01

    Functionally-responsive amphiphilic core-shell nanoscopic objects, capable of either complete or partial inversion processes, were produced by the supramolecular assembly of pH-responsive block copolymers, without or with covalent crosslinking of the shell layer, respectively. A new type of well-defined, dual-functionalized boronic acid- and amino-based diblock copolymer poly(3-acrylamidophenylboronic acid)30-block-poly(acrylamidoethylamine)25 (PAPBA30-b-PAEA25) was synthesized by sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and then assembled into cationic micelles in aqueous solution at pH 5.5. The micelles were further cross-linked throughout the shell domain comprised of poly(acrylamidoethylamine) by reaction with a bis-activated ester of 4,15-dioxo-8,11-dioxa-5,14-diazaoctadecane-1,18-dioic acid, upon increase of the pH to 7, to different cross-linking densities (2%, 5% and 10%), forming well-defined shell cross-linked nanoparticles (SCKs) with hydrodynamic diameters of ca. 50 nm. These smart micelles and SCKs presented switchable cationic, zwitterionic and anionic properties, and existed as stable nanoparticles with high positive surface charge at low pH (pH = 2, zeta potential ~ +40 mV) and strong negative surface charge at high pH (pH = 12, zeta potential ~ −35 mV). 1H NMR spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), and zeta potential, were used to characterize the chemical compositions, particle sizes, morphologies and surface charges. Precipitation occurred near the isoelectric points (IEP) of the polymer/particle solutions, and the IEP values could be tuned by changing the shell cross-linking density. The block copolymer micelles were capable of full reversible morphological inversion as a function of pH, by orthogonal protonation of the PAEA and hydroxide association with the PAPBA units, whereas the

  11. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.

    PubMed Central

    Sonnenberg, M G; Belisle, J T

    1997-01-01

    A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis Kat

  12. Pointing to others: How the target gender influences pointing performance.

    PubMed

    Cleret de Langavant, Laurent; Jacquemot, Charlotte; Cruveiller, Virginie; Dupoux, Emmanuel; Bachoud-Lévi, Anne-Catherine

    2016-01-01

    Pointing is a communicative gesture that allows individuals to share information about surrounding objects with other humans. Patients with heterotopagnosia are specifically impaired in pointing to other humans' body parts but not in pointing to themselves or to objects. Here, we describe a female patient with heterotopagnosia who was more accurate in pointing to men's body parts than to women's body parts. We replicated this gender effect in healthy participants with faster reaction times for pointing to men's body parts than to women's body parts. We discuss the role of gender stereotypes in explaining why it is more difficult to point to women than to men. PMID:27593456

  13. pH-dependent immobilization of proteins on surfaces functionalized by plasma-enhanced chemical vapor deposition of poly(acrylic acid)- and poly(ethylene oxide)-like films.

    PubMed

    Belegrinou, Serena; Mannelli, Ilaria; Lisboa, Patricia; Bretagnol, Frederic; Valsesia, Andrea; Ceccone, Giacomo; Colpo, Pascal; Rauscher, Hubert; Rossi, François

    2008-07-15

    The interaction of the proteins bovine serum albumin (BSA), lysozyme (Lys), lactoferrin (Lf), and fibronectin (Fn) with surfaces of protein-resistant poly(ethylene oxide) (PEO) and protein-adsorbing poly(acrylic acid) (PAA) fabricated by plasma-enhanced chemical vapor deposition has been studied with quartz crystal microbalance with dissipation monitoring (QCM-D). We focus on several parameters which are crucial for protein adsorption, i.e., the isoelectric point (pI) of the proteins, the pH of the solution, and the charge density of the sorbent surfaces, with the zeta-potential as a measure for the latter. The measurements reveal adsorption stages characterized by different segments in the plots of the dissipation vs frequency change. PEO remains protein-repellent for BSA, Lys, and Lf at pH 4-8.5, while weak adsorption of Fn was observed. On PAA, different stages of protein adsorption processes could be distinguished under most experimental conditions. BSA, Lys, Lf, and Fn generally exhibit a rapid initial adsorption phase on PAA, often followed by slower processes. The evaluation of the adsorption kinetics also reveals different adsorption stages, whereas the number of these stages does not always correspond to the structurally different phases as revealed by the D- f plots. The results presented here, together with information obtained in previous studies by other groups on the properties of these proteins and their interaction with surfaces, allow us to develop an adsorption scenario for each of these proteins, which takes into account electrostatic protein-surface and protein-protein interaction, but also the pH-dependent properties of the proteins, such as shape and exposure of specific domains. PMID:18549295

  14. 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...

  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. 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...

  17. 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...

  18. 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...

  19. Amino acids

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/002222.htm Amino acids To use the sharing features on this page, please enable JavaScript. Amino acids are organic compounds that combine to form proteins . ...

  20. Mefenamic Acid

    MedlinePlus

    Mefenamic acid is used to relieve mild to moderate pain, including menstrual pain (pain that happens before or during a menstrual period). Mefenamic acid is in a class of medications called NSAIDs. ...

  1. Aminocaproic Acid

    MedlinePlus

    Aminocaproic acid is used to control bleeding that occurs when blood clots are broken down too quickly. This type ... the baby is ready to be born). Aminocaproic acid is also used to control bleeding in the ...

  2. Ascorbic Acid

    MedlinePlus

    Ascorbic acid is used to prevent and treat scurvy, a disease caused by a lack of vitamin C in ... Ascorbic acid comes in extended-release (long-acting) capsules and tablets, lozenges, syrup, chewable tablets, and liquid drops to ...

  3. Acid mucopolysaccharides

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003368.htm Acid mucopolysaccharides To use the sharing features on this page, please enable JavaScript. Acid mucopolysaccharides is a test that measures the amount ...

  4. Ethacrynic Acid

    MedlinePlus

    Ethacrynic acid, a 'water pill,' is used to treat swelling and fluid retention caused by various medical problems. It ... Ethacrynic acid comes as a tablet to take by mouth. It is usually taken once or twice a day ...

  5. 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.

  6. Valproic Acid

    MedlinePlus

    Valproic acid is used alone or with other medications to treat certain types of seizures. Valproic acid is also used to treat mania (episodes of ... to relieve headaches that have already begun. Valproic acid is in a class of medications called anticonvulsants. ...

  7. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... provide the address or location of the control point where station records will be kept. (b) When the address or location of a control point where station records are kept is to be changed, the licensee...

  8. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... provide the address or location of the control point where station records will be kept. (b) When the address or location of a control point where station records are kept is to be changed, the licensee...

  9. BACTERIAL OXIDATION OF DIPICOLINIC ACID

    PubMed Central

    Kobayashi, Yasuo; Arima, Kei

    1962-01-01

    Kobayashi, Yasuo (University of Tokyo, Tokyo, Japan) and Kei Arima. Bacterial oxidation of dipicolinic acid. II. Identification of α-ketoglutaric acid and 3-hydroxydipicolinic acid and some properties of cell-free extracts. J. Bacteriol. 84:765–771. 1962—When a dipicolinic acid (DPA)-decomposing bacterium, Achromobacter strain 1–2, was incubated at 30 C with shaking in a DPA solution containing 10−3m arsenite, a keto acid was accumulated. The 2,4-dinitrophenylhydrazone of this acid was synthesized and identified as α-ketoglutaric acid by paper chromatography, visible absorption spectrum, infrared analysis, elemental analysis, and mixed melting point. During this incubation, oxalic acid equivalent to the consumed dipicolinic acid was produced. A fluorescent material was also isolated from culture fluid and identified as 3-hydroxydipicolinic acid by paper chromatography and the ultraviolet absorption spectrum. Further, cell-free extracts were prepared by sonic oscillation. Ferrous ion and a reduced di- or triphosphopyridine nucleotide-generating system were proven to be required for enzymic oxidation of DPA. And 3-hydroxydipicolinic acid was also oxidized by this preparation. From the results obtained, a possible metabolic pathway of dipicolinic acid was proposed. PMID:14033954

  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. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... provide the address or location of the control point where station records will be kept. (b) When the address or location of a control point where station records are kept is to be changed, the licensee must request a modification of the station license. (c) Control points not collocated with station records...

  12. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... provide the address or location of the control point where station records will be kept. (b) When the address or location of a control point where station records are kept is to be changed, the licensee must request a modification of the station license. (c) Control points not collocated with station records...

  13. 47 CFR 80.41 - Control points and dispatch points.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... provide the address or location of the control point where station records will be kept. (b) When the address or location of a control point where station records are kept is to be changed, the licensee must request a modification of the station license. (c) Control points not collocated with station records...

  14. Fatty acids - trans fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The data supporting a negative effect of dietary trans fatty acids on cardiovascular disease risk is consistent. The primary dietary sources of trans fatty acids include partially hydrogenated fat and rudiment fat. The adverse effect of trans fatty acids on plasma lipoprotein profiles is consisten...

  15. Freezing point and melting point of barnacle muscle fibers.

    PubMed

    Caillé, J P

    1983-10-01

    The freezing point and the melting point of myoplasm were measured with two experimental models. In all samples, a supercooled stage was reached by lowering the temperature of the sample to approximately - 7 degrees C, and the freezing of the sample was mechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43 degrees C), and the melting point indicated that the intracellular and the extracellular compartments are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a structural component similar to that of the intact muscle fiber, but the melting point was lower than the freezing and the melting points of the embedding solution. This was interpreted as evidence that the counterions around the contractile filaments occupied a nonnegligible fraction of the intracellular compartment. PMID:6640420

  16. 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.

  17. Utilization of acid tars

    SciTech Connect

    Frolov, A.F.; Denisova, T.L.; Aminov, A.N.

    1987-01-01

    Freshly produced acid tar (FPAT), obtained as refinery waste in treating petroleum oils with sulfuric acid and oleum, contains 80% or more sulfuric acid. Of such tars, pond acid tars, which contain up to 80% neutral petroleum products and sulfonated resins, are more stable, and have found applications in the production of binders for paving materials. In this article the authors are presenting results obtained in a study of the composition and reactivity of FPAT and its stability in storage in blends with asphalts obtained in deasphalting operations, and the possibility of using the FPAT in road construction has been examined. In this work, wastes were used which were obtained in treating the oils T-750, KhF-12, I-8A, and MS-14. Data on the change in group chemical composition of FPAT are shown, and the acidity, viscosity, needle penetration, and softening point of acid tars obtained from different grades of oils are plotted as functions of the storage time. It is also shown that the fresh and hardened FPATs differ in their solubilities in various solvents.

  18. Optimize acid gas removal

    SciTech Connect

    Nicholas, D.M.; Wilkins, J.T.

    1983-09-01

    Innovative design of physical solvent plants for acid gas removal can materially reduce both installation and operating costs. A review of the design considerations for one physical solvent process (Selexol) points to numerous arrangements for potential improvement. These are evaluated for a specific case in four combinations that identify an optimum for the case in question but, more importantly, illustrate the mechanism for use for such optimization elsewhere.

  19. 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.

  20. 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.

  1. Point-based manifold harmonics.

    PubMed

    Liu, Yang; Prabhakaran, Balakrishnan; Guo, Xiaohu

    2012-10-01

    This paper proposes an algorithm to build a set of orthogonal Point-Based Manifold Harmonic Bases (PB-MHB) for spectral analysis over point-sampled manifold surfaces. To ensure that PB-MHB are orthogonal to each other, it is necessary to have symmetrizable discrete Laplace-Beltrami Operator (LBO) over the surfaces. Existing converging discrete LBO for point clouds, as proposed by Belkin et al., is not guaranteed to be symmetrizable. We build a new point-wisely discrete LBO over the point-sampled surface that is guaranteed to be symmetrizable, and prove its convergence. By solving the eigen problem related to the new operator, we define a set of orthogonal bases over the point cloud. Experiments show that the new operator is converging better than other symmetrizable discrete Laplacian operators (such as graph Laplacian) defined on point-sampled surfaces, and can provide orthogonal bases for further spectral geometric analysis and processing tasks. PMID:22879345

  2. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.

    PubMed

    Langbein, L; Heid, H W; Moll, I; Franke, W W

    1993-12-01

    Differentiation of human plantar and palmar epidermis is characterized by the suprabasal synthesis of a major special intermediate-sized filament (IF) protein, the type I (acidic) cytokeratin 9 (CK 9). Using partial amino acid (aa) sequence information obtained by direct Edman sequencing of peptides resulting from proteolytic digestion of purified CK 9, we synthesized several redundant primers by 'back-translation'. Amplification by polymerase chain reaction (PCR) of cDNAs obtained by reverse transcription of mRNAs from human foot sole epidermis, including 5'-primer extension, resulted in multiple overlapping cDNA clones, from which the complete cDNA (2353 bp) could be constructed. This cDNA encoded the CK 9 polypeptide with a calculated molecular weight of 61,987 and an isoelectric point at about pH 5.0. The aa sequence deduced from cDNA was verified in several parts by comparison with the peptide sequences and showed the typical structure of type I CKs, with a head (153 aa), and alpha-helical coiled-coil-forming rod (306 aa), and a tail (163 aa) domain. The protein displayed the highest homology to human CK 10, not only in the highly conserved rod domain but also in large parts of the head and the tail domains. On the other hand, the aa sequence revealed some remarkable differences from CK 10 and other CKs, even in the most conserved segments of the rod domain. The nuclease digestion pattern seen on Southern blot analysis of human genomic DNA indicated the existence of a unique CK 9 gene. Using CK 9-specific riboprobes for hybridization on Northern blots of RNAs from various epithelia, a mRNA of about 2.4 kb in length could be identified only in foot sole epidermis, and a weaker cross-hybridization signal was seen in RNA from bovine heel pad epidermis at about 2.0 kb. A large number of tissues and cell cultures were examined by PCR of mRNA-derived cDNAs, using CK 9-specific primers. But even with this very sensitive signal amplification, only palmar

  3. 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

  4. Acid Deposition

    EPA Science Inventory

    This indicator presents acid deposition trends in the contiguous U.S. from 1989 to 2007. Data are broken down by wet and dry deposition and deposition of nitrogen and sulfur compounds. Acid deposition is particularly damaging to lakes, streams, and forests and the plants and a...

  5. Acid rain

    SciTech Connect

    White, J.C. )

    1988-01-01

    This book presents the proceedings of the third annual conference sponsored by the Acid Rain Information Clearinghouse (ARIC). Topics covered include: Legal aspects of the source-receptor relationship: an energy perspective; Scientific uncertainty, agency inaction, and the courts; and Acid rain: the emerging legal framework.

  6. Acid rain

    SciTech Connect

    Elsworth, S.

    1985-01-01

    This book was written in a concise and readable style for the lay public. It's purpose was to make the public aware of the damage caused by acid rain and to mobilize public opinion to favor the elimination of the causes of acid rain.

  7. Magnetic suspension and pointing system

    NASA Technical Reports Server (NTRS)

    Anderson, W. W.; Groom, N. J. (Inventor)

    1978-01-01

    An apparatus is reported for accurate pointing of instruments on a carrier vehicle and for isolation of the instruments from the vehicle's motion disturbances. The apparatus includes two assemblies with connecting interfaces. The first assembly is attached to the carrier vehicle and consists of an azimuth gimbal and an elevation gimbal which provide coarse pointing by allowing two rotations of the instruments relative to the carrier vehicle. The second or vernier pointing assembly is made up of magnetic suspension and fine pointing actuators, roll motor segments, and an instrument mounting plase which provides appropriate magnetic circuits for the actuators and the roll motor segments. The vernier pointing assembly provides attitude fine pointing and roll positioning of the instruments as well as six degree-of-freedom isolation from carrier motion disturbances.

  8. 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.

  9. Acid rain

    SciTech Connect

    Sweet, W.

    1980-06-20

    Acid precipitation includes not only rain but also acidified snow, hail and frost, as well as sulfur and nitrogen dust. The principal source of acid precipitation is pollution emitted by power plants and smelters. Sulfur and nitrogen compounds contained in the emissions combine with moisture to form droplets with a high acid content - sometimes as acidic as vinegar. When sufficiently concentrated, these acids can kill fish and damage material structures. Under certain circumstances they may reduce crop and forest yields and cause or aggravate respiratory diseases in humans. During the summer, especially, pollutants tend to collect over the Great Lakes in high pressure systems. Since winds typically are westerly and rotate clockwise around high pressure systems, the pollutants gradually are dispersed throughout the eastern part of the continent.

  10. Asparagusic acid.

    PubMed

    Mitchell, Stephen C; Waring, Rosemary H

    2014-01-01

    Asparagusic acid (1,2-dithiolane-4-carboxylic acid) is a simple sulphur-containing 5-membered heterocyclic compound that appears unique to asparagus, though other dithiolane derivatives have been identified in non-food species. This molecule, apparently innocuous toxicologically to man, is the most probable culprit responsible for the curious excretion of odorous urine following asparagus ingestion. The presence of the two adjacent sulphur atoms leads to an enhanced chemical reactivity, endowing it with biological properties including the ability to substitute potentially for α-lipoic acid in α-keto-acid oxidation systems. This brief review collects the scattered data available in the literature concerning asparagusic acid and highlights its properties, intermediary metabolism and exploratory applications. PMID:24099657

  11. 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.

  12. Antimicrobial activities of bacteriocins E 50-52 and B 602 against antobiotic resistant strains involved in nosocomial infections

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antimicrobial spectra of previously published bacteriocins (BCN) E 50-52 and B 602 was determined. The amino acid sequences, molecular weights and the isoelectric points of both E 50-52 and B 602 BCN were consistent with class IIa characteristics, contained 39 and 29 amino acid residues, molecul...

  13. Molecular cloning and characterization of two hypersensitive induced reaction genes from wheat infected by stripe rust pathogen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequenc...

  14. 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...

  15. 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]…

  16. MASSACHUSETTS DEP EELGRASS VERIFIED POINTS

    EPA Science Inventory

    Field verified points showing presence or absence of submerged rooted vascular plants along Massachusetts coastline. In addition to the photo interpreted eelgrass coverage (EELGRASS), this point coverage (EGRASVPT) was generated based on field-verified sites as well as all field...

  17. 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…

  18. 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.

  19. Acid fog

    SciTech Connect

    Hileman, B.

    1983-03-01

    Fog in areas of southern California previously thought to be pollution-free has been shown to have a pH as low as 1.69. It has been found to be most acidic after smoggy days, suggesting that it forms on the aerosol associated with the previously exiting smog. Studies on Whiteface Mountain in the Adirondacks show that fog water is often 10 times as acidic as rainwater. As a result of their studies, California plans to spend $4 million on acid deposition research in the coming year. (JMT)

  20. Planetary system detection by POINTS

    NASA Technical Reports Server (NTRS)

    Reasenberg, Robert D.

    1993-01-01

    The final report and semiannual reports 1, 2, and 3 in response to the study of 'Planetary System Detection by POINTS' is presented. The grant covered the period from 15 Jun. 1988 through 31 Dec. 1989. The work during that period comprised the further development and refinement of the POINTS concept. The status of the POINTS development at the end of the Grant period was described by Reasenberg in a paper given at the JPL Workshop on Space Interferometry, 12-13 Mar. 1990, and distributed as CfA Preprint 3138. That paper, 'POINTS: a Small Astrometric Interferometer,' follows as Appendix-A. Our proposal P2276-7-09, dated July 1990, included a more detailed description of the state of the development of POINTS at the end of the tenure of Grant NAGW-1355. That proposal, which resulted in Grant NAGW-2497, is included by reference.

  1. Folic acid

    MedlinePlus

    ... in the blood vessel to keep it open. Bipolar disorder. Taking folic acid does not appear to improve the antidepressant effects of lithium in people with bipolar disorder. However, taking folate with the medication valproate improves ...

  2. Mefenamic Acid

    MedlinePlus

    ... as mefenamic acid may cause ulcers, bleeding, or holes in the stomach or intestine. These problems may ... like coffee grounds, blood in the stool, or black and tarry stools.Keep all appointments with your ...

  3. ACID RAIN

    EPA Science Inventory

    Acid precipitation has become one of the major environmental problems of this decade. It is a challenge to scientists throughout the world. Researchers from such diverse disciplines as plant pathology, soil science, bacteriology, meteorology and engineering are investigating diff...

  4. Acid Precipitation

    ERIC Educational Resources Information Center

    Likens, Gene E.

    1976-01-01

    Discusses the fact that the acidity of rain and snow falling on parts of the U.S. and Europe has been rising. The reasons are still not entirely clear and the consequences have yet to be well evaluated. (MLH)

  5. Carnosic acid.

    PubMed

    Birtić, Simona; Dussort, Pierre; Pierre, François-Xavier; Bily, Antoine C; Roller, Marc

    2015-07-01

    Carnosic acid (salvin), which possesses antioxidative and antimicrobial properties, is increasingly exploited within the food, nutritional health and cosmetics industries. Since its first extraction from a Salvia species (∼70 years ago) and its identification (∼50 years ago), numerous articles and patents (∼400) have been published on specific food and medicinal applications of Rosmarinus and Salvia plant extracts abundant in carnosic acid. In contrast, relevant biochemical, physiological or molecular studies in planta have remained rare. In this overview, recent advances in understanding of carnosic acid distribution, biosynthesis, accumulation and role in planta, and its applications are summarised. We also discuss the deficiencies in our understanding of the relevant biochemical processes, and suggest the molecular targets of carnosic acid. Finally, future perspectives and studies related to its potential roles are highlighted. PMID:25639596

  6. Aminocaproic Acid

    MedlinePlus

    Amicar® Oral Solution ... Aminocaproic acid comes as a tablet and a solution (liquid) to take by mouth. It is usually ... it at room temperature and away from excess heat and moisture (not in the bathroom). Throw away ...

  7. Tranexamic Acid

    MedlinePlus

    ... is used to treat heavy bleeding during the menstrual cycle (monthly periods) in women. Tranexamic acid is in ... tablets for more than 5 days in a menstrual cycle or take more than 6 tablets in a ...

  8. Cannabinoid acids analysis.

    PubMed

    Lercker, G; Bocci, F; Frega, N; Bortolomeazzi, R

    1992-03-01

    The cannabinoid pattern of vegetable preparations from Cannabis sativa (hashish, marijuana) allows to recognize the phenotype of the plants, to be used as drug or for fiber. Cannabinoid determination by analytical point of view has represented some problems caused by the complex composition of the hexane extract. Capillary gas chromatography of the hexane extracts of vegetable samples, shows the presence of rather polar constituents that eluted, with noticeable interactions, only on polar phase. The compounds can be methylated by diazomethane and silanized (TMS) by silylating reagents. The methyl and methyl-TMS derivatives are analyzed by high resolution gas chromatography (HRGC) and by gas chromatography-mass spectrometry (GC-MS). The identification of the compounds shows their nature of cannabinoid acids, which the main by quantitative point of view results the cannabidiolic acid (CBDA). It is known that the cannabinoid acids are thermally unstable and are transformed in the corresponding cannabinoids by decarboxilation. This is of interest in forensic analysis with the aim to establish the total amount of THC in the Cannabis preparations, as the active component. PMID:1503600

  9. Acidic precipitation

    SciTech Connect

    Martin, H.C.

    1987-01-01

    At the International Symposium on Acidic Precipitation, over 400 papers were presented, and nearly 200 of them are included here. They provide an overview of the present state of the art of acid rain research. The Conference focused on atmospheric science (monitoring, source-receptor relationships), aquatic effects (marine eutrophication, lake acidification, impacts on plant and fish populations), and terrestrial effects (forest decline, soil acidification, etc.).

  10. Pivots for Pointing: Visually-Monitored Pointing Has Higher Arm Elevations than Pointing Blindfolded

    ERIC Educational Resources Information Center

    Wnuczko, Marta; Kennedy, John M.

    2011-01-01

    Observers pointing to a target viewed directly may elevate their fingertip close to the line of sight. However, pointing blindfolded, after viewing the target, they may pivot lower, from the shoulder, aligning the arm with the target as if reaching to the target. Indeed, in Experiment 1 participants elevated their arms more in visually monitored…

  11. Davies Critical Point and Tunneling

    NASA Astrophysics Data System (ADS)

    La, Hoseong

    2012-04-01

    From the point of view of tunneling, the physical meaning of the Davies critical point of a second-order phase transition in the black hole thermodynamics is clarified. At the critical point, the nonthermal contribution vanishes so that the black hole radiation is entirely thermal. It separates two phases: one with radiation enhanced by the nonthermal contribution, the other suppressed by the nonthermal contribution. We show this in both charged and rotating black holes. The phase transition is also analyzed in the cases in which emissions of charges and angular momenta are incorporated.

  12. NOTE: Do acupuncture points exist?

    NASA Astrophysics Data System (ADS)

    Yan, Xiaohui; Zhang, Xinyi; Liu, Chenglin; Dang, Ruishan; Huang, Yuying; He, Wei; Ding, Guanghong

    2009-05-01

    We used synchrotron x-ray fluorescence analysis to probe the distribution of four chemical elements in and around acupuncture points, two located in the forearm and two in the lower leg. Three of the four acupuncture points showed significantly elevated concentrations of elements Ca, Fe, Cu and Zn in relation to levels in the surrounding tissue, with similar elevation ratios for Cu and Fe. The mapped distribution of these elements implies that each acupuncture point seems to be elliptical with the long axis along the meridian.

  13. Screening of Bothrops snake venoms for L-amino acid oxidase activity.

    PubMed

    Pessatti, M; Fontana, J D; Furtado, M F; Guimãraes, M F; Zanette, L R; Costa, W T; Baron, M

    1995-01-01

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use of biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom. PMID:7668847

  14. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    SciTech Connect

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F.

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  15. Detecting determinism from point processes

    NASA Astrophysics Data System (ADS)

    Andrzejak, Ralph G.; Mormann, Florian; Kreuz, Thomas

    2014-12-01

    The detection of a nonrandom structure from experimental data can be crucial for the classification, understanding, and interpretation of the generating process. We here introduce a rank-based nonlinear predictability score to detect determinism from point process data. Thanks to its modular nature, this approach can be adapted to whatever signature in the data one considers indicative of deterministic structure. After validating our approach using point process signals from deterministic and stochastic model dynamics, we show an application to neuronal spike trains recorded in the brain of an epilepsy patient. While we illustrate our approach in the context of temporal point processes, it can be readily applied to spatial point processes as well.

  16. NULL convention floating point multiplier.

    PubMed

    Albert, Anitha Juliette; Ramachandran, Seshasayanan

    2015-01-01

    Floating point multiplication is a critical part in high dynamic range and computational intensive digital signal processing applications which require high precision and low power. This paper presents the design of an IEEE 754 single precision floating point multiplier using asynchronous NULL convention logic paradigm. Rounding has not been implemented to suit high precision applications. The novelty of the research is that it is the first ever NULL convention logic multiplier, designed to perform floating point multiplication. The proposed multiplier offers substantial decrease in power consumption when compared with its synchronous version. Performance attributes of the NULL convention logic floating point multiplier, obtained from Xilinx simulation and Cadence, are compared with its equivalent synchronous implementation. PMID:25879069

  17. NULL Convention Floating Point Multiplier

    PubMed Central

    Ramachandran, Seshasayanan

    2015-01-01

    Floating point multiplication is a critical part in high dynamic range and computational intensive digital signal processing applications which require high precision and low power. This paper presents the design of an IEEE 754 single precision floating point multiplier using asynchronous NULL convention logic paradigm. Rounding has not been implemented to suit high precision applications. The novelty of the research is that it is the first ever NULL convention logic multiplier, designed to perform floating point multiplication. The proposed multiplier offers substantial decrease in power consumption when compared with its synchronous version. Performance attributes of the NULL convention logic floating point multiplier, obtained from Xilinx simulation and Cadence, are compared with its equivalent synchronous implementation. PMID:25879069

  18. Hermit Points on a Box

    ERIC Educational Resources Information Center

    Hess, Richard; Grinstead, Charles; Grindstead, Marshall; Bergstrand, Deborah

    2008-01-01

    Suppose that we are given a rectangular box in 3-space. Given any two points on the surface of this box, we can define the surface distance between them to be the length of the shortest path between them on the surface of the box. This paper determines the pairs of points of maximum surface distance for all boxes. It is often the case that these…

  19. Point-Force Energy Coupling

    NASA Astrophysics Data System (ADS)

    Burton, Tristan; Squires, Kyle

    2005-11-01

    Fully resolved simulations of particle-laden turbulent flows are computationally expensive even with a single particle. Therefore, simulations of flows with realistic numbers of particles typically treat the disperse phase as point-particles and models are used to account for the interaction between the phases. The particle trajectories are determined using a Lagrangian particle equation of motion that accounts for the fluid forces. The effect of the particulate phase on the fluid is included using point-force momentum coupling, where the opposite of the force applied to each particle by the fluid is distributed back to fluid grid points in a local region. In this work, we perform direct numerical simulation (DNS) of a particle moving at a prescribed constant or time-dependent velocity through a stationary fluid, and use the resulting force history in a corresponding point-force simulation to study point-force energy coupling. The energy input from the moving particle and the fluid dissipation in the DNS are compared to corresponding quantities in the unresolved calculation. A range of particle Reynolds numbers and ratios of the particle diameter to the unresolved grid spacing are considered to determine the conditions under which point-force momentum coupling provides accurate energy coupling.

  20. Characterizing configurations of fire ignition points through spatiotemporal point processes

    NASA Astrophysics Data System (ADS)

    Comas, C.; Costafreda-Aumedes, S.; Vega-Garcia, C.

    2014-04-01

    Human-caused forest fires are usually regarded as unpredictable but often exhibit trends towards clustering in certain locations and periods. Characterizing such configurations is crucial for understanding spatiotemporal fire dynamics and implementing preventive actions. Our objectives were to analyse the spatiotemporal point configuration and to test for spatiotemporal interaction. We characterized the spatiotemporal structure of 984 fire ignition points in a study area of Galicia, Spain, during 2007-2011 by the K-Ripley's function. Our results suggest the presence of spatiotemporal structures for time lags of less than two years and ignition point distances in the range 0-12 km. Ignition centre points at time lags of less than 100 days are aggregated for any inter-event distance. This cluster structure loses strength as the time lag increases, and at time lags of more than 365 days this cluster structure is not significant for any lag distance. Our results also suggest spatiotemporal interdependencies at time lags of less than 100 days and inter-event distances of less than 10 km. At time lags of up to 365 days spatiotemporal components are independent for any point distance. These results suggest that risk conditions occur locally and are short-lived in this study area.

  1. An Emprical Point Error Model for Tls Derived Point Clouds

    NASA Astrophysics Data System (ADS)

    Ozendi, Mustafa; Akca, Devrim; Topan, Hüseyin

    2016-06-01

    The random error pattern of point clouds has significant effect on the quality of final 3D model. The magnitude and distribution of random errors should be modelled numerically. This work aims at developing such an anisotropic point error model, specifically for the terrestrial laser scanner (TLS) acquired 3D point clouds. A priori precisions of basic TLS observations, which are the range, horizontal angle and vertical angle, are determined by predefined and practical measurement configurations, performed at real-world test environments. A priori precision of horizontal (𝜎𝜃) and vertical (𝜎𝛼) angles are constant for each point of a data set, and can directly be determined through the repetitive scanning of the same environment. In our practical tests, precisions of the horizontal and vertical angles were found as 𝜎𝜃=±36.6𝑐𝑐 and 𝜎𝛼=±17.8𝑐𝑐, respectively. On the other hand, a priori precision of the range observation (𝜎𝜌) is assumed to be a function of range, incidence angle of the incoming laser ray, and reflectivity of object surface. Hence, it is a variable, and computed for each point individually by employing an empirically developed formula varying as 𝜎𝜌=±2-12 𝑚𝑚 for a FARO Focus X330 laser scanner. This procedure was followed by the computation of error ellipsoids of each point using the law of variance-covariance propagation. The direction and size of the error ellipsoids were computed by the principal components transformation. The usability and feasibility of the model was investigated in real world scenarios. These investigations validated the suitability and practicality of the proposed method.

  2. Robot positioning based on point-to-point motion capability

    SciTech Connect

    Park, Y. S.; Cho, H. S.; Koh, K. C.

    2000-03-20

    This paper presents an optimal search method for determining the base location of a robot manipulator so that the robot can have a designated point-to-point (PTP) motion capabilities. Based on the topological characterization of the manipulator workspace and the definitions of various p-connectivity, a computational method is developed for enumerating various PTP motion capabilities into quantitative cost functions. Then an unconstrained search by minimizing the cost function yields the task feasible location of the robot base. This methodology is useful for placement of mobile manipulators and robotic workcell layout design.

  3. Salicylic acids

    PubMed Central

    Hayat, Shamsul; Irfan, Mohd; Wani, Arif; Nasser, Alyemeni; Ahmad, Aqil

    2012-01-01

    Salicylic acid is well known phytohormone, emerging recently as a new paradigm of an array of manifestations of growth regulators. The area unleashed yet encompassed the applied agriculture sector to find the roles to strengthen the crops against plethora of abiotic and biotic stresses. The skipped part of integrated picture, however, was the evolutionary insight of salicylic acid to either allow or discard the microbial invasion depending upon various internal factors of two interactants under the prevailing external conditions. The metabolic status that allows the host invasion either as pathogenesis or symbiosis with possible intermediary stages in close systems has been tried to underpin here. PMID:22301975

  4. Acid rain information book. Draft final report

    SciTech Connect

    1980-12-01

    Acid rain is one of the most widely publicized environmental issues of the day. The potential consequences of increasingly widespread acid rain demand that this phenomenon be carefully evaluated. Reveiw of the literature shows a rapidly growing body of knowledge, but also reveals major gaps in understanding that need to be narrowed. This document discusses major aspects of the acid rain phenomenon, points out areas of uncertainty, and summarizes current and projected research by responsible government agencies and other concerned organizations.

  5. GRATIS: Pointing and Tracking System

    NASA Astrophysics Data System (ADS)

    Seiffert, M. D.; Lubin, P. M.; Craig, W. W.; McLean, R.; Harrison, F.

    1992-12-01

    The Gamma-Ray Arc Minute Telescope Imaging System (GRATIS; see companion paper by Harrison et al.) requires a balloon-borne stabilized platform capable of 20 arc second absolute pointing accuracy. We have developed a system which uses computer-based inertial-guidance control of an azimuth-elevation pointing mechanism for the telescope. An innovative computer-based star pattern recognition system automatically generates drift corrections from an image acquired by a Peltier cooled CCD camera. The inertial guidance system provides three axis pointing information with approximately 12 arc seconds precision. This is a true inertial guidance system with gyros, accelerometers, and an integral navigational processor. The gyros have high relative pointing precision, but a slow drift component degrades their absolute accuracy. Control of the elevation position is accomplished through a torque motor that is directly coupled to the telescope. Azimuth control is accomplished through an active zero-stiction bearing at the top of the gondola and a reaction wheel at the bottom. The pointing system has been fully constructed and tested and has been mated with the telescope. We present the results of an extensive series of tracking tests.

  6. Folic acid

    MedlinePlus

    ... disease called vitiligo, and an inherited disease called Fragile-X syndrome. It is also used for reducing harmful side ... to blood clots (ischemic stroke). Inherited disease called Fragile-X syndrome.Taking folic acid by mouth does not improve ...

  7. Acid rain

    SciTech Connect

    Not Available

    1984-06-01

    An overview is presented of acid rain and the problems it causes to the environment worldwide. The acidification of lakes and streams is having a dramatic effect on aquatic life. Aluminum, present in virtually all forest soils, leaches out readily under acid conditions and interferes with the gills of all fish, some more seriously than others. There is evidence of major damage to forests in European countries. In the US, the most severe forest damage appears to be in New England, New York's Adirondacks, and the central Appalachians. This small region is part of a larger area of the Northeast and Canada that appears to have more acid rainfall than the rest of the country. It is downwind from major coal burning states, which produce about one quarter of US SO/sub 2/ emissions and one sixth of nitrogen oxide emissions. Uncertainties exist over the causes of forest damage and more research is needed before advocating expensive programs to reduce rain acidity. The President's current budget seeks an expansion of research funds from the current $30 million per year to $120 million.

  8. Formic acid

    Integrated Risk Information System (IRIS)

    Formic acid ; CASRN 64 - 18 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  9. Selenious acid

    Integrated Risk Information System (IRIS)

    Selenious acid ; CASRN 7783 - 00 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  10. Benzoic acid

    Integrated Risk Information System (IRIS)

    Benzoic acid ; CASRN 65 - 85 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effec

  11. Trichloroacetic acid

    Integrated Risk Information System (IRIS)

    Trichloroacetic acid ( TCA ) ; CASRN 76 - 03 - 9 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Nonca

  12. Dichloroacetic acid

    Integrated Risk Information System (IRIS)

    Dichloroacetic acid ; CASRN 79 - 43 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogeni

  13. Acrylic acid

    Integrated Risk Information System (IRIS)

    Acrylic acid ( CASRN 79 - 10 - 7 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  14. Cacodylic acid

    Integrated Risk Information System (IRIS)

    Cacodylic acid ; CASRN 75 - 60 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  15. Phosphoric acid

    Integrated Risk Information System (IRIS)

    Phosphoric acid ; CASRN 7664 - 38 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  16. Stearic Acid

    ERIC Educational Resources Information Center

    Young, Jay A.

    2004-01-01

    A chemical laboratory information profile (CLIP) is presented for the chemical, stearic acid. The profile lists the chemical's physical and harmful characteristics, exposure limits, and symptoms of major exposure, for the benefit of teachers and students, who use the chemical in the laboratory.

  17. Remotely Sensed Ground Control Points

    NASA Astrophysics Data System (ADS)

    Hummel, P.

    2016-06-01

    Accurate ground control is required to georeferenced airborne and spaceborne images. The production of ortho-photogrammetric data requires ground control that is traditionally provided as Ground Control Points (GCPs) by GNSS measurements in the field. However, it can be difficult to acquire accurate ground control points due to required turn-around time, high costs or impossible access. CompassData, Inc. a specialist in ground control, has expanded its service to deliver Remotely Sensed Ground Control Points (RSGCPs®). TerraSAR-X and TanDEM-X are two satellites with such high accuracy of their orbital positions and SAR data that RSGCPs® can be produced to a sub-meter quality depending on certain parameters and circumstances. The technology and required parameters are discussed in this paper as well as the resulting accuracies.

  18. Tipping Points in Texas Rivers

    NASA Astrophysics Data System (ADS)

    Phillips, Jonathan

    2016-04-01

    Anticipating geomorphic tipping points requires that we learn from the past. Major geomorphic changes in coastal plain rivers of Texas resulting in river metamorphosis or regime shifts were identified, and the major driving factors determined. Nine fluvial tipping points were identified from contemporary observations, historical records, and Quaternary reconstructions. Two of the tipping points (between general aggrading and degrading valley states) are associated with reversals in a fundamental system control (sea-level). One (stable or aggrading vs. degrading channels) is associated with an abrupt change in sediment supply due to dam construction, and two others (changes from meandering to anastomosing channel patterns, and different anastomosis styles) are similarly related to changes in sediment supply and/or transport capacity, but with additional elements of historical contingency. Three tipping points are related to avulsions. One, from a regime dominated to reoccupation of former channels to one dominated by progradation into flood basins, is driven by progressive long term filling of incised valleys. Another, nodal avulsions, are driven by disturbances associated with tectonics or listric faults. The third, avulsions and related valley metamorphosis in unfilled incised valleys, is due to fundamental dynamical instabilities within the fluvial system. This synthesis and analysis suggests that geomorphic tipping points are sometimes associated with general extrinsic or intrinsic (to the fluvial system) environmental change, independent of any disturbances or instabilities. Others are associated with natural (e.g., tectonic) or human (dams) disturbances, and still others with intrinsic geomorphic instabilities. This suggests that future tipping points will be equally diverse with respect to their drivers.

  19. Point estimates for probability moments

    PubMed Central

    Rosenblueth, Emilio

    1975-01-01

    Given a well-behaved real function Y of a real random variable X and the first two or three moments of X, expressions are derived for the moments of Y as linear combinations of powers of the point estimates y(x+) and y(x-), where x+ and x- are specific values of X. Higher-order approximations and approximations for discontinuous Y using more point estimates are also given. Second-moment approximations are generalized to the case when Y is a function of several variables. PMID:16578731

  20. Inflection point inflation and reheating

    NASA Astrophysics Data System (ADS)

    Choi, Soo-Min; Lee, Hyun Min

    2016-06-01

    We revisit the inflection point inflation with an extended discussion to large field values and consider the reheating effects on the inflationary predictions. Parametrizing the reheating dynamics in terms of the reheating temperature and the equation of state during reheating, we show how the observationally favored parameter space of inflection point inflation is affected by reheating dynamics. Consequently, we apply the general results to the inflation models with non-minimal coupling, such as the SM Higgs inflation and the B-L Higgs inflation.

  1. EPA FACILITY POINT LOCATION FILES

    EPA Science Inventory

    Data includes locations of facilities from which pollutants are discharged. The epapoints.tar.gz file is a gzipped tar file of 14 Arc/Info export files and text documents. The .txt files define the attributes located in the INFO point coverage files. Projections are defined in...

  2. Turning Points: Opportunity or Trouble?

    ERIC Educational Resources Information Center

    Jackson, Anthony W.

    1991-01-01

    Reviews recommendations of the Carnegie report "Turning Points: Preparing American Youth for the 21st Century" with respect to the internal restructuring of middle schools and ways of integrating community and parental support. Discusses suggestions concerning small learning communities, core academic programs, ensuring student success, expert…

  3. The Mean as Balance Point

    ERIC Educational Resources Information Center

    O'Dell, Robin S.

    2012-01-01

    There are two primary interpretations of the mean: as a leveler of data (Uccellini 1996, pp. 113-114) and as a balance point of a data set. Typically, both interpretations of the mean are ignored in elementary school and middle school curricula. They are replaced with a rote emphasis on calculation using the standard algorithm. When students are…

  4. Decimal Fractions: An Important Point

    ERIC Educational Resources Information Center

    Martinie, Sherri L.

    2014-01-01

    How can a simple dot--the decimal point--be the source of such frustration for students and teachers? As the author worked through her own frustrations, she found that her students seemed to fall into groups in terms of misconceptions that they revealed when talking about and working with decimals. When asking students to illustrate their thinking…

  5. Critical points of metal vapors

    SciTech Connect

    Khomkin, A. L. Shumikhin, A. S.

    2015-09-15

    A new method is proposed for calculating the parameters of critical points and binodals for the vapor–liquid (insulator–metal) phase transition in vapors of metals with multielectron valence shells. The method is based on a model developed earlier for the vapors of alkali metals, atomic hydrogen, and exciton gas, proceeding from the assumption that the cohesion determining the basic characteristics of metals under normal conditions is also responsible for their properties in the vicinity of the critical point. It is proposed to calculate the cohesion of multielectron atoms using well-known scaling relations for the binding energy, which are constructed for most metals in the periodic table by processing the results of many numerical calculations. The adopted model allows the parameters of critical points and binodals for the vapor–liquid phase transition in metal vapors to be calculated using published data on the properties of metals under normal conditions. The parameters of critical points have been calculated for a large number of metals and show satisfactory agreement with experimental data for alkali metals and with available estimates for all other metals. Binodals of metals have been calculated for the first time.

  6. High Points of Human Genetics

    ERIC Educational Resources Information Center

    Stern, Curt

    1975-01-01

    Discusses such high points of human genetics as the study of chromosomes, somatic cell hybrids, the population formula: the Hardy-Weinberg Law, biochemical genetics, the single-active X Theory, behavioral genetics and finally how genetics can serve humanity. (BR)

  7. A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

    PubMed Central

    Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

    2014-01-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

  8. Molten fatty acid based microemulsions.

    PubMed

    Noirjean, Cecile; Testard, Fabienne; Dejugnat, Christophe; Jestin, Jacques; Carriere, David

    2016-06-21

    We show that ternary mixtures of water (polar phase), myristic acid (MA, apolar phase) and cetyltrimethylammonium bromide (CTAB, cationic surfactant) studied above the melting point of myristic acid allow the preparation of microemulsions without adding a salt or a co-surfactant. The combination of SANS, SAXS/WAXS, DSC, and phase diagram determination allows a complete characterization of the structures and interactions between components in the molten fatty acid based microemulsions. For the different structures characterized (microemulsion, lamellar or hexagonal phases), a similar thermal behaviour is observed for all ternary MA/CTAB/water monophasic samples and for binary MA/CTAB mixtures without water: crystalline myristic acid melts at 52 °C, and a thermal transition at 70 °C is assigned to the breaking of hydrogen bounds inside the mixed myristic acid/CTAB complex (being the surfactant film in the ternary system). Water determines the film curvature, hence the structures observed at high temperature, but does not influence the thermal behaviour of the ternary system. Myristic acid is partitioned in two "species" that behave independently: pure myristic acid and myristic acid associated with CTAB to form an equimolar complex that plays the role of the surfactant film. We therefore show that myristic acid plays the role of a solvent (oil) and a co-surfactant allowing the fine tuning of the structure of oil and water mixtures. This solvosurfactant behaviour of long chain fatty acid opens the way for new formulations with a complex structure without the addition of any extra compound. PMID:27241163

  9. Characterization of papaya peptidase A as an enzyme of extreme basicity.

    PubMed

    Kaarsholm, N C; Schack, P

    1983-01-01

    Papaya peptidase A, a papain-like enzyme, has formerly been shown to contain a larger excess of basic amino acids and to have a higher isoelectric point than any of the other enzymes in the papaya latex. Determinations of the free electrophoretic mobility as a function of pH now establishes the isoelectric point of papaya peptidase A as 11.7 and that of succinylated papaya peptidase A as 3.8. Although the specific activity of the enzyme appears only slightly affected by the succinylation, the accompanying change in the charge/mobility ratio seems to indicate a relatively large conformational change upon succinylation. PMID:6362305

  10. Hydroxycarboxylic acids and salts

    DOEpatents

    Kiely, Donald E; Hash, Kirk R; Kramer-Presta, Kylie; Smith, Tyler N

    2015-02-24

    Compositions which inhibit corrosion and alter the physical properties of concrete (admixtures) are prepared from salt mixtures of hydroxycarboxylic acids, carboxylic acids, and nitric acid. The salt mixtures are prepared by neutralizing acid product mixtures from the oxidation of polyols using nitric acid and oxygen as the oxidizing agents. Nitric acid is removed from the hydroxycarboxylic acids by evaporation and diffusion dialysis.

  11. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  12. Sensing with Superconducting Point Contacts

    PubMed Central

    Nurbawono, Argo; Zhang, Chun

    2012-01-01

    Superconducting point contacts have been used for measuring magnetic polarizations, identifying magnetic impurities, electronic structures, and even the vibrational modes of small molecules. Due to intrinsically small energy scale in the subgap structures of the supercurrent determined by the size of the superconducting energy gap, superconductors provide ultrahigh sensitivities for high resolution spectroscopies. The so-called Andreev reflection process between normal metal and superconductor carries complex and rich information which can be utilized as powerful sensor when fully exploited. In this review, we would discuss recent experimental and theoretical developments in the supercurrent transport through superconducting point contacts and their relevance to sensing applications, and we would highlight their current issues and potentials. A true utilization of the method based on Andreev reflection analysis opens up possibilities for a new class of ultrasensitive sensors. PMID:22778630

  13. EBTR design-point selection

    SciTech Connect

    Krakowski, R.A.; Bathke, C.G.

    1981-01-01

    The procedure used to select the design point for the ELMO Bumpy Torus Reactor (EBTR) study is described. The models used in each phase of the selection process are described, with an emphasis placed on the parametric design curves produced by each model. The tradeoffs related to burn physics, stability/equilibrium, electron-ring physics, and magnetics design are discussed. The resulting design point indicates a plasma with a 35-m major radius and a 1-m minor radium operating at an average core-plasma beta of 0.17, which at approx. 30 keV produces an average neutron wall loading of 1.4 MW/m/sup 2/ while maintaining key magnet (< 10 T) and total power (less than or equal to 4000 MWt) constraints.

  14. Point contacts in encapsulated graphene

    SciTech Connect

    Handschin, Clevin; Fülöp, Bálint; Csonka, Szabolcs; Makk, Péter; Blanter, Sofya; Weiss, Markus; Schönenberger, Christian; Watanabe, Kenji; Taniguchi, Takashi

    2015-11-02

    We present a method to establish inner point contacts with dimensions as small as 100 nm on hexagonal boron nitride (hBN) encapsulated graphene heterostructures by pre-patterning the top-hBN in a separate step prior to dry-stacking. 2- and 4-terminal field effect measurements between different lead combinations are in qualitative agreement with an electrostatic model assuming point-like contacts. The measured contact resistances are 0.5–1.5 kΩ per contact, which is quite low for such small contacts. By applying a perpendicular magnetic field, an insulating behaviour in the quantum Hall regime was observed, as expected for inner contacts. The fabricated contacts are compatible with high mobility graphene structures and open up the field for the realization of several electron optical proposals.

  15. What are cirrus point sources?

    NASA Technical Reports Server (NTRS)

    Heiles, Carl; Mccarthy, Patrick J.; Reach, William; Strauss, Michael A.

    1987-01-01

    Most cirrus point sources are associated with interstellar gas. A subset of these was isolated, together with other sources showing large band 4 to 3 flux density ratios, that are not associated with interstellar gas. Most of the point sources are associated with diffuse cirrus emissions. The sources appear to be distributed randomly on the sky, with the exception of six clusters, one of which is not associated with any known object. Six sources out of seventeen that were observed for redshifted H I at Arecibo were found to be associated with relatively nondescript external galaxies. Most of the sources do not appear on the Palomar Sky Survey. Deep optical observations of eight fields revealed some fairly distant galaxies, one object with a very peculiar optical spectrum, and several blank fields.

  16. Lensless reflective point diffraction interferometer.

    PubMed

    Zhu, Wenhua; Chen, Lei; Zheng, Donghui; Yang, Ying; Han, Zhigang; Li, Jinpeng

    2016-07-01

    A lensless reflective point diffraction interferometer (LRPDI) is proposed for dynamic wavefront measurement. The point diffraction interferometer is integrated on a small substrate with properly designed thin film, which is used for generating the interferogram with high carrier frequency at a CCD target. By lensless imaging, the complex amplitude at the CCD target can be propagated to the conjugated plane of the exit pupil of an incident wavefront, which not only avoids the edge diffraction in the interferogram, but also eliminates systematic error. The accuracy of LRPDI is demonstrated by simulation and experiment, and a precision better than 1/150 wavelength is achieved. The new design with lensless imaging processing is suitable for dynamic wavefront measurement. PMID:27409204

  17. Femtosecond photoelectron point projection microscope

    SciTech Connect

    Quinonez, Erik; Handali, Jonathan; Barwick, Brett

    2013-10-15

    By utilizing a nanometer ultrafast electron source in a point projection microscope we demonstrate that images of nanoparticles with spatial resolutions of the order of 100 nanometers can be obtained. The duration of the emission process of the photoemitted electrons used to make images is shown to be of the order of 100 fs using an autocorrelation technique. The compact geometry of this photoelectron point projection microscope does not preclude its use as a simple ultrafast electron microscope, and we use simple analytic models to estimate temporal resolutions that can be expected when using it as a pump-probe ultrafast electron microscope. These models show a significant increase in temporal resolution when comparing to ultrafast electron microscopes based on conventional designs. We also model the microscopes spectroscopic abilities to capture ultrafast phenomena such as the photon induced near field effect.

  18. Maximum Power Point Regulator System

    NASA Astrophysics Data System (ADS)

    Simola, J.; Savela, K.; Stenberg, J.; Tonicello, F.

    2011-10-01

    The target of the study done under the ESA contract No.17830/04/NL/EC (GSTP4) for Maximum Power Point Regulator System (MPPRS) was to investigate, design and test a modular power system (a core PCU) fulfilling requirement for maximum power transfer even after a single failure in the Power System by utilising a power concept without any potential and credible single point failure. The studied MPPRS concept is of a modular construction, able to track the MPP individually on each SA sections, maintaining its functionality and full power capability after a loss of a complete MPPR module (by utilizingN+1module).Various add-on DCDC converter topology candidates were investigated and redundancy, failure mechanisms and protection aspects were studied

  19. Freely suspended quantum point contacts

    NASA Astrophysics Data System (ADS)

    Rössler, C.; Herz, M.; Bichler, M.; Ludwig, S.

    2010-05-01

    We present a versatile design of freely suspended quantum point contacts with particular large one-dimensional subband quantization energies of up to Δɛ≈10 meV. The nanoscale bridges embedding a two-dimensional electron system are fabricated from AlGaAs/GaAs heterostructures by electron-beam lithography and etching techniques. Narrow constrictions define quantum point contacts that are capacitively controlled via local in-plane side gates. Employing transport spectroscopy, we investigate the transition from electrostatic subbands to Landau quantization in a perpendicular magnetic field. The large subband quantization energies allow us to utilize a wide magnetic field range and thereby observe a large exchange split spin-gap of the two lowest Landau-levels.

  20. Viviani Polytopes and Fermat Points

    ERIC Educational Resources Information Center

    Zhou, Li

    2012-01-01

    Given a set of oriented hyperplanes P = {p1, . . . , pk} in R[superscript n], define v : R[superscript n] [right arrow] R by v(X) = the sum of the signed distances from X to p[subscript 1], . . . , p[subscript k], for any point X [is a member of] R[superscript n]. We give a simple geometric characterization of P for which v is constant, leading to…

  1. Multiple Fixed-Point Cell

    NASA Astrophysics Data System (ADS)

    Edler, F.; Ederer, P.

    2014-07-01

    The paper describes the construction and investigation of multiple fixed-point cells usable for the calibration of thermocouples at temperatures above 1100 C. These fixed-point cells made of pure graphite are characterized by a simple construction as well as by a flexible application. The cylindrical basic mount is equipped with a central hole for the insertion of a thermocouple, and with eight drill holes containing exchangeable cartridges which surround the central bore axially symmetrically. The cartridges are filled with different metal-carbon (Me-C) eutectics: cobalt-carbon (Co-C), nickel-carbon (Ni-C), palladium-carbon (Pd-C), and rhodium-carbon (Rh-C). The melting temperatures of the different Me-C eutectics of the cartridges were compared to the melting temperatures of commonly used Me-C eutectic fixed-point cells of the Physikalisch-Technische Bundesanstalt by using a Pt/Pd thermocouple (Co-C, Ni-C) and Type B thermocouples (Pd-C, Rh-C). The uncertainties () of the emfs measured at the inflection points of the melting curves are in the order of a few V which correspond to temperature equivalents between 0.3 K and 0.6 K. Furthermore, the difference between the melting temperatures of the Co-C and Ni-C cartridges was found to be 4.2 K by using simultaneously two sets of four cartridges filled with the two materials and placed alternately in the eight outer holes of one basic mount.

  2. The politician's point of view

    NASA Astrophysics Data System (ADS)

    Mahran, Maher

    1993-03-01

    The politician's point of view refers to studies which prove that neither a socio-economic development plan which ignores population questions, nor population education in isolation, can succeed. Educated people tend to have fewer children and to provide better education for them, while girls marrying before the age of 20 have little hope of continuing education and are subject to other social pressures. Examples are given from population education programs in Egypt.

  3. Turning points in reactor design

    SciTech Connect

    Beckjord, E.S.

    1995-09-01

    This article provides some historical aspects on nuclear reactor design, beginning with PWR development for Naval Propulsion and the first commercial application at Yankee Rowe. Five turning points in reactor design and some safety problems associated with them are reviewed: (1) stability of Dresden-1, (2) ECCS, (3) PRA, (4) TMI-2, and (5) advanced passive LWR designs. While the emphasis is on the thermal-hydraulic aspects, the discussion is also about reactor systems.

  4. The Critical Point Facility (CPF)

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Critical Point Facility (CPF) is an ESA multiuser facility designed for microgravity research onboard Spacelab. It has been conceived and built to offer investigators opportunities to conduct research on critical point phenomena in microgravity. This facility provides the high precision and stability temperature standards required in this field of research. It has been primarily designed for the purpose of optical investigations of transparent fluids. During a Spacelab mission, the CPF automatically processes several thermostats sequentially, each thermostat corresponding to an experiment. The CPF is now integrated in Spacelab at Kennedy Space Center, in preparation for the International Microgravity Lab. mission. The CPF was designed to submit transparent fluids to an adequate, user defined thermal scenario, and to monitor their behavior by using thermal and optical means. Because they are strongly affected by gravity, a good understanding of critical phenomena in fluids can only be gained in low gravity conditions. Fluids at the critical point become compressed under their own weight. The role played by gravity in the formation of interfaces between distinct phases is not clearly understood.

  5. 7 CFR 989.105 - Inspection point.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Inspection point. 989.105 Section 989.105 Agriculture... CALIFORNIA Administrative Rules and Regulations Definitions § 989.105 Inspection point. Inspection point... handler, and which is so designated by the Committee. The inspection point(s) of the handler shall...

  6. 49 CFR 236.334 - Point detector.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Point detector. 236.334 Section 236.334... Instructions § 236.334 Point detector. Point detector shall be maintained so that when switch mechanism is... switch point. Point detector circuit controller shall be maintained so that the contacts will not...

  7. 49 CFR 236.334 - Point detector.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Point detector. 236.334 Section 236.334... Instructions § 236.334 Point detector. Point detector shall be maintained so that when switch mechanism is... switch point. Point detector circuit controller shall be maintained so that the contacts will not...

  8. 7 CFR 989.105 - Inspection point.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Inspection point. 989.105 Section 989.105 Agriculture... CALIFORNIA Administrative Rules and Regulations Definitions § 989.105 Inspection point. Inspection point... handler, and which is so designated by the Committee. The inspection point(s) of the handler shall...

  9. The influence of casein haplotype on morphometric characteristics of fat globules and fatty acid composition of milk in Italian Holstein cows.

    PubMed

    Perna, Annamaria; Intaglietta, Immacolata; Simonetti, Amalia; Gambacorta, Emilio

    2016-04-01

    The aim of this work was to investigate the effect of casein haplotypes (αS1-, β-, and κ-caseins) on morphometric characteristics of fat globules and fatty acid composition of Italian Holstein milk. Casein haplotypes were determined by isoelectric focusing; milk fat globule size was measured by using a fluorescence microscope; and fatty acid profile was determined by gas chromatography. Casein haplotype significantly affected the fat globule size, the percentage incidence of each globule size class on total measured milk fat globules, and fatty acid composition. A higher incidence of smaller milk fat globules was associated with the BB-A(2)A(2)-BB genotype (αS1-, β-, and κ-casein haplotypes, respectively), whereas small globules were not detected in BB-A(2)A(1)-AA milk, but that milk had the highest percentage of large globules. A higher content of monounsaturated fatty acids was associated with the BB-A(2)A(2)-AB genotype, whereas higher contents of conjugated linoleic acid and docosahexaenoic acid were detected in BB-A(1)A(1)-AA milk. Our results indicate that casein haplotype could affect fat characteristics and, therefore, the nutritional and technological quality of milk. PMID:26874415

  10. Point-point and point-line moving-window correlation spectroscopy and its applications

    NASA Astrophysics Data System (ADS)

    Zhou, Qun; Sun, Suqin; Zhan, Daqi; Yu, Zhiwu

    2008-07-01

    In this paper, we present a new extension of generalized two-dimensional (2D) correlation spectroscopy. Two new algorithms, namely point-point (P-P) correlation and point-line (P-L) correlation, have been introduced to do the moving-window 2D correlation (MW2D) analysis. The new method has been applied to a spectral model consisting of two different processes. The results indicate that P-P correlation spectroscopy can unveil the details and re-constitute the entire process, whilst the P-L can provide general feature of the concerned processes. Phase transition behavior of dimyristoylphosphotidylethanolamine (DMPE) has been studied using MW2D correlation spectroscopy. The newly proposed method verifies that the phase transition temperature is 56 °C, same as the result got from a differential scanning calorimeter. To illustrate the new method further, a lysine and lactose mixture has been studied under thermo perturbation. Using the P-P MW2D, the Maillard reaction of the mixture was clearly monitored, which has been very difficult using conventional display of FTIR spectra.

  11. Methylmalonic acid blood test

    MedlinePlus

    ... acid is a substance produced when proteins, called amino acids, in the body break down. The health care ... Cederbaum S, Berry GT. Inborn errors of carbohydrate, ammonia, amino acid, and organic acid metabolism. In: Gleason CA, Devaskar ...

  12. Folic acid - test

    MedlinePlus

    Folic acid is a type of B vitamin. This article discusses the test to measure the amount of folic acid in the blood. ... that may interfere with test results, including folic acid supplements. Drugs that can decrease folic acid measurements ...

  13. Uric acid urine test

    MedlinePlus

    The uric acid urine test measures the level of uric acid in urine. Uric acid level can also be checked using a blood ... help determine the cause of a high uric acid level in the blood. It may also be ...

  14. Methylmalonic acid blood test

    MedlinePlus

    The methylmalonic acid blood test measures the amount of methylmalonic acid in the blood. ... Methylmalonic acid is a substance produced when proteins, called amino acids, in the body break down. The health care ...

  15. Folic Acid and Pregnancy

    MedlinePlus

    ... 5 Things to Know About Zika & Pregnancy Folic Acid and Pregnancy KidsHealth > For Parents > Folic Acid and ... before conception and during early pregnancy . About Folic Acid Folic acid, sometimes called folate, is a B ...

  16. Structure of seven organic salts assembled from 2,6-diaminopyridine with monocarboxylic acids, dicarboxylic acids, and tetracarboxylic acids

    NASA Astrophysics Data System (ADS)

    Gao, Xingjun; Zhang, Huan; Wen, Xianhong; Liu, Bin; Jin, Shouwen; Wang, Daqi

    2015-08-01

    Studies concentrating on non-covalent interactions between the organic base of 2,6-diaminopyridine, and carboxylic acids have led to an increased understanding of the role 2,6-diaminopyridine in binding with carboxylic acid derivatives. Here anhydrous and hydrated multi-component organic acid-base salts of 2,6-diaminopyridine have been prepared with the carboxylic acids as nicotinic acid, o-chlorobenzoic acid, 1,3-benzodioxole-5-carboxylic acid, 3,5-dinitrosalicylic acid, 4-nitro-phthalic acid, 1,4-cyclohexanedicarboxylic acid, and butane-1,2,3,4-tetracarboxylic acid. The seven crystalline compounds were characterized by X-ray diffraction analysis, infrared (IR), melting point (mp), and elemental analysis. All structures adopted the hetero R22(8) supramolecular synthons. The supramolecular architectures bear extensive Nsbnd H⋯N, Osbnd H⋯N, Osbnd H⋯O, Nsbnd H⋯O, and CH⋯O associations as well as other nonbonding contacts as CHsbnd N, CH2sbnd O, π-π, C-π, O-π, Cl-π, Clsbnd O, and Osbnd O interactions. The role of weak and strong hydrogen bonding in the crystal packing is ascertained.

  17. Non-Gimbaled Antenna Pointing

    NASA Technical Reports Server (NTRS)

    Vigil, Jeannine S.

    1997-01-01

    The small satellite community has been interested in accessing fixed ground stations for means of space-to-ground transmissions, although a problem arises from the limited global coverage. There is a growing interest for using the Space Network (SN) or Tracking and Data Relay Satellites (TDRS) as the primary support for communications because of the coverage it provides. This thesis will address the potential for satellite access of the Space Network with a non-gimbaled antenna configuration and low-power, coded transmission. The non-gimbaled antenna and the TDRS satellites, TDRS-East, TDRS-West, and TDRS-Zone of Exclusion, were configured in an orbital analysis software package called Satellite Tool Kit to emulate the three-dimensional position of the satellites. The access potential, which is the average number of contacts per day and the average time per contact, were obtained through simulations run over a 30-day period to gain all the possible orientations. The orbital altitude was varied from 600 km through 1200 km with the results being a function of orbital inclination angles varying from 20 deg through 100 deg and pointing half-angles of I0 deg through 40 deg. To compare the validity of the simulations, Jet Propulsion Laboratory granted the use of the TOPEX satellite. The TOPEX satellite was configured to emulate a spin-stabilized antenna with its communications antenna stowed in the zenith-pointing direction. This mimicked the antenna pointing spin-stabilized satellite in the simulations. To make valid comparisons, the TOPEX orbital parameters were entered into Satellite Tool Kit and simulated over five test times provided by Jet Propulsion Laboratory.

  18. Discontinuous dynamics with grazing points

    NASA Astrophysics Data System (ADS)

    Akhmet, M. U.; Kıvılcım, A.

    2016-09-01

    Discontinuous dynamical systems with grazing solutions are discussed. The group property, continuation of solutions, continuity and smoothness of solutions are thoroughly analyzed. A variational system around a grazing solution which depends on near solutions is constructed. Orbital stability of grazing cycles is examined by linearization. Small parameter method is extended for analysis of grazing orbits, and bifurcation of cycles is observed in an example. Linearization around an equilibrium grazing point is discussed. The results can be extended on functional differential equations, partial differential equations and others. Appropriate illustrations are depicted to support the theoretical results.

  19. Breaking Points in Quartic Maps

    NASA Astrophysics Data System (ADS)

    Romera, M.; Pastor, G.; Martin, A.; Orue, A. B.; Montoya, F.; Danca, M.-F.

    Dynamical systems, whether continuous or discrete, are used by physicists in order to study nonlinear phenomena. In the case of discrete dynamical systems, one of the most used is the quadratic map depending on a parameter. However, some phenomena can depend alternatively on two values of the same parameter. We use the quadratic map xn+1 = 1 - axn2 when the parameter alternates between two values during the iteration process. In this case, the orbit of the alternate system is the sum of the orbits of two quartic maps. The bifurcation diagrams of these maps present breaking points at which there is an abrupt change in their evolution.

  20. Understanding Acid Rain

    ERIC Educational Resources Information Center

    Damonte, Kathleen

    2004-01-01

    The term acid rain describes rain, snow, or fog that is more acidic than normal precipitation. To understand what acid rain is, it is first necessary to know what an acid is. Acids can be defined as substances that produce hydrogen ions (H+), when dissolved in water. Scientists indicate how acidic a substance is by a set of numbers called the pH…

  1. New bioactive fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many oxygenated fatty acids are bioactive compounds. Nocardia cholesterolicum and Flavobacterium DS5 convert oleic acid to 10 hydroxy stearic acid and linoleic acid to 10-hydroxy-12(Z)-octadecanoic acid. Pseudomonas aeruginosa PR3 converts oleic acid to the new compounds, 7,10-dihydroxy-8(E)-octad...

  2. New Bioactive Fatty Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many oxygenated fatty acids are bioactive compounds. Nocardia cholesterolicum and Flavobacterium DS5 convert oleic acid to 10 hydroxy stearic acid and linoleic acid to 10-hydroxy-12(Z)-octadecanoic acid. Pseudomonas aeruginosa PR3 converts oleic acid to new compounds, 7,10-dihydroxy-8(E)-octadecen...

  3. Satellite dual antenna pointing system

    NASA Technical Reports Server (NTRS)

    Keigler, John E. (Inventor); Hartshorne, Frank A. (Inventor)

    1986-01-01

    A satellite antenna pointing system for separately pointing separated transmit and receive high gain antenna systems includes means for separately and sequentially applying a beacon signal to the transmit and receive antenna systems and a broad beam width antenna which has a coverage area greater than the overall coverage region of the spot beam antenna systems. The system includes ground stations located at or near the periphery of the overall coverage region adapted to receive these beacon signals. At a central control station these beacon signals are compared to provide first signals proportional to the ratio of said beacon signals received from said transmit antenna system and said broad beam width antenna and second signals proportional to the ratio of said beacon signals received from said satellite receive antenna system and said broad beam width antenna. The central station generates from said first signals transmit antenna control signals which are sent to the satellite to control the orientation of said transmit antenna system. Likewise, the central control station generates from the second signals receiver antenna control signals which are applied to the satellite to control the orientation of the satellite receive antenna system.

  4. Early Time Points Perfusion Imaging

    PubMed Central

    Kwong, Kenneth K.; Reese, Timothy G.; Nelissen, Koen; Wu, Ona; Chan, Suk-Tak; Benner, Thomas; Mandeville, Joseph B.; Foley, Mary; Vanduffel, Wim; Chesler, David A.

    2010-01-01

    The aim was to investigate the feasibility of making relative cerebral blood flow (rCBF) maps from MR images acquired with short TR by measuring the initial arrival amount of Gd-DTPA evaluated within a time window before any contrast agent has a chance to leave the tissue. We named this rCBF measurement technique utilizing the early data points of the Gd-DTPA bolus the “early time points” method (ET), based on the hypothesis that early time point signals were proportional to rCBF. Simulation data were used successfully to examine the ideal behavior of ET while monkey’s MRI results offered encouraging support to the utility of ET for rCBF calculation. A better brain coverage for ET could be obtained by applying the Simultaneous Echo Refocusing (SER) EPI technique. A recipe to run ET was presented, with attention paid to the noise problem around the time of arrival (TOA) of the contrast agent. PMID:20851196

  5. Extended Automatic Pointing Assistive Program--A Pointing Assistance Program to Help People with Developmental Disabilities Improve Their Pointing Efficiency

    ERIC Educational Resources Information Center

    Shih, Ching-Hsiang; Li, Chia-Chun; Shih, Ching-Tien; Lin, Kun-Tsan; Lo, Ching-Shui

    2010-01-01

    The latest research adopted software technology to improve pointing performance is through an Automatic Pointing Assistive Program (APAP). However, APAP has some limitations. This study evaluated whether two children with developmental disabilities would be able to improve their pointing performance through an Extended Automatic Pointing Assistive…

  6. Points of regulation for auxin action.

    PubMed

    Zazimalova, E; Napier, R M

    2003-03-01

    There have been few examples of the application of our growing knowledge of hormone action to crop improvement. In this review we discuss what is known about the critical points regulating auxin action. We examine auxin metabolism, transport, perception and signalling and identify genes and proteins that might be keys to regulation, particularly the rate-limiting steps in various pathways. Certain mutants show that substrate flow in biosynthesis can be limiting. To date there is little information available on the genes and proteins of catabolism. There have been several auxin transport proteins and some elegant transport physiology described recently, and the potential for using transport proteins to manage free indole-3-acetic acid (IAA) concentrations is discussed. Free IAA is very mobile, and so while it may be more practical to control auxin action through managing the receptor and signalling pathways, the candidate genes and proteins through which this can be done remain largely unknown. From the available evidence, it is clear that the reason for so few commercial applications arising from the control of auxin action is that knowledge is still limited. PMID:12789411

  7. Regioselective chemical modification of monoclonal antibodies

    DOEpatents

    Ranadive, G.; Rozenzweig, H.S.; Epperly, M.; Bloomer, W.

    1993-05-04

    A method is presented of selectively modifying an immunoglobulin having at least one Fab region and at least one Fc region. Each region has an isoelectric point where the isoelectric point of the Fab fragment of the immunoglobulin is different from the isoelectric point of the Fc fragment of the immunoglobulin. The method comprises of a modification of the immunoglobulin at a pH between the respective isoelectric points of the Fab and Fc fragments of the immunoglobulin.

  8. Regioselective chemical modification of monoclonal antibodies

    DOEpatents

    Ranadive, Girish; Rosenzweig, Howard S.; Epperly, Michael; Bloomer, William

    1993-01-01

    A method of selectively modifying an immunoglobulin having at least one Fab region and at least one Fc region, each region having an isoelectric point wherein said isoelectric point of the Fab fragment of said immunoglobulin is different than the isoelectric point of the Fc fragment of the immunoglobulin, said method comprising modification of the immunoglobulin at a pH between the respective isoelectric points of the Fab and Fc fragments of the immunoglobulin.

  9. Coulometric titration of acids in non-aqueous solvents.

    PubMed

    Fritz, J S; Gainer, F E

    1968-09-01

    Coulometric titrations of mineral acids, sulphonic acids, carboxylic acids, enols, imides and phenols have been carried out in t-butanol or in acetone with electrically generated tetrabutylammonium hydroxide. Either a potentiometric titration or a visual indicator end-point may be used. The amount of acid titrated ranges from 10 to 60 muequiv, and the precision and accuracy of the method are excellent. PMID:18960387

  10. Synthesis of Hydroxymethylenebisphosphonic Acid Derivatives in Different Solvents.

    PubMed

    Nagy, Dávid Illés; Grün, Alajos; Garadnay, Sándor; Greiner, István; Keglevich, György

    2016-01-01

    The syntheses of hydroxymethylenebisphosphonic acid derivatives (dronic acid derivatives) starting from the corresponding substituted acetic acids and P-reagents, mainly phosphorus trichloride and phosphorous acid are surveyed according to the solvents applied. The nature of the solvent is a critical point due to the heterogeneity of the reaction mixtures. This review sheds light on the optimum choice and ratio of the P-reactants, and on the optimum conditions. PMID:27529200

  11. Point Source Location Sensitivity Analysis

    NASA Astrophysics Data System (ADS)

    Cox, J. Allen

    1986-11-01

    This paper presents the results of an analysis of point source location accuracy and sensitivity as a function of focal plane geometry, optical blur spot, and location algorithm. Five specific blur spots are treated: gaussian, diffraction-limited circular aperture with and without central obscuration (obscured and clear bessinc, respectively), diffraction-limited rectangular aperture, and a pill box distribution. For each blur spot, location accuracies are calculated for square, rectangular, and hexagonal detector shapes of equal area. The rectangular detectors are arranged on a hexagonal lattice. The two location algorithms consist of standard and generalized centroid techniques. Hexagonal detector arrays are shown to give the best performance under a wide range of conditions.

  12. Dioxin. A case in point.

    PubMed

    Tedeschi, L G

    1980-06-01

    The dangers posed by herbicidal chemicals to organisms and the environment are pointed out in this review of their development beginning in World War II. In particular, the toxic results of the spraying of dioxin (Agent Orange) in Vietnam are detailed, with corroborating data from clinical and field studies and reports of related industrial accidents. Despite a recent ban imposed on all dioxin-containing herbicides by the Environmental Protection Agency, widespread domestic use of herbicides continues and acceptance of the dangers is incomplete. As a result, forensic scientists must remain alert to potential hazards stemming from the use of these contaminants in their communities at least until strict federal regulatory legislation can be enacted. PMID:7246507

  13. Correlations in avalanche critical points.

    PubMed

    Cerruti, Benedetta; Vives, Eduard

    2009-07-01

    Avalanche dynamics and related power-law statistics are ubiquitous in nature, arising in phenomena such as earthquakes, forest fires, and solar flares. Very interestingly, an analogous behavior is associated with many condensed-matter systems, such as ferromagnets and martensites. Bearing it in mind, we study the prototypical random-field Ising model at T=0. We find a finite correlation between waiting intervals and the previous avalanche size. This correlation is not found in other models for avalanches but it is experimentally found in earthquakes and in forest fires. Our study suggests that this effect occurs in critical points that are at the end of a first-order discontinuity separating two regimes: one with high activity from another with low activity. PMID:19658651

  14. Discovering new points of differentiation.

    PubMed

    MacMillan, I C; McGrath, R G

    1997-01-01

    Most profitable strategies are built on differentiation: offering customers something they value that competitors don't have. But most companies concentrate only on their products or services. In fact, a company can differentiate itself every point where it comes in contact with its customers--from the moment customers realize they need a product or service to the time when they dispose of it. The authors believe that if companies open up their thinking to their customer's entire experience with a product or service--the consumption chain--they can uncover opportunities to position their offerings in ways that neither they nor their competitors though possible. The authors show how even a mundane product such as candles can be successfully differentiated. By analyzing its customers' experiences and exploring various options, Blyth Industries, for example, has grown from a $2 million U.S. candle manufacturer into a global candle and accessory business with nearly $500 million in sales and a market value of $1.2 billion. Finding ways to differentiate one's company is a skill that can be nurtured, the authors contend. In this Manager's Tool Kit, they have designed a two-part approach that can help companies continually identify new points of differentiation and develop the ability to generate successful differentiation strategies. "Mapping the Consumption Chain" captures the customer's total experience with a product or service. "Analyzing Your Customer's Experience" shows managers how directed brainstorming about each step in the consumption chain can elicit numerous ways to differentiate any offering. PMID:10168333

  15. Four-Point-Latching Microactuator

    NASA Technical Reports Server (NTRS)

    Toda, Risaku; Yang, Eui-Hyeok

    2008-01-01

    An experimental inchworm-type linear microactuator is depicted. This microactuator is a successor to one described in "MEMS-Based Piezoelectric/Electrostatic Inchworm Actuator" (NPO-30672), NASA Tech Briefs, Vol. 27, No. 6 (June 2003), page 68. Both actuators are based on the principle of using a piezoelectric transducer operated in alternation with electrostatically actuated clutches to cause a slider to move in small increments. However, the design of the present actuator incorporates several improvements over that of the previous one. The most readily apparent improvement is in geometry and, consequently, in fabrication: In the previous actuator, the inchworm motion was perpendicular to the broad faces of a flat silicon wafer on which the actuator was fabricated, and fabrication involved complex processes to form complex three-dimensional shapes in and on the wafer. In the present actuator, the inchworm motion is parallel to the broad faces of a wafer on which it is fabricated. The components needed to produce the in-plane motion are nearly planar in character and, consequently, easier to fabricate. Other advantages of the present design are described, including that the previous actuator contained two clutches (denoted 'holders' in the cited prior article), the present actuator contains four clutches. The operational sequence of the previous two-clutch actuator is similar. However, the two-clutch configuration is susceptible to tilt of the slider and a consequent large increase in drag. Hence, the primary operational advantages of the present four-point-latching design over the prior two-point-latching design are less drag and greater control robustness arising from greater stability of the orientation of the slider.

  16. The Herschel Point Source Catalogue

    NASA Astrophysics Data System (ADS)

    Marton, Gabor; Schulz, Bernhard; Altieri, Bruno; Calzoletti, Luca; Kiss, Csaba; Lim, Tanya; Lu, Nanyao; Paladini, Roberta; Papageorgiou, Andreas; Pearson, Chris; Rector, John; Shupe, David; Valtchanov, Ivan; Verebélyi, Erika; Xu, Kevin

    2015-08-01

    The Herschel Space Observatory was the fourth cornerstone mission in the European Space Agency (ESA) science programme with excellent broad band imaging capabilities in the submillimetre and far-infrared part of the spectrum. Although the spacecraft finished its observations in 2013, it left a large legacy dataset that is far from having been fully scrutinized and still has potential for new scientific discoveries. This is specifically true for the photometric observations of the PACS and SPIRE instruments that scanned >10% of the sky at 70, 100, 160, 250, 350 and 500 microns. Some source catalogs have already been produced by individual observing programs, but there are many observations that would never be analyzed for their full source content. To maximize the science return of the SPIRE and PACS data sets, our international team of instrument experts is in the process of building the Herschel Point Source Catalog (HPSC) from all scan map observations. Our homogeneous source extraction enables a systematic and unbiased comparison of sensitivity across the different Herschel fields that single programs will generally not be able to provide. The extracted point sources will contain individual YSOs of our Galaxy, unresolved YSO clusters in resolved nearby galaxies and unresolved galaxies of the local and distant Universe that are related to star formation. Such a huge dataset will help scientists better understand the evolution from interstellar clouds to individual stars. Furthermore the analysis of stellar clusters and the star formation on galactic scales will add more details to the understanding of star formation laws through time.We present our findings on comparison of different source detection and photometric tools. First results of the extractions are shown along with the description of our pipelines and catalogue entries. We also provide an additional science product, the structure noise map, that is used for the quality assessment of the catalogue in

  17. University Students' Grasp of Inflection Points

    ERIC Educational Resources Information Center

    Tsamir, Pessia; Ovodenko, Regina

    2013-01-01

    This paper describes university students' grasp of inflection points. The participants were asked what inflection points are, to mark inflection points on graphs, to judge the validity of related statements, and to find inflection points by investigating (1) a function, (2) the derivative, and (3) the graph of the derivative. We found four…

  18. Accurate pointing of tungsten welding electrodes

    NASA Technical Reports Server (NTRS)

    Ziegelmeier, P.

    1971-01-01

    Thoriated-tungsten is pointed accurately and quickly by using sodium nitrite. Point produced is smooth and no effort is necessary to hold the tungsten rod concentric. The chemically produced point can be used several times longer than ground points. This method reduces time and cost of preparing tungsten electrodes.

  19. 47 CFR 90.467 - Dispatch points.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... radio station operated from a dispatch point or points shall assume full responsibility for the use and... comply with the policy: (a) A dispatch point may be linked to the transmitter(s) being operated by... telephone position in the public, switched, telephone network will be treated as a dispatch point within...

  20. 49 CFR 236.738 - Detector, point.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Detector, point. 236.738 Section 236.738... Detector, point. A circuit controller which is part of the switch operating mechanism and operated by a rod connected to a switch, derail or movable point frog to indicate that the point is within a...