Sample records for gigas purification crystallization

  1. Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

    PubMed

    Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

    The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Purification, crystal growth and characterization of CdSe single crystals

    NASA Astrophysics Data System (ADS)

    Burger, A.; Henderson, D. O.; Morgan, S. H.; Silberman, E.

    1991-02-01

    CdSe single crystals have been grown from the stoichiometric melt and from Se rich solutions. Here we report the first mid and far infrared spectra of CdSe crystals free of any known impurity bands. Previous studies of the lattice vibrational properties of CdSe crystals have shown the presence of two bands at 538 and 270 cm -1. Modifications in the purification and crystal growth conditions lead us to assign these two bands to a sulfur impurity. Low temperature photoluminescence spectra are also presented and discussed.

  3. Purification of a Multidrug Resistance Transporter for Crystallization Studies

    PubMed Central

    Alegre, Kamela O.; Law, Christopher J.

    2015-01-01

    Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

  4. Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas).

    PubMed

    Fu, Xue-Yan; Xue, Chang-Hu; Miao, Ben-Chun; Liang, Jun-Ni; Li, Zhao-Jie; Cui, Feng-Xia

    2006-02-08

    Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.

  5. Protein purification and crystallization artifacts: The tale usually not told.

    PubMed

    Niedzialkowska, Ewa; Gasiorowska, Olga; Handing, Katarzyna B; Majorek, Karolina A; Porebski, Przemyslaw J; Shabalin, Ivan G; Zasadzinska, Ewelina; Cymborowski, Marcin; Minor, Wladek

    2016-03-01

    The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building. © 2016 The Protein Society.

  6. GigaDB: announcing the GigaScience database.

    PubMed

    Sneddon, Tam P; Li, Peter; Edmunds, Scott C

    2012-07-12

    With the launch of GigaScience journal, here we provide insight into the accompanying database GigaDB, which allows the integration of manuscript publication with supporting data and tools. Reinforcing and upholding GigaScience's goals to promote open-data and reproducibility of research, GigaDB also aims to provide a home, when a suitable public repository does not exist, for the supporting data or tools featured in the journal and beyond.

  7. GigaDB: announcing the GigaScience database

    PubMed Central

    2012-01-01

    With the launch of GigaScience journal, here we provide insight into the accompanying database GigaDB, which allows the integration of manuscript publication with supporting data and tools. Reinforcing and upholding GigaScience’s goals to promote open-data and reproducibility of research, GigaDB also aims to provide a home, when a suitable public repository does not exist, for the supporting data or tools featured in the journal and beyond. PMID:23587345

  8. Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

    PubMed Central

    Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

    2011-01-01

    Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

  9. The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

    1998-01-01

    Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

  10. Fast and scalable purification of a therapeutic full-length antibody based on process crystallization.

    PubMed

    Smejkal, Benjamin; Agrawal, Neeraj J; Helk, Bernhard; Schulz, Henk; Giffard, Marion; Mechelke, Matthias; Ortner, Franziska; Heckmeier, Philipp; Trout, Bernhardt L; Hekmat, Dariusch

    2013-09-01

    The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies. Copyright © 2013 Wiley Periodicals, Inc.

  11. TlBr purification and single crystal growth for the detector applications

    NASA Astrophysics Data System (ADS)

    Kozlov, Vasilij; Heikkilä, Mikko; Kostamo, Pasi; Lipsanen, Harri; Leskelä, Markku

    2011-05-01

    The combination of distillation, Bridgman-Stockbarger, hydrothermal recrystallisation and travelling molten zone (TMZ) methods were used for TlBr purification. Grown crystals were characterised by XRD rocking curve and FTIR spectroscopy methods, and by electrical measurements made from 200 to 300 K.

  12. From Egg to Crystal: A Practical on Purification, Characterization, and Crystallization of Lysozyme for Bachelor Students

    ERIC Educational Resources Information Center

    Olieric, Vincent; Schreiber, Angelique; Lorber, Bernard; Putz, Joern

    2007-01-01

    A practical hands-on course encompassing enzyme purification, biochemical characterization, and crystallization that completed the course work of 350 second-year bachelor students enrolled in molecular biology/biochemistry was given at the Universite Louis Pasteur of Strasbourg (France). The experimental part of the practical dealt entirely with…

  13. Purification, growth, and characterization of Zn(x)Cd(1-x)Se crystals

    NASA Astrophysics Data System (ADS)

    Silberman, E.; Burger, A.; Chen, W.; Henderson, D. O.; Morgan, S. H.; Springer, John M.; Yao, Y.

    The purification of starting materials which were used in the growth of Zn(x)Cd(1-x)Se (x = 0.2) single crystals using the traveling solution method (TSM) is reported. Up to 13 cm long single crystals and as grown resistivities of 6 x 10(exp 12) ohm/cm could be achieved. Infrared and Raman spectra of Zn(0.2)Cd(0.8)Se are also presented and discussed.

  14. Purification, growth, and characterization of Zn(x)Cd(1-x)Se crystals

    NASA Technical Reports Server (NTRS)

    Silberman, E.; Burger, A.; Chen, W.; Henderson, D. O.; Morgan, S. H.; Springer, John M.; Yao, Y.

    1989-01-01

    The purification of starting materials which were used in the growth of Zn(x)Cd(1-x)Se (x = 0.2) single crystals using the traveling solution method (TSM) is reported. Up to 13 cm long single crystals and as grown resistivities of 6 x 10(exp 12) ohm/cm could be achieved. Infrared and Raman spectra of Zn(0.2)Cd(0.8)Se are also presented and discussed.

  15. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  16. Purification, crystallization and preliminary X-ray diffraction analysis of royal palm tree (Roystonea regia) peroxidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Watanabe, Leandra; Nascimento, Alessandro S.; Zamorano, Laura S.

    2007-09-01

    The purification, crystallization, X-ray diffraction data acquisition and molecular-replacement results of royal palm tree (R. regia) peroxidase are described. Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005 ▶), Biomacromolecules, 6, 1360–1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 116.83, c = 92.24 Å, and contain one protein molecule per asymmetric unit. The V{sub M} value and solvent content are 4.07 Å{sup 3} Da{sup −1} and 69.8%, respectively.« less

  17. Electromigration process for the purification of molten silicon during crystal growth

    DOEpatents

    Lovelace, Alan M. Administrator of the National Aeronautics and Space; Shlichta, Paul J.

    1982-01-01

    A process for the purification of molten materials during crystal growth by electromigration of impurities to localized dirty zones. The process has particular applications for silicon crystal growth according to Czochralski techniques and edge-defined film-fed growth (EFG) conditions. In the Czochralski crystal growing process, the impurities are electromigrated away from the crystallization interface by applying a direct electrical current to the molten silicon for electromigrating the charged impurities away from the crystal growth interface. In the EFG crystal growth process, a direct electrical current is applied between the two faces which are used in forming the molten silicon into a ribbon. The impurities are thereby migrated to one side only of the crystal ribbon. The impurities may be removed or left in place. If left in place, they will not adversely affect the ribbon when used in solar collectors. The migration of the impurity to one side only of the silicon ribbon is especially suitable for use with asymmetric dies which preferentially crystallize uncharged impurities along one side or face of the ribbon.

  18. Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lane, Michael Douglas; Nam, Hyun-Joo; Padron, Eric

    2005-06-01

    The production, purification, crystallization and preliminary X-ray crystallographic analysis of adeno-associated virus serotype 8 is reported. Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Åmore » resolution using synchrotron radiation and belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.« less

  19. Large-scale crystallization of proteins for purification and formulation.

    PubMed

    Hekmat, Dariusch

    2015-07-01

    Since about 170 years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.

  20. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    PubMed Central

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  1. Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

    PubMed Central

    Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

    1993-01-01

    A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

  2. Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin.

    PubMed

    Jaimohan, S M; Naresh, M D; Arumugam, V; Mandal, A B

    2009-10-01

    Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemoglobins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 A resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 A, beta = 109.35 degrees . Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

  3. An effective method for thallium bromide purification and research on crystal properties

    NASA Astrophysics Data System (ADS)

    Zheng, Zhiping; Meng, Fang; Gong, Shuping; Quan, Lin; Wang, Jing; Zhou, Dongxiang

    2012-06-01

    Thallium bromide (TlBr) is a promising candidate for room-temperature X- and gamma-ray detectors in view of its excellent intrinsic features. However, material purity and crystal quality concerns still limit the use of TlBr crystals as detectors. In this work, a combination of hydrothermal recrystallization (HR) and vacuum distillation (VD) methods were applied to purify TlBr salts prior to crystal growth. Trace impurities at the ppb/ppm level were determined by inductively coupled plasma mass spectroscopy (ICP-MS). The results showed that the impurity concentrations of the TlBr salt decreased significantly after HR and VD purification, and high performance of the resultant TlBr crystal in areas such as electrical and optical properties was achieved. The combination of HR and VD methods could fabricate purer material, with an order of magnitude higher resistivity and better optical quality, than HR or VD method used separately. The possible technological considerations affecting the parameters of the crystals are investigated.

  4. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombicmore » space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.« less

  5. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  6. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lu, Feifei; Gao, Feng; Li, Honglin

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

  7. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, R.K.; Bowman, K.A.; Mazgaj, R.M.; Cochran, C.N.

    1983-10-25

    A method is described for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm. 2 figs.

  8. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, Robert K.; Bowman, Kenneth A.; Mazgaj, Robert M.; Cochran, C. Norman

    1983-10-25

    A method for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm.

  9. Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin

    PubMed Central

    Jaimohan, S. M.; Naresh, M. D.; Arumugam, V.; Mandal, A. B.

    2009-01-01

    Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemo­globins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 Å resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 Å, β = 109.35°. Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit. PMID:19851014

  10. GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the pacific oyster Crassostrea gigas.

    PubMed

    Riviere, Guillaume; Klopp, Christophe; Ibouniyamine, Nabihoudine; Huvet, Arnaud; Boudry, Pierre; Favrel, Pascal

    2015-12-02

    The Pacific oyster, Crassostrea gigas, is one of the most important aquaculture shellfish resources worldwide. Important efforts have been undertaken towards a better knowledge of its genome and transcriptome, which makes now C. gigas becoming a model organism among lophotrochozoans, the under-described sister clade of ecdysozoans within protostomes. These massive sequencing efforts offer the opportunity to assemble gene expression data and make such resource accessible and exploitable for the scientific community. Therefore, we undertook this assembly into an up-to-date publicly available transcriptome database: the GigaTON (Gigas TranscriptOme pipeliNe) database. We assembled 2204 million sequences obtained from 114 publicly available RNA-seq libraries that were realized using all embryo-larval development stages, adult organs, different environmental stressors including heavy metals, temperature, salinity and exposure to air, which were mostly performed as part of the Crassostrea gigas genome project. This data was analyzed in silico and resulted into 56621 newly assembled contigs that were deposited into a publicly available database, the GigaTON database. This database also provides powerful and user-friendly request tools to browse and retrieve information about annotation, expression level, UTRs, splice and polymorphism, and gene ontology associated to all the contigs into each, and between all libraries. The GigaTON database provides a convenient, potent and versatile interface to browse, retrieve, confront and compare massive transcriptomic information in an extensive range of conditions, tissues and developmental stages in Crassostrea gigas. To our knowledge, the GigaTON database constitutes the most extensive transcriptomic database to date in marine invertebrates, thereby a new reference transcriptome in the oyster, a highly valuable resource to physiologists and evolutionary biologists.

  11. Purification and crystal growth of NPB via imidazolium based ionic liquids

    NASA Astrophysics Data System (ADS)

    Oh, Yong-Taeg; Shin, Dong-Chan

    2018-04-01

    Here we report the production of high purity and crystallinity organic electronic material of NPB (N,N‧-Di-[(1-naphthyl)-N,N‧-diphenyl]-1,1‧-biphenyl-4,4‧-diamine (C44H32N2) through solution recrystallization within imidazolium based ionic liquids. When low purity NPB was recrystallized at 170 °C within C8MIM[TFSI], its purity was drastically improved from 82% to 99.92%. These recrystallized NPB crystals showed 0.040° FWHM (Full Width Half Maximum) of X-ray (1 1 1) diffraction peak. Such small FWHM angle indicates single-crystal like crystallinity. Initial NPB powder was dissolved at 100 °C and recrystallized at temperature above 110 °C. At higher temperature of 170 °C, a small number of bigger crystals were formed compared to those at 110 °C. This can be well explained by the classical nucleation and growth theory. Therefore, solution recrystallization process using ionic liquid might be promising for mass production of organic electronic materials by replacing the widely-used sublimation purification method.

  12. Purification of proteins from solutions containing residual host cell proteins via preparative crystallization.

    PubMed

    Hekmat, Dariusch; Breitschwerdt, Peter; Weuster-Botz, Dirk

    2015-09-01

    To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP). Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%. Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.

  13. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    DOE PAGES

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; ...

    2014-08-20

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

  14. Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi

    CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

  15. Purification, characterization and crystallization of the human 80S ribosome

    PubMed Central

    Khatter, Heena; Myasnikov, Alexander G.; Mastio, Leslie; Billas, Isabelle M. L.; Birck, Catherine; Stella, Stefano; Klaholz, Bruno P.

    2014-01-01

    Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work. PMID:24452798

  16. Purification of organic nonlinear optical materials for bulk crystal growth from melt

    NASA Astrophysics Data System (ADS)

    Gebre, Tesfaye; Bhat, Kamala N.; Batra, Ashok K.; Lal, Ravindra B.; Aggarwal, Mohan D.; Penn, Benjamin G.; Frazier, Donald O.

    2002-10-01

    The techniques developed for purification of nonlinear optical organic materials, such as benzil, 2-methyl-4-nitroaniline (MNA), Dicyanovinyl anisole (DIVA) and its derivatives, nitrophenyl prolinol (NPP) and other Schiff's base compounds, include Kugelrohy method, physical vapor transport, zone refining and recrystallization from the solvent are described. Purity of the materials is tested using differential thermal analysis, gas chromatograph/Mass detector, Fourier Transform Infrared spectroscopy and melting point measurements. The purified materials were later used in the growth of single crystal by Bridgman-Stockbarger and Czochralski techniques.

  17. Effect of additives on the purification of urease

    NASA Astrophysics Data System (ADS)

    Yu, X.; Wang, J.; Ulrich, J.

    2015-12-01

    The effect of additives on the purification of proteins was investigated. The target protein studied here is the enzyme urease. Studies on the purification of urease from jack bean meal were carried out. 32% (v/v) acetone was utilized to extract urease from the jack bean meal. Further purification by crystallization with the addition of 2-mercaptoethanol and EDTA disodium salt dehydrate was carried out. It was found out that the presence of additives can affect the selectivity of the crystallization. Increases in both purity and yield of the urease after crystallization were observed in the presence of additives, which were proven using both SDS-PAGE and activity. Urease crystals with a yield of 69.9% and a purity of 85.1% were obtained in one crystallization step in the presence of additives. Furthermore, the effect of additives on the thermodynamics and kinetics of urease crystallization was studied.

  18. GigaDB: promoting data dissemination and reproducibility

    PubMed Central

    Sneddon, Tam P.; Si Zhe, Xiao; Edmunds, Scott C.; Li, Peter; Goodman, Laurie; Hunter, Christopher I.

    2014-01-01

    Often papers are published where the underlying data supporting the research are not made available because of the limitations of making such large data sets publicly and permanently accessible. Even if the raw data are deposited in public archives, the essential analysis intermediaries, scripts or software are frequently not made available, meaning the science is not reproducible. The GigaScience journal is attempting to address this issue with the associated data storage and dissemination portal, the GigaScience database (GigaDB). Here we present the current version of GigaDB and reveal plans for the next generation of improvements. However, most importantly, we are soliciting responses from you, the users, to ensure that future developments are focused on the data storage and dissemination issues that still need resolving. Database URL: http://www.gigadb.org PMID:24622612

  19. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

    PubMed

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

    TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

  20. Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Garrote, Ana M.; Redondo, Pilar; Montoya, Guillermo, E-mail: gmontoya@cnio.es

    2014-02-19

    The C-terminal kinase domain of TLK2 (a human tousled-like kinase) has been cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- ormore » hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å{sup 3} Da{sup −1} and a solvent content of 73.23%.« less

  1. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

    PubMed

    Jagadeesan, G; Malathy, P; Gunasekaran, K; Harikrishna Etti, S; Aravindhan, S

    2014-11-01

    Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

  2. Purification, crystallization and preliminary X-ray diffraction of the C-terminal bromodomain from human BRD2

    PubMed Central

    Umehara, Takashi; Wakamori, Masatoshi; Tanaka, Akiko; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2007-01-01

    BRD2 is a bromodomain-containing BET-family protein that associates with acetylated histones throughout the cell cycle. Although the tertiary structures of the bromodomains involved in histone acetyl transfer are already known, the structures of the BET-type bromodomains, which are required for tight association with acetylated chromatin, are poorly understood. Here, the expression, purification and crystallization of the C-terminal bromodomain of human BRD2 are reported. The protein was crystallized by the sitting-drop vapour-diffusion method in the orthorhombic space group P21212, with unit-cell parameters a = 71.78, b = 52.60, c = 32.06 Å and one molecule per asymmetric unit. The crystal diffracted beyond 1.80 Å resolution using synchrotron radiation. PMID:17620725

  3. Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Renzi, Fabiana; Panetta, Gianna; Vallone, Beatrice

    Recombinant His-tagged XendoU, a eukaryotic endoribonuclease, appeared to aggregate in the presence of divalent cations. Monodisperse protein which yielded crystals diffracting to 2.2 Å was obtained by addition of EDTA. XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potentialmore » drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3{sub 1}21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.« less

  4. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jagadeesan, G.; Malathy, P.; Gunasekaran, K.

    2014-10-25

    The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to themore » trigonal system P3{sub 1}21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.« less

  5. Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas).

    PubMed

    Perfetto, Rosa; Del Prete, Sonia; Vullo, Daniela; Sansone, Giovanni; Barone, Carmela M A; Rossi, Mosè; Supuran, Claudiu T; Capasso, Clemente

    2017-08-28

    The carbonic anhydrase (CA, EC 4.2.1.1) superfamily of metalloenzymes catalyzes the hydration of carbon dioxide to bicarbonate and protons. The catalytically active form of these enzymes incorporates a metal hydroxide derivative, the formation of which is the rate-determining step of catalytic reaction, being affected by the transfer of a proton from a metal-coordinated water molecule to the environment. Here, we report the cloning, expression, and purification of a particular CA, i.e., nacrein-like protein encoded in the genome of the Pacific oyster Magallana gigas (previously known as Crassostrea gigas ). Furthermore, the amino acid sequence, kinetic constants, and anion inhibition profile of the recombinant enzyme were investigated for the first time. The new protein, CgiNAP2X1, is highly effective as catalyst for the CO₂ hydration reaction, based on the measured kinetic parameters, i.e., k cat = 1.0 × 10⁶ s -1 and k cat / K M = 1.2 × 10⁸ M -1 ·s -1 . CgiNAP2X1 has a putative signal peptide, which probably allows an extracellular localization of the protein. The inhibition data demonstrated that the best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with K I s in the range of 76-87 μM. These studies may add new information on the physiological role of the molluskan CAs in the biocalcification processes.

  6. High-level Expression Purification Crystallization and Preliminary X-ray Crystallographic Studies of the Receptor Binding Domain of botulinum neurotoxin Serotype D

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Y Zhang; X Gao; G Buchko

    2011-12-31

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less

  7. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Y.; Robinson, H.; Gao, X.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8,more » b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.« less

  8. Expression, Purification, Crystallization of Two Major Envelope Proteins from White Spot Syndrome Virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang,X.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapor-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 Mmore » sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 {angstrom} resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 {angstrom}. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 {angstrom}, and diffracts to 2.0 {angstrom} resolution.« less

  9. Synthesis, purification and bulk crystal growth of radiation detector materials using melt growth technique

    NASA Astrophysics Data System (ADS)

    Surabhi, Raja Rahul Reddy

    In the past decade, there has been new and increased usage of radiation-detection technologies for applications in homeland security, non-proliferation, and national defense. Most of these applications require a portable device with high gamma-ray energy resolution and detection efficiency, compact size, room-temperature operation, and low cost. Consequently, there is a renewed understanding of the material limitations for these technologies and a great demand to develop next-generation radiation-detection materials that can operate at room temperature. Mercuric iodide (HgI2), Lead iodide (PbI2), and CdZnTe (CZT) are the current leading candidates for radiation detector applications. This is because of their high atomic number and large band gap that makes them particularly well suited for fabrication of high resolution and high efficiency compact devices. PbI2 is a promising material for room temperature nuclear radiation detectors, characterized by its wide band gap (EG=2.32eV) and high-density (rho=6.2g/cm3). It has been reported that PbI2 crystal detectors are able to detect gamma-ray in the range of 1KeV-1MeV, with good energy resolution. However, PbI 2 detectors have not been studied in detail because of non-availability of high quality single crystals. This study presents the synthesis, purification, growth and characterization of PbI2 single crystals grown. In this research, solid-state synthesis technique has been utilized for obtaining PbI2 as a starting material. For the first time, a unique low-temperature purification technique has been developed to obtain high-purity starting material. The crystals were grown using 2-zone Bridgman-Stockbarger (B.S) technique wherein growth rate and temperature gradient at the solid-liquid interface were optimized. Single crystals of PbI2 were successfully grown in quartz glass ampoule under different growth conditions. Material purity was determined by measuring the elemental concentration using the Inductively

  10. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gupta, Vibha; Gupta, Rakesh K.; Ram Lal Anand College, University of Delhi, Benito Juarez Road, New Delhi 110021

    2008-05-01

    The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein productmore » was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4{sub 2}, with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å.« less

  11. Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

    PubMed

    Plaga, W; Lottspeich, F; Oesterhelt, D

    1992-04-01

    An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

  12. Stochasticity of convection in Giga-LES data

    NASA Astrophysics Data System (ADS)

    De La Chevrotière, Michèle; Khouider, Boualem; Majda, Andrew J.

    2016-09-01

    The poor representation of tropical convection in general circulation models (GCMs) is believed to be responsible for much of the uncertainty in the predictions of weather and climate in the tropics. The stochastic multicloud model (SMCM) was recently developed by Khouider et al. (Commun Math Sci 8(1):187-216, 2010) to represent the missing variability in GCMs due to unresolved features of organized tropical convection. The SMCM is based on three cloud types (congestus, deep and stratiform), and transitions between these cloud types are formalized in terms of probability rules that are functions of the large-scale environment convective state and a set of seven arbitrary cloud timescale parameters. Here, a statistical inference method based on the Bayesian paradigm is applied to estimate these key cloud timescales from the Giga-LES dataset, a 24-h large-eddy simulation (LES) of deep tropical convection (Khairoutdinov et al. in J Adv Model Earth Syst 1(12), 2009) over a domain comparable to a GCM gridbox. A sequential learning strategy is used where the Giga-LES domain is partitioned into a few subdomains, and atmospheric time series obtained on each subdomain are used to train the Bayesian procedure incrementally. Convergence of the marginal posterior densities for all seven parameters is demonstrated for two different grid partitions, and sensitivity tests to other model parameters are also presented. A single column model simulation using the SMCM parameterization with the Giga-LES inferred parameters reproduces many important statistical features of the Giga-LES run, without any further tuning. In particular it exhibits intermittent dynamical behavior in both the stochastic cloud fractions and the large scale dynamics, with periods of dry phases followed by a coherent sequence of congestus, deep, and stratiform convection, varying on timescales of a few hours consistent with the Giga-LES time series. The chaotic variations of the cloud area fractions were

  13. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  14. Expression, purification, crystallization and preliminary X-ray diffraction analysis of α-11 giardin from Giardia lamblia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pathuri, Puja; Nguyen, Emily Tam; Luecke, Hartmut, E-mail: hudel@uci.edu

    2006-11-01

    α-11 giardin from the intestinal protozoan parasite, G. lamblia has been cloned, expressed, purified and crystallized under two different conditions and in two different space groups. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group and crystals obtained in the second condition diffracted to 2.93 Å and belong to a primitive monoclinic space group. α-11 Giardin, a protein from the annexin superfamily, is a 35.0 kDa protein from the intestinal protozoan parasite Giardia lamblia which triggers a form of diarrhea called giardiasis. Here, the cloning, expression, purification and the crystallization of α-11more » giardin under two different conditions and in two different space groups is reported. Crystals from the first condition diffracted to 1.1 Å and belong to a primitive orthorhombic space group, while crystals from the second condition, which included calcium in the crystallization solution, diffracted to 2.93 Å and belong to a primitive monoclinic space group. Determination of the detailed atomic structure of α-11 giardin will provide a better insight into its biological function and might establish whether this class of proteins is a potential drug target against giardiasis.« less

  15. Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

    NASA Technical Reports Server (NTRS)

    Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

    2004-01-01

    Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

  16. Screening of detergents for solubilization, purification and crystallization of membrane proteins: a case study on succinate:ubiquinone oxidoreductase from Escherichia coli.

    PubMed

    Shimizu, Hironari; Nihei, Coh-ichi; Inaoka, Daniel Ken; Mogi, Tatushi; Kita, Kiyoshi; Harada, Shigeharu

    2008-09-01

    Succinate:ubiquinone oxidoreductase (SQR) was solubilized and purified from Escherichia coli inner membranes using several different detergents. The number of phospholipid molecules bound to the SQR molecule varied greatly depending on the detergent combination that was used for the solubilization and purification. Crystallization conditions were screened for SQR that had been solubilized and purified using 2.5%(w/v) sucrose monolaurate and 0.5%(w/v) Lubrol PX, respectively, and two different crystal forms were obtained in the presence of detergent mixtures composed of n-alkyl-oligoethylene glycol monoether and n-alkyl-maltoside. Crystallization took place before detergent phase separation occurred and the type of detergent mixture affected the crystal form.

  17. Purification, crystallization and preliminary crystallographic studies of the TLDc domain of oxidation resistance protein 2 from zebrafish

    PubMed Central

    Alsarraf, Husam M. A. B.; Laroche, Fabrice; Spaink, Herman; Thirup, Søren; Blaise, Mickael

    2011-01-01

    Cell metabolic processes are constantly producing reactive oxygen species (ROS), which have deleterious effects by triggering, for example, DNA damage. Numerous enzymes such as catalase, and small compounds such as vitamin C, provide protection against ROS. The TLDc domain of the human oxidation resistance protein has been shown to be able to protect DNA from oxidative stress; however, its mechanism of action is still not understood and no structural information is available on this domain. Structural information on the TLDc domain may therefore help in understanding exactly how it works. Here, the purification, crystallization and preliminary crystallographic studies of the TLDc domain from zebrafish are reported. Crystals belonging to the orthorhombic space group P21212 were obtained and diffracted to 0.97 Å resolution. Selenomethionine-substituted protein could also be crystallized; these crystals diffracted to 1.1 Å resolution and the structure could be solved by SAD/MAD methods. PMID:22102041

  18. Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

    NASA Astrophysics Data System (ADS)

    Garber, M. B.; Agalarov, S. Ch.; Eliseikina, I. A.; Sedelnikova, S. E.; Tishchenko, S. V.; Shirokov, V. A.; Yusupov, M. M.; Reshetnikova, L. S.; Trakhanov, S. D.; Tukalo, M. A.; Yaremchuk, A. D.

    1991-03-01

    An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

  19. A biomineralization study of the Indo-Pacific giant clam Tridacna gigas

    NASA Astrophysics Data System (ADS)

    Gannon, M. E.; Pérez-Huerta, A.; Aharon, P.; Street, S. C.

    2017-06-01

    The giant clam, Tridacna gigas, is an important faunal component of reef ecosystems of the Indo-Pacific region. In addition to its ecological role, shells of this bivalve species are useful bioarchives for past climate and environmental reconstructions. However, the biomineralization processes involved in shell aragonite deposition are insufficiently understood. Here, we present a study of the shell microstructure of modern specimens from Palm Island, Great Barrier Reef (GBR), Australia, and Huon Peninsula, Papua New Guinea (PNG), using a combination of petrography, scanning electron microscopy, electron backscatter diffraction, Raman spectroscopy and stable carbon isotope ratios. Daily growth increments were recognizable in all specimens through ontogeny, and counting these growth lines provides a robust specimen age estimate. For the internal layers, paired increments of organized aragonitic needles and compact, oblong crystals were recognized in a specimen from PNG, whereas specimens from GBR were composed of shield-like crystals that were not definable at the microscale. The combination of nutrient availability, rainfall and solar irradiance are likely to be the most significant factors controlling shell growth and may explain the observed differences in microstructure. The external layer, identical in all specimens, was composed of dendritic microstructure that is significantly enriched in 13C compared to the internal layer, suggesting different metabolic controls on layer deposition. We propose that the mineralization of the internal and external layers is independent from each other and associated with the activity of specific mantles. Future studies using T. gigas shells as bioarchives should consider the microstructure as it reflects the environment in which the individual lived and the differences in mineralization pathways of internal and external layers.

  20. Purification, isolation, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 from Drosophila melanogaster

    NASA Astrophysics Data System (ADS)

    Boyko, K. M.; Nikolaeva, A. Yu.; Kachalova, G. S.; Bonchuk, A. N.; Popov, V. O.

    2017-11-01

    The spatial organization of the genome is controlled by a special class of architectural proteins, including proteins containing BTB domains that are able to dimerize or multimerize. The centrosomal protein 190 is one of such architectural proteins. The purification, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 are reported. The crystallization conditions were found by the vapor-diffusion technique. The crystals diffracted to 1.5 Å resolution and belonged to sp. gr. P3221. The structure was solved by the molecular replacement method. The structure refinement is currently underway.

  1. Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4.

    PubMed

    Xu, Xiang; Zhang, Xiaoai; Lu, Guangwen; Cai, Yongping

    2012-08-01

    Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, β = 94.7°. Preliminary analysis of the diffraction data was also performed.

  2. Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4

    PubMed Central

    Xu, Xiang; Zhang, Xiaoai; Lu, Guangwen; Cai, Yongping

    2012-01-01

    Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Å resolution and belonged to space group P21, with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, β = 94.7°. Preliminary analysis of the diffraction data was also performed. PMID:22869128

  3. Screening of detergents for solubilization, purification and crystallization of membrane proteins: a case study on succinate:ubiquinone oxidoreductase from Escherichia coli

    PubMed Central

    Shimizu, Hironari; Nihei, Coh-ichi; Inaoka, Daniel Ken; Mogi, Tatushi; Kita, Kiyoshi; Harada, Shigeharu

    2008-01-01

    Succinate:ubiquinone oxidoreductase (SQR) was solubilized and purified from Escherichia coli inner membranes using several different detergents. The number of phospholipid molecules bound to the SQR molecule varied greatly depending on the detergent combination that was used for the solubilization and purification. Crystallization conditions were screened for SQR that had been solubilized and purified using 2.5%(w/v) sucrose monolaurate and 0.5%(w/v) Lubrol PX, respectively, and two different crystal forms were obtained in the presence of detergent mixtures composed of n-alkyl-oligoethylene glycol monoether and n-alkyl-maltoside. Crystallization took place before detergent phase separation occurred and the type of detergent mixture affected the crystal form. PMID:18765923

  4. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tang, Xuhua; Hew, Choy Leong, E-mail: dbshewcl@nus.edu.sg

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals ofmore » SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.« less

  5. Population structure of the giant garter snake, Thamnophis gigas

    USGS Publications Warehouse

    Paquin, M.M.; Wylie, G.D.; Routman, E.J.

    2006-01-01

    The giant garter snake, Thamnophis gigas, is a threatened species endemic to California's Central Valley. We tested the hypothesis that current watershed boundaries have caused genetic differentiation among populations of T. gigas. We sampled 14 populations throughout the current geographic range of T. gigas and amplified 859 bp from the mitochondrial gene ND4 and one nuclear microsatellite locus. DNA sequence variation from the mitochondrial gene indicates there is some genetic structuring of the populations, with high F ST values and unique haplotypes occurring at high frequency in several populations. We found that clustering populations by watershed boundary results in significant between-region genetic variance for mtDNA. However, analysis of allele frequencies at the microsatellite locus NSU3 reveals very low F ST values and little between-region variation in allele frequencies. The discordance found between mitochondrial and microsatellite data may be explained by aspects of molecular evolution and/or T. gigas life history characteristics. Differences in effective population size between mitochondrial and nuclear DNA, or male-biased gene flow, result in a lower migration rate of mitochondrial haplotypes relative to nuclear alleles. However, we cannot exclude homoplasy as one explanation for homogeneity found for the single microsatellite locus. The mitochondrial nucleotide sequence data supports conservation practices that identify separate management units for T. gigas. ?? Springer 2006.

  6. A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trindade, Inês B.; Fonseca, Bruno M.; Matias, Pedro M.

    The gene encoding a putative siderophore-interacting protein from the marine bacterium S. frigidimarina was successfully cloned, followed by expression and purification of the gene product. Optimized crystals diffracted to 1.35 Å resolution and preliminary crystallographic analysis is promising with respect to structure determination and increased insight into the poorly understood molecular mechanisms underlying iron acquisition. Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI-RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this proteinmore » are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.« less

  7. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gao, Jinlan; Li, Xiaolu; Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People's Republic of China

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGEmore » after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .« less

  8. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  9. Gigas Sulci

    NASA Image and Video Library

    2016-11-03

    Today's VIS image shows a small portion of Gigas Sulci. Located in the Tharsis region, these small linear ridges follow two trends at angle to each other, one toward the upper left corner and the other generally across from right to left. Small dunes are located between the ridges. The ridges were most likely formed by tectonic activity. Orbit Number: 65422 Latitude: 9.32915 Longitude: 231.265 Instrument: VIS Captured: 2016-09-12 16:56 http://photojournal.jpl.nasa.gov/catalog/PIA21157

  10. Secretory immunoglobulin purification from whey by chromatographic techniques.

    PubMed

    Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

    2017-08-15

    Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Heterologous Expression of the Desulfovibrio gigas [NiFe] Hydrogenase in Desulfovibrio fructosovorans MR400

    PubMed Central

    Rousset, Marc; Magro, Valérie; Forget, Nicole; Guigliarelli, Bruno; Belaich, Jean-Pierre; Hatchikian, E. Claude

    1998-01-01

    The ability of Desulfovibrio fructosovorans MR400 ΔhynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon. PMID:9733707

  12. Heterologous expression of the Desulfovibrio gigas [NiFe] hydrogenase in Desulfovibrio fructosovorans MR400.

    PubMed

    Rousset, M; Magro, V; Forget, N; Guigliarelli, B; Belaich, J P; Hatchikian, E C

    1998-09-01

    The ability of Desulfovibrio fructosovorans MR400 DeltahynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.

  13. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DiMattia,M.; Govindasamy, L.; Levy, H.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c =more » 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.« less

  14. Purification, crystallization and preliminary X-ray diffraction studies on goat (Capra hircus) hemoglobin - a low oxygen affinity species.

    PubMed

    Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Balasubramanian, Moovarkumudalvan; Ponnuswamy, Mondikalipudur Nanjappa Gounder

    2009-01-01

    Hemoglobin is a vital protein present in almost all higher species. It is a transport protein involved in carrying oxygen from lungs to tissues and carbon dioxide back to lungs by an intrinsically coordinated manner. Even though a good amount of work has been carried out in this direction there exists scarcity of structural insight on low oxygen affinity species. Attempts are being made to unravel the structural insight of this low oxygen affinity species. Goat blood plasma was collected, treated with EDTA to avoid blood clotting and purification was accomplished using DEAE-anion chromatographic column. The goat hemoglobin was crystallized using 50mM of phosphate buffer at pH 6.7 with 1M NaCl and PEG 3350 as precipitant by hanging drop vapor diffusion method. Crystals obtained are screened and suitable crystals are taken for data collection using mar345dtb as image plate detector system. Goat hemoglobin crystal diffracted up to 2.61 A resolution. Goat hemoglobin crystallizes in orthorhombic space group P212(1)2(1) as a whole biological molecule in the asymmetric unit with cell dimensions a=53.568A, b=67.365A, c=154.183A.

  15. Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus

    PubMed Central

    Tsukazaki, Tomoya; Mori, Hiroyuki; Fukai, Shuya; Numata, Tomoyuki; Perederina, Anna; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo; Vassylyev, Dmitry G.; Nureki, Osamu; Ito, Koreaki

    2006-01-01

    Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 Å upon X-ray irradiation, revealing that they belonged to space group P43212. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 Å resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery. PMID:16582489

  16. Semiconductor grade, solar silicon purification project

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Rosler, R. R.; Thompson, S. W.; Chaney, R. E.

    1979-01-01

    Experimental apparatus and procedures used in the development of a 3-step SiF2(x) polymer transport purification process are described. Both S.S.M.S. and E.S. analysis demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). Recent electrical analysis via crystal growth reveals that the product contains compensated phosphorus and boron. The low projected product cost and short energy payback time suggest that the economics of this process will result in a cost less than the goal of $10/Kg(1975 dollars). The process appears to be readily scalable to a major silicon purification facility.

  17. A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

    PubMed Central

    Vasquez, Hebert Ely; Hashimoto, Kyotaro; Yoshida, Asami; Hara, Kenji; Imai, Chisato Chris; Kitamura, Hitoshi; Satuito, Cyril Glenn

    2013-01-01

    Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et2O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics. PMID:24349261

  18. Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas.

    PubMed

    Wu, Shengjun; Huang, Xiaolian

    2017-02-01

    Oligosaccharides were prepared from Crassostrea gigas by hydrolysis of polysaccharide in C. gigas with peroxide oxygen (H2O2). The hydrolysates were cleared of protein, filtered, ultrafiltered and precipitated with absolute ethanol to give C. gigas oligosaccharides (CGOs). Factors affecting CGO yields, i.e., reaction time, temperature, and H2O2 concentration, were optimised as follows: 2.96h reaction time, 84.71°C reaction temperature, and 2.46% H2O2 concentration. Under these conditions, the maximum yield of CGOs reached 10.61%. The CGOs were then partially characterised by Fourier transform infrared spectroscopy, UV spectroscopy, monosaccharide composition, and antioxidant activities. Results indicate that CGOs possessed strong hydroxyl radical activity, 2,2-diphenyl-β-picrylhydrazyl-radical-scavenging activity and reducing capacity at a concentration of 100μg/mL. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Advancing Genomics through the Global Invertebrate Genomics Alliance (GIGA).

    PubMed

    Voolstra, Christian R; Wörheide, Gert; Lopez, Jose V

    2017-03-01

    The Global Invertebrate Genomics Alliance (GIGA), a collaborative network of diverse scientists, marked its second anniversary with a workshop in Munich, Germany, where international attendees focused on discussing current progress, milestones and bioinformatics resources. The community determined the recruitment and training talented researchers as one of the most pressing future needs and identified opportunities for network funding. GIGA also promotes future research efforts to prioritize taxonomic diversity and create new synergies. Here, we announce the generation of a central and simple data repository portal with a wide coverage of available sequence data, via the compagen platform, in parallel with more focused and specialized organism databases to globally advance invertebrate genomics. Therefore this article serves the objectives of GIGA by disseminating current progress and future prospects in the science of invertebrate genomics with the aim of promotion and facilitation of interdisciplinary and international research.

  20. Purification of silicon for photovoltaic applications

    NASA Astrophysics Data System (ADS)

    Delannoy, Yves

    2012-12-01

    Solar grade silicon, as a starting material for crystallization to produce solar cells, is discussed here in terms of impurities whose maximum content is estimated from recent literature and conferences. A review of the production routes for each category of solar-grade silicon (undoped, compensated or heavily compensated) is proposed with emphasis on the metallurgical route. Some recent results are proposed concerning segregation, showing that directional solidification systems can be used for solidification even at high solidification rate (15 cm/h). Results on inductive plasma purification, where boron is evacuated as HBO in a gas phase blown from an inductive plasma torch, are shown to apply as well to arc plasmas and purification by moist gas. Special attention is paid to the history of impurities in the purification processes, showing that impure auxiliary phases (silicon tetrachloride, slag, aluminum, etc.) often need their own purification process to enable their recycling, which has to be considered to evaluate the cost (financial, energetic and environmental) of the purification route.

  1. Water purification using organic salts

    DOEpatents

    Currier, Robert P.

    2004-11-23

    Water purification using organic salts. Feed water is mixed with at least one organic salt at a temperature sufficiently low to form organic salt hydrate crystals and brine. The crystals are separated from the brine, rinsed, and melted to form an aqueous solution of organic salt. Some of the water is removed from the aqueous organic salt solution. The purified water is collected, and the remaining more concentrated aqueous organic salt solution is reused.

  2. Advancing Genomics through the Global Invertebrate Genomics Alliance (GIGA)

    PubMed Central

    Voolstra, Christian R.; Wörheide, Gert; Lopez, Jose V.

    2017-01-01

    The Global Invertebrate Genomics Alliance (GIGA), a collaborative network of diverse scientists, marked its second anniversary with a workshop in Munich, Germany, where international attendees focused on discussing current progress, milestones and bioinformatics resources. The community determined the recruitment and training talented researchers as one of the most pressing future needs and identified opportunities for network funding. GIGA also promotes future research efforts to prioritize taxonomic diversity and create new synergies. Here, we announce the generation of a central and simple data repository portal with a wide coverage of available sequence data, via the compagen platform, in parallel with more focused and specialized organism databases to globally advance invertebrate genomics. Therefore this article serves the objectives of GIGA by disseminating current progress and future prospects in the science of invertebrate genomics with the aim of promotion and facilitation of interdisciplinary and international research. PMID:28603454

  3. Purification, crystallization and preliminary X-ray analysis of phycocyanin and phycoerythrin from Porphyra yezoensis Ueda

    PubMed Central

    Cai, Chuner; Wu, Lian; Li, Chunxia; He, Peimin; Li, Jie; Zhou, Jiahai

    2011-01-01

    Porphyra yezoensis is one of the most important and widely cultured seaweeds in China. Phycobiliproteins exhibit excellent spectroscopic properties and play versatile roles in the biomedical, food, cosmetics and chemical synthetic dye industries. Here, the purification and crystallization of phycoerythrin and phycocyanin, two phycobiliproteins extracted from P. yezoensis, are described. Using a novel protocol including co-precipitation with ammonium sulfate and hydroxyapatite column chromatography, both phycobiliproteins were produced on a large scale with improved quality and yield compared with those previously reported. Native PAGE analysis indicated that phycoerythrin and phycocyanin exist as (αβ)3 heterohexamers in solution. The crystals of phycoerythrin diffracted to 2.07 Å resolution and belonged to space group R3. The unit-cell parameters referred to hexagonal axes are a = b = 187.7, c = 59.7 Å, with nine (αβ)2 heterotetramers per unit cell. The crystals of phycocyanin diffracted to 2.70 Å resolution in space group P21. Matthews coefficient analysis shows that 10–19 (αβ) heterodimers of phycocyanin in the asymmetric unit would be reasonable. A self-rotation function calculation clarified this ambiguity and indicated that 12 (αβ) heterodimers of phycocyanin are assembled in the asymmetric unit. PMID:21543866

  4. Systems Harmonization and Convergence - the GIGAS Approach

    NASA Astrophysics Data System (ADS)

    Marchetti, P. G.; Biancalana, A.; Coene, Y.; Uslander, T.

    2009-04-01

    0.1 Background The GIGAS1 Support Action promotes the coherent and interoperable development of the GMES, INSPIRE and GEOSS initiatives through their concerted adoption of standards, protocols, and open architectures. 0.2 Preparing for Coordinated Data Access The GMES Coordinated Data Access System is under design and implementation2. This objective has motivated the definition of the interoperability standards between the contributing missions. The following elements have been addressed with associated papers submitted to OGC: The EO Product Metadata has been based on the OGC Geographic Markup Language, addressing sensor characteristics for optical, radar and atmospheric products. Collection and service discovery: an ISO extension package for CSW ebRim has been proposed. Catalogue Service (CSW): an Earth Observation extension package of the CSW ebRim has been proposed. Feasibility Analysis and Order: an Order interface control document and an Earth Observation profile of the Sensor Planning Service have been proposed. Online Data Access: an Earth Observation profile of the Web Map Services (WMS) for visualization and evaluation purposes has been proposed. Identity (user) management: the objective in the long term is to allow for a single sign-on to the Coordinated Data Access system by users registered in the various Earth Observation ground segments by providing a federated identity across participating ground segments, exploiting OASIS standards. 0.3 The GIGAS proposed harmonization approach The approach proposed by GIGAS is based on three elements: Technology watch Comparative analysis Shaping of initiatives and standards This paper concentrates on the methodology for technology watch and comparative analysis. The complexity of the GIGAS scenario involving huge systems (i.e. GEOSS, INSPIRE, GMES etc.) entails the interaction with different heterogeneous partners, each with a specific competence, expertise and know-how. 0.3.1 Technology watch The methodology

  5. Neuroprotective and Cognitive Enhancement Potentials of Angelica gigas Nakai Root: A Review

    PubMed Central

    Sowndhararajan, Kandhasamy; Kim, Songmun

    2017-01-01

    Angelica gigas Nakai is an important medicinal plant with health promoting properties that is used to treat many disorders. In traditional herbal medicine, the root of this plant is used to promote blood flow, to treat anemia, and is used as sedative or tonic agent. The root contains various bioactive metabolites; in particular, decursin and decursinol (pyranocoumarin type components) have been reported to possess various pharmacological properties. Recently, several in vitro and in vivo studies have reported that the crude extracts and isolated components from the root of A. gigas exhibited neuroprotective and cognitive enhancement effects. Neuronal damage or death is the most important factor for many neurodegenerative diseases. In addition, recent studies have clearly demonstrated the possible mechanisms behind the neuroprotective action of extracts/compounds from the root of A. gigas. In the present review, we summarized the neuroprotective and cognitive enhancement effects of extracts and individual compounds from A. gigas root. PMID:28452965

  6. Neuroprotective and Cognitive Enhancement Potentials of Angelica gigas Nakai Root: A Review.

    PubMed

    Sowndhararajan, Kandhasamy; Kim, Songmun

    2017-04-28

    Angelica gigas Nakai is an important medicinal plant with health promoting properties that is used to treat many disorders. In traditional herbal medicine, the root of this plant is used to promote blood flow, to treat anemia, and is used as sedative or tonic agent. The root contains various bioactive metabolites; in particular, decursin and decursinol (pyranocoumarin type components) have been reported to possess various pharmacological properties. Recently, several in vitro and in vivo studies have reported that the crude extracts and isolated components from the root of A. gigas exhibited neuroprotective and cognitive enhancement effects. Neuronal damage or death is the most important factor for many neurodegenerative diseases. In addition, recent studies have clearly demonstrated the possible mechanisms behind the neuroprotective action of extracts/compounds from the root of A. gigas . In the present review, we summarized the neuroprotective and cognitive enhancement effects of extracts and individual compounds from A. gigas root.

  7. Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huché, Frédéric, E-mail: huche@pasteur.fr; Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15; Delepelaire, Philippe

    2006-01-01

    The expression, purification, and crystallization in space group P2{sub 1}2{sub 1}2{sub 1} of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å. Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized.more » Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å.« less

  8. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fulton, Zara; Crellin, Paul K.; Brammananth, Rajini

    2008-05-28

    Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5.more » A complete data set was collected to 1.8 {angstrom} resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.« less

  9. [Extraction, isolation and purification for ginkgolide B].

    PubMed

    Zhang, Chenfeng; Li, Minghui; Tang, Yun; Zhang, Yanhai; Shi, Min; Sheng, Longsheng

    2010-08-01

    To establish a simple extraction, isolation and purification method for ginkgolide B from ginkgo leaf. The optimum conditions of extraction, isolation and purification were studied by taking the transfer rate of ginkgolide B as index. Ginkgo leaf was extracted with 70% ethanol for three times, the extracts were concentrated to remove ethanol and diluted by water till the crude drug density reached 0.1 g x mL(-1). The dilution was adsorbed with HPD-450 macroporous resin. The impurities were eluted with 20% ethanol and ginkgolide B was eluted with 80% ethanol. Then the 80% ethanol eluant was concentrated and crystallized. Finally the crude crystals were recrystallized with isopropanol. The purity of the ginkgolide B recrystallization was 95%. The process was stable and easy to operate, which was suited to industrialized production.

  10. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fulton, Zara; Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800; Crellin, Paul K.

    2008-05-01

    MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicatedmore » in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.« less

  11. Innovative uses of GigaPan Technology for Onsite and Distance Education

    NASA Astrophysics Data System (ADS)

    Bentley, C.; Schott, R. C.; Piatek, J. L.; Richards, B.

    2013-12-01

    GigaPans are gigapixel panoramic images that can be viewed at a wide range of magnifications, allowing users to explore them in various degrees of detail from the smallest scale to the full image extent. In addition to panoramic images captured with the GigaPan camera mount ('Dry Falls' - http://www.gigapan.com/gigapans/89093), users can also upload annotated images (For example, 'Massanutten sandstone slab with trace fossils (annotated)', http://www.gigapan.com/gigapans/124295) and satellite images (For example, 'Geology vs. Topography - State of Connecticut', http://www.gigapan.com/gigapans/111265). Panoramas with similar topics have been gathered together on the site in galleries, both user-generated and site-curated (For example, http://www.gigapan.com/galleries?categories=geology&page=1). Further innovations in display technology have also led to the development of improved viewers (for example, the annotations in the image linked above can be explored via paired viewers at http://coursecontent.nic.edu/bdrichards/gigapixelimages/callanview) GigaPan panoramas can be created through use of the GigaPan robotic camera mount and a digital camera (different models of the camera mount are available and work with a wide range of cameras). The camera mount can be used to create high-resolution pans ranging in scale from hand sample to outcrop up to landscape via the stitching software included with the robotic mount. The software can also be used to generate GigaPan images from other sources, such as thin section or satellite images, so these images can also be viewed with the online viewer. GigaPan images are typically viewed via a web-based interface that allows the user to interact with the image from the limits of the image detail up to the full panorama. After uploading, information can be added to panoramas with both text captions and geo-referencing (geo-located panoramas can then be viewed in Google Earth). Users can record specific locations and zoom levels in

  12. Molecular and functional characterization of a fads2 orthologue in the Amazonian teleost, Arapaima gigas.

    PubMed

    Lopes-Marques, Mónica; Ozório, Rodrigo; Amaral, Ricardo; Tocher, Douglas R; Monroig, Óscar; Castro, L Filipe C

    2017-01-01

    The Brazilian teleost Arapaima gigas is an iconic species of the Amazon. In recent years a significant effort has been put into the farming of arapaima to mitigate overfishing threats. However, little is known regarding the nutritional requirements of A. gigas in particular those for essential fatty acids including the long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ability to biosynthesize LC-PUFA is dependent upon the gene repertoire of fatty acyl desaturases (Fads) and elongases (Elovl), as well as their fatty acid specificities. In the present study we characterized both molecularly and functionally an orthologue of the desaturase fatty acid desaturase 2 (fads2) from A. gigas. The isolated sequence displayed the typical desaturase features, a cytochrome b 5 -domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. Functional characterization of A. gigas fads2 showed that, similar to other teleosts, the A. gigas fads2 exhibited a predominant Δ6 activity complemented with some capacity for Δ8 desaturation. Given that A. gigas belongs to one of the oldest teleostei lineages, the Osteoglossomorpha, these findings offer a significant insight into the evolution LC-PUFA biosynthesis in teleosts. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. GIGA: a simple, efficient algorithm for gene tree inference in the genomic age.

    PubMed

    Thomas, Paul D

    2010-06-09

    Phylogenetic relationships between genes are not only of theoretical interest: they enable us to learn about human genes through the experimental work on their relatives in numerous model organisms from bacteria to fruit flies and mice. Yet the most commonly used computational algorithms for reconstructing gene trees can be inaccurate for numerous reasons, both algorithmic and biological. Additional information beyond gene sequence data has been shown to improve the accuracy of reconstructions, though at great computational cost. We describe a simple, fast algorithm for inferring gene phylogenies, which makes use of information that was not available prior to the genomic age: namely, a reliable species tree spanning much of the tree of life, and knowledge of the complete complement of genes in a species' genome. The algorithm, called GIGA, constructs trees agglomeratively from a distance matrix representation of sequences, using simple rules to incorporate this genomic age information. GIGA makes use of a novel conceptualization of gene trees as being composed of orthologous subtrees (containing only speciation events), which are joined by other evolutionary events such as gene duplication or horizontal gene transfer. An important innovation in GIGA is that, at every step in the agglomeration process, the tree is interpreted/reinterpreted in terms of the evolutionary events that created it. Remarkably, GIGA performs well even when using a very simple distance metric (pairwise sequence differences) and no distance averaging over clades during the tree construction process. GIGA is efficient, allowing phylogenetic reconstruction of very large gene families and determination of orthologs on a large scale. It is exceptionally robust to adding more gene sequences, opening up the possibility of creating stable identifiers for referring to not only extant genes, but also their common ancestors. We compared trees produced by GIGA to those in the TreeFam database, and they

  14. Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

    PubMed

    van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

    2015-06-01

    Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marland, Zara; Beddoe, Travis; Zaker-Tabrizi, Leyla

    2005-12-01

    A lipoprotein implicated in mycobacterial cell-wall biosynthesis, LpqW, was expressed in E. coli. Crystals were obtained that diffracted to 2.4 Å resolution. Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acidmore » lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%(w/v) PEG 4000, 8–16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way.« less

  16. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein

    PubMed Central

    Rahaman, Siti Nurulnabila A.; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

    2016-01-01

    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (V M) value of 2.27 Å3 Da−1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%. PMID:26919524

  17. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

    PubMed

    Rahaman, Siti Nurulnabila A; Mat Yusop, Jastina; Mohamed-Hussein, Zeti-Azura; Ho, Kok Lian; Teh, Aik-Hong; Waterman, Jitka; Ng, Chyan Leong

    2016-03-01

    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

  18. GIGA: a simple, efficient algorithm for gene tree inference in the genomic age

    PubMed Central

    2010-01-01

    Background Phylogenetic relationships between genes are not only of theoretical interest: they enable us to learn about human genes through the experimental work on their relatives in numerous model organisms from bacteria to fruit flies and mice. Yet the most commonly used computational algorithms for reconstructing gene trees can be inaccurate for numerous reasons, both algorithmic and biological. Additional information beyond gene sequence data has been shown to improve the accuracy of reconstructions, though at great computational cost. Results We describe a simple, fast algorithm for inferring gene phylogenies, which makes use of information that was not available prior to the genomic age: namely, a reliable species tree spanning much of the tree of life, and knowledge of the complete complement of genes in a species' genome. The algorithm, called GIGA, constructs trees agglomeratively from a distance matrix representation of sequences, using simple rules to incorporate this genomic age information. GIGA makes use of a novel conceptualization of gene trees as being composed of orthologous subtrees (containing only speciation events), which are joined by other evolutionary events such as gene duplication or horizontal gene transfer. An important innovation in GIGA is that, at every step in the agglomeration process, the tree is interpreted/reinterpreted in terms of the evolutionary events that created it. Remarkably, GIGA performs well even when using a very simple distance metric (pairwise sequence differences) and no distance averaging over clades during the tree construction process. Conclusions GIGA is efficient, allowing phylogenetic reconstruction of very large gene families and determination of orthologs on a large scale. It is exceptionally robust to adding more gene sequences, opening up the possibility of creating stable identifiers for referring to not only extant genes, but also their common ancestors. We compared trees produced by GIGA to those in

  19. Anti-tumor activities of decursinol angelate and decursin from Angelica gigas.

    PubMed

    Lee, Sanghyun; Lee, Yeon Sil; Jung, Sang Hoon; Shin, Kuk Hyun; Kim, Bak-Kwang; Kang, Sam Sik

    2003-09-01

    The in vivo anti-tumor activities of decursinol angelate (1) and decursin (2) isolated from the roots of Angelica gigas were investigated. These two compounds, when administered consecutively for 9 days at 50 and 100 mg/kg i.p. in mice, caused a significant increase in the life span and a significant decrease in the tumor weight and volume of mice inoculated with Sarcoma-180 tumor cells. These results suggest that decursinol angelate (1) and decursin (2) from A. gigas have anti-tumor activities.

  20. Fishery biology of jumbo flying squid Dosidicus gigas off Costa Rica Dome

    NASA Astrophysics Data System (ADS)

    Chen, Xinjun; Li, Jianghua; Liu, Bilin; Li, Gang; Lu, Huajie

    2014-06-01

    The jumbo flying squid ( Dosidicus gigas) population was surveyed with the help of Chinese squid jigging vessels off the Costa Rica Dome (4°-11°N, 90°-100°W) in 2009 and 2010. The daily catch of D. gigas in the two survey cruises ranged from 0 to 5.5 t and was mostly obtained from the areas bounded by 6°-9°N and 91°-94°W and by 6°30'-7°30'N and 96°-97°W. The sea surface temperature in the areas yielding the most catch ranged from 27.5 to 29°C. The sex ratio of the total catch was 3.75:1 (female: male). The mantle length of the squid ranged from 211 to 355 mm (male) and from 204 to 429 mm (female) with an average of 297.9 and 306.7 mm, respectively. In the relationship of the mantle length (mm) and body weight (g) of the squid, there was no significant difference between sexes. The female and male were at a similar maturity, and most individuals are maturing or have matured with a few females being spent. The size (mantle length) and age at the first sexual maturity were 297 mm and 195 d in females, and less than 211 mm and 130 d in males, respectively. Most of the sampled stomachs (70.6%) had no food remains. The major preys of the squids were fish, cephalopods and crustaceans, with the most abundant Myctophum orientale and D. gigas. The preys in more than 65% of the non-empty sampled stomachs evidenced the cannibalism of D. gigas. The results improved current understanding of the fishery biology of D. gigas off the Costa Rica Dome, which may facilitate the assessment and management of relative fishery resources.

  1. GigaPan Technology to Enhance In-Class and In-Field Learning in Community College Settings

    NASA Astrophysics Data System (ADS)

    Villalobos, J. I.; Bentley, C.

    2014-12-01

    Community college students account for over 40% of all undergraduates in the United States as well as the majority of minority and non-traditional students attending undergraduate courses. Implementing innovative, cost effective, and formative pedagogies to the diverse backgrounds of students that typically enroll at a community is often a challenge. Interactive pedagogies in geology pose a unique challenge considering that students gain the most long-term knowledge when topics covered in a course are exposed to them in outdoor settings where they are allowed to explore and make connections. The ability to expose students to real world examples is challenging to many community college faculty considering that that many; lack funds or means for transportation of students, do not have administrative support on such endeavors, teach evening or night classes, or have a high percentage of students who are physically limited or have obligations to work and family. A joint collaborative between El Paso Community College (EPCC) and Northern Virginia Community College (NOVA) has explored the usage of GigaPan technology to create multi-layered online material to minimize these issues faced by many community college faculty and students. The primary layer of the online material is GigaPans of local geological sites that highlight large-scale structures in the El Paso, Texas region that are commonly used in local field trips and lab book material. The second layer is of Macro-GigaPans of hand samples of key outcrops from the primarily GigaPans which facilitate student learning, exploration, and ability to make connections by exploring smaller scale features of the primary layer. A third layer of online material, GigaPans of thin sections of hand samples (from secondary layers), and curriculum based on the GigaPans was then created to assist students in evaluating proposed hypotheses on the primary layers' geological origin. GigaPan cirriculum was utilized in introductory

  2. Mitochondrial genome of the endangered marine gastropod Strombus gigas Linnaeus, 1758 (Mollusca: Gastropoda).

    PubMed

    Márquez, Edna J; Castro, Erick R; Alzate, Juan F

    2016-01-01

    The queen conch Strombus gigas is an endangered marine gastropod of significant economic importance across the Greater Caribbean region. This work reports for the first time the complete mitochondrial genome of S. gigas, obtained by FLX 454 pyrosequencing. The mtDNA genome encodes for 13 proteins, 22 tRNAs and 2 ribosomal RNAs. In addition, the coding sequences and gene synteny were similar to other previously reported mitogenomes of gastropods.

  3. Initial Data of Digital Correlation ECE with a Giga Hertz Sampling Digitizer

    NASA Astrophysics Data System (ADS)

    Tsuchiya, Hayato; Inagaki, Shigeru; Tokuzawa, Tokihiko; Nagayama, Yoshio

    2015-03-01

    The proposed Digital Correlation ECE (DCECE) technique is applied in Large Helical Device. DCECE is realized by the use of the Giga Hertz Sampling Digitizer. The waveform of intermediate frequency band of ECE, whose frequency is several giga hertz, can be discretized and saved directly. The discretized IF data can be used for the analysis of correlation ECE with arbitrary parameter of spatial resolution and temporal resolution. In this paper, the characteristic of DCECE and initial Data in LHD is introduced.

  4. Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

    PubMed

    Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

    2011-02-01

    Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

  5. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level withmore » high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.« less

  6. Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rong, Hui; Li, Yan; Lou, Xiao-hua

    2007-02-01

    A novel cardiotoxin-like basic protein from Naja naja atra was crystallized and diffraction data were collected to 2.35 Å resolution. A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data setmore » was collected to 2.35 Å resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 43.2, c = 147.9 Å. There are two molecules in the crystallographic asymmetric unit.« less

  7. Identification of the Molecular Clockwork of the Oyster Crassostrea gigas

    PubMed Central

    Perrigault, Mickael; Tran, Damien

    2017-01-01

    Molecular clock system constitutes the origin of biological rhythms that allow organisms to anticipate cyclic environmental changes and adapt their behavior and physiology. Components of the molecular clock are largely conserved across a broad range of species but appreciable diversity in clock structure and function is also present especially in invertebrates. The present work aimed at identify and characterize molecular clockwork components in relationship with the monitoring of valve activity behavior in the oyster Crassostrea gigas. Results provided the characterization of most of canonical clock gene including clock, bmal/cycle, period, timeless, vertebrate-type cry, rev-erb, ror as well as other members of the cryptochrome/photolyase family (plant-like cry, 6–4 photolyase). Analyses of transcriptional variations of clock candidates in oysters exposed to light / dark regime and to constant darkness led to the generation of a putative and original clockwork model in C. gigas, intermediate of described systems in vertebrates and insects. This study is the first characterization of a mollusk clockwork. It constitutes essential bases to understand interactions of the different components of the molecular clock in C. gigas as well as the global mechanisms associated to the generation and the synchronization of biological rhythms in oysters. PMID:28072861

  8. Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

    PubMed

    Worobec, E A; Martin, N L; McCubbin, W D; Kay, C M; Brayer, G D; Hancock, R E

    1988-04-07

    A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

  9. Shell shape variation of queen conch Strombus gigas (Mesograstropoda: Strombidae) from Southwest Caribbean.

    PubMed

    Márquez, Edna Judith; Restrepo-Escobar, Natalia; Montoya-Herrera, Francisco Luis

    2016-12-01

    The endangered species Strombus gigas is a marine gastropod of significant economic importance through the Greater Caribbean region. In contrast to phenotypic plasticity, the role of genetics on shell variations in S. gigas has not been addressed so far, despite its importance in evolution, management and conservation of this species. This work used geometric morphometrics to investigate the phenotypic variation of 219 shells of S. gigas from eight sites of the Colombian Southwest Caribbean. Differences in mean size between sexes and among sites were contrasted by analysis of variance. Allometry was tested by multivariate regression and the hypothesis of common slope was contrasted by covariance multivariate analysis. Differences in the shell shape among sites were analyzed by principal component analysis. Sexual size dimorphism was not significant, whereas sexual shape dimorphism was significant and variable across sites. Differences in the shell shape among sites were concordant with genetic differences based on microsatellite data, supporting its genetic background. Besides, differences in the shell shape between populations genetically similar suggest a role of phenotypic plasticity in the morphometric variation of the shell shape. These outcomes evidence the role of genetic background and phenotypic plasticity in the shell shape of S. gigas. Thus, geometric morphometrics of shell shape may constitute a complementary tool to explore the genetic diversity of this species.

  10. Toxic effects of heavy metal Cu2+ on the pacific oyster Crassostrea gigas

    NASA Astrophysics Data System (ADS)

    Gao, Ceng; Zhang, Xinxin; Li, Xiumei; Tang, Xuexi

    2017-05-01

    The effects of different concentrations of heavy metal ions on the survival of the Pacific oyster Crassostrea gigas were studied by using experimental ecology method in 96 h. The results showed that the LC50 of copper ion was 21.748mg/L and the safe concentration was 2.1748mg/L mg/L. Under the condition of laboratory, under laboratory conditions, the research of Cu2+ Stress on the C. gigas gill and digestive gland and adductor muscle tissue SOD, GPx and the induction of CAT activity. The results showed that the activities of SOD, GPx and CAT in the C. gigas were significantly changed by copper ion + stress. The results showed that in the low concentration Cu2+ treatment could induce the three kinds of enzymes, in the high concentration Cu2+ treatment group, SOD and CAT and GPx on the inhibition of the effect. The sensitivity of the three antioxidant enzymes to copper ion showed a certain difference. The sensitivity of the three kinds of tissue enzymes to Cu2+ treatment was digestive gland> fascia> gill. The experimental results show that the single factor for copper in water pollutants, the C. gigas digestive gland tissue SOD, GPX and CAT activity has certain significance to it, but will it as index applied to the actual water need further study.

  11. A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

    PubMed Central

    Trindade, Inês B.; Fonseca, Bruno M.; Matias, Pedro M.; Louro, Ricardo O.; Moe, Elin

    2016-01-01

    Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing. PMID:27599855

  12. Cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase and pyranocoumarin biosynthesis in Angelica gigas.

    PubMed

    Park, Jee Hee; Park, Nam Il; Xu, Hui; Park, Sang Un

    2010-08-27

    Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.

  13. The quorum-quenching lactonase from Alicyclobacter acidoterrestris : purification, kinetic characterization, crystallization and crystallographic analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bergonzi, Celine; Schwab, Michael; Chabriere, Eric

    Lactonases comprise a class of enzymes that hydrolyze lactones, including acyl-homoserine lactones (AHLs); the latter are used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases have therefore been demonstrated to quench AHL-based bacterial communication. In particular, lactonases are capable of inhibiting bacterial behaviors that depend on these chemicals, such as the formation of biofilms or the expression of virulence factors. A novel representative from the metallo-β-lactamase superfamily, named AaL, was isolated from the thermoacidophilic bacteriumAlicyclobacter acidoterrestris. Kinetic characterization proves AaL to be a proficient lactonase, with catalytic efficiencies (k cat/K m) against AHLs in the region of 10 5Mmore » -1s -1. AaL exhibits a very broad substrate specificity. Its structure is expected to reveal the molecular determinants for its substrate binding and specificity, as well as to provide grounds for future protein-engineering projects. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection of AaL at 1.65Å resolution are reported.« less

  14. Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pegos, Vanessa R.; Hey, Louis; LaMirande, Jacob

    Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP fromPolaromonasJS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently knownmore » PBPs. Here, it is shown that the PBP fromPolaromonasJS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP fromPolaromonasJS666 are reported.« less

  15. The Global Invertebrate Genomics Alliance (GIGA): Developing Community Resources to Study Diverse Invertebrate Genomes

    PubMed Central

    2014-01-01

    Over 95% of all metazoan (animal) species comprise the “invertebrates,” but very few genomes from these organisms have been sequenced. We have, therefore, formed a “Global Invertebrate Genomics Alliance” (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture. PMID:24336862

  16. Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai

    NASA Astrophysics Data System (ADS)

    Seo, Hye-Young; Kim, Jun-Hyoung; Song, Hyun-Pa; Kim, Dong-Ho; Byun, Myung-Woo; Kwon, Joog-Ho; Kim, Kyong-Su

    2007-11-01

    The study was carried out to determine the effects of gamma irradiation on the volatile flavor components including essential oils, of Angelica gigas Nakai. The volatile organic compounds from non- and irradiated A. gigas Nakai at doses of 1, 3, 5, 10 and 20 kGy were extracted by a simultaneous steam distillation and extraction (SDE) method and identified by GC/MS analysis. A total of 116 compounds were identified and quantified from non- and irradiated A. gigas Nakai. The major volatile compounds were identified 2,4,6-trimethyl heptane, α-pinene, camphene, α-limonene, β-eudesmol, α-murrolene and sphatulenol. Among these compounds, the amount of essential oils in non-irradiated sample were 77.13%, and the irradiated samples at doses of 1, 3, 5, 10 and 20 kGy were 84.98%, 83.70%, 83.94%, 82.84% and 82.58%, respectively. Oxygenated terpenes such as β-eudesmol, α-eudesmol, and verbenone were increased after irradiation but did not correlate with the irradiation dose. The yields of active substances such as essential oil were increased after irradiation; however, the yields of essential oils and the irradiation dose were not correlated. Thus, the profile of composition volatiles of A. gigas Nakai did not change with irradiation.

  17. The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes.

    PubMed

    Bracken-Grissom, Heather; Collins, Allen G; Collins, Timothy; Crandall, Keith; Distel, Daniel; Dunn, Casey; Giribet, Gonzalo; Haddock, Steven; Knowlton, Nancy; Martindale, Mark; Medina, Mónica; Messing, Charles; O'Brien, Stephen J; Paulay, Gustav; Putnam, Nicolas; Ravasi, Timothy; Rouse, Greg W; Ryan, Joseph F; Schulze, Anja; Wörheide, Gert; Adamska, Maja; Bailly, Xavier; Breinholt, Jesse; Browne, William E; Diaz, M Christina; Evans, Nathaniel; Flot, Jean-François; Fogarty, Nicole; Johnston, Matthew; Kamel, Bishoy; Kawahara, Akito Y; Laberge, Tammy; Lavrov, Dennis; Michonneau, François; Moroz, Leonid L; Oakley, Todd; Osborne, Karen; Pomponi, Shirley A; Rhodes, Adelaide; Santos, Scott R; Satoh, Nori; Thacker, Robert W; Van de Peer, Yves; Voolstra, Christian R; Welch, David Mark; Winston, Judith; Zhou, Xin

    2014-01-01

    Over 95% of all metazoan (animal) species comprise the "invertebrates," but very few genomes from these organisms have been sequenced. We have, therefore, formed a "Global Invertebrate Genomics Alliance" (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture.

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike proteinmore » is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.« less

  19. A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

    PubMed Central

    Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

    2009-01-01

    Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively. PMID:19851019

  20. Guidelines for the successful generation of protein–ligand complex crystals

    PubMed Central

    Müller, Ilka

    2017-01-01

    With continuous technical improvements at synchrotron facilities, data-collection rates have increased dramatically. This makes it possible to collect diffraction data for hundreds of protein–ligand complexes within a day, provided that a suitable crystal system is at hand. However, developing a suitable crystal system can prove challenging, exceeding the timescale of data collection by several orders of magnitude. Firstly, a useful crystallization construct of the protein of interest needs to be chosen and its expression and purification optimized, before screening for suitable crystallization and soaking conditions can start. This article reviews recent publications analysing large data sets of crystallization trials, with the aim of identifying factors that do or do not make a good crystallization construct, and gives guidance in the design of an expression construct. It provides an overview of common protein-expression systems, addresses how ligand binding can be both help and hindrance for protein purification, and describes ligand co-crystallization and soaking, with an emphasis on troubleshooting. PMID:28177304

  1. Effects of Purification on the Crystallization of Lysozyme

    NASA Technical Reports Server (NTRS)

    Ewing, Felecia L.; Forsythe, Elizabeth L.; Van Der Woerd, Mark; Pusey, Marc L.

    1996-01-01

    We have additionally purified a commercial lysozyme preparation by cation exchange chromatography, followed by recrystallization. This material is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 C. Protein used directly from the bottle, prepared by dialysis against distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by size exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower pHs. The origin of the bundled rod habit was postulated to be a thermally dependent tetragonal- orthorhombic change in the protein structure. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneous forms of lysozyme may be responsible. These results demonstrate three classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic techniques; and (3) heterogeneous forms of the protein, which can be removed in this case by cation exchange chromatography. Of these, heterogeneous forms of the lysozyme apparently have the greatest affect on its crystallization behavior.

  2. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gowda, Giri; Sagurthi, Someswar Rao; Savithri, H. S.

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1},more » with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.« less

  3. Crystallization using reverse micelles and water-in-oil microemulsion systems: the highly selective tool for the purification of organic compounds from complex mixtures.

    PubMed

    Kljajic, Alen; Bester-Rogac, Marija; Klobcar, Andrej; Zupet, Rok; Pejovnik, Stane

    2013-02-01

    The active pharmaceutical ingredient orlistat is usually manufactured using a semi-synthetic procedure, producing crude product and complex mixtures of highly related impurities with minimal side-chain structure variability. It is therefore crucial for the overall success of industrial/pharmaceutical application to develop an effective purification process. In this communication, we present the newly developed water-in-oil reversed micelles and microemulsion system-based crystallization process. Physiochemical properties of the presented crystallization media were varied through surfactants and water composition, and the impact on efficiency was measured through final variation of these two parameters. Using precisely defined properties of the dispersed water phase in crystallization media, a highly efficient separation process in terms of selectivity and yield was developed. Small-angle X-ray scattering, high-performance liquid chromatography, mass spectrometry, and scanning electron microscopy were used to monitor and analyze the separation processes and orlistat products obtained. Typical process characteristics, especially selectivity and yield in regard to reference examples, were compared and discussed. Copyright © 2012 Wiley Periodicals, Inc.

  4. Towards Protein Crystallization as a Process Step in Downstream Processing of Therapeutic Antibodies: Screening and Optimization at Microbatch Scale

    PubMed Central

    Zang, Yuguo; Kammerer, Bernd; Eisenkolb, Maike; Lohr, Katrin; Kiefer, Hans

    2011-01-01

    Crystallization conditions of an intact monoclonal IgG4 (immunoglobulin G, subclass 4) antibody were established in vapor diffusion mode by sparse matrix screening and subsequent optimization. The procedure was transferred to microbatch conditions and a phase diagram was built showing surprisingly low solubility of the antibody at equilibrium. With up-scaling to process scale in mind, purification efficiency of the crystallization step was investigated. Added model protein contaminants were excluded from the crystals to more than 95%. No measurable loss of Fc-binding activity was observed in the crystallized and redissolved antibody. Conditions could be adapted to crystallize the antibody directly from concentrated and diafiltrated cell culture supernatant, showing purification efficiency similar to that of Protein A chromatography. We conclude that crystallization has the potential to be included in downstream processing as a low-cost purification or formulation step. PMID:21966480

  5. Expression, purification and crystallization of Helicobacter pyloril-asparaginase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dhavala, Prathusha; Krasotkina, Julya; Dubreuil, Christine

    2008-08-01

    l-Asparaginase from H. pylori was overexpressed in E. coli, purified and crystallized. The crystals belonged to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule in the asymmetric unit. A complete data set to 1.6 Å resolution was collected using synchrotron radiation. The l-asparaginases from Escherichia coli and Erwinia chrysanthemi are effective drugs that have been used in the treatment of acute childhood lymphoblastic leukaemia for over 30 years. However, despite their therapeutic potential, they can cause serious side effects as a consequence of their intrinsic glutaminase activity, which leadsmore » to l-glutamine depletion in the blood. Consequently, new asparaginases with low glutaminase activity, fewer side effects and high activity towards l-asparagine are highly desirable as better alternatives in cancer therapy. l-Asparaginase from Helicobacter pylori was overexpressed in E. coli and purified for structural studies. The enzyme was crystallized at pH 7.0 in the presence of 16–19%(w/v) PEG 4000 and 0.1 M magnesium formate. Data were collected to 1.6 Å resolution at 100 K from a single crystal at a synchrotron-radiation source. The crystals belong to space group I222, with unit-cell parameters a = 63.6, b = 94.9, c = 100.2 Å and one molecule of l-asparaginase in the asymmetric unit. Elucidation of the crystal structure will provide insight into the active site of the enzyme and a better understanding of the structure–activity relationship in l-asparaginases.« less

  6. Giga-z: A 100,000 Object Superconducting Spectrophotometer for LSST Follow-up

    NASA Astrophysics Data System (ADS)

    Marsden, Danica W.; Mazin, Benjamin A.; O'Brien, Kieran; Hirata, Chris

    2013-09-01

    We simulate the performance of a new type of instrument, a Superconducting Multi-Object Spectrograph (SuperMOS), that uses microwave kinetic inductance detectors (MKIDs). MKIDs, a new detector technology, feature good quantum efficiency in the UVOIR, can count individual photons with microsecond timing accuracy, and, like X-ray calorimeters, determine their energy to several percent. The performance of Giga-z, a SuperMOS designed for wide field imaging follow-up observations, is evaluated using simulated observations of the COSMOS mock catalog with an array of 100,000 R 423 nm = E/ΔE = 30 MKID pixels. We compare our results against a simultaneous simulation of LSST observations. In 3 yr on a dedicated 4 m class telescope, Giga-z could observe ≈2 billion galaxies, yielding a low-resolution spectral energy distribution spanning 350-1350 nm for each; 1000 times the number measured with any currently proposed LSST spectroscopic follow-up, at a fraction of the cost and time. Giga-z would provide redshifts for galaxies up to z ≈ 6 with magnitudes mi <~ 25, with accuracy σΔz/(1 + z) ≈ 0.03 for the whole sample, and σΔz/(1 + z) ≈ 0.007 for a select subset. We also find catastrophic failure rates and biases that are consistently lower than for LSST. The added constraint on dark energy parameters for WL + CMB by Giga-z using the FoMSWG default model is equivalent to multiplying the LSST Fisher matrix by a factor of α = 1.27 (wp ), 1.53 (wa ), or 1.98 (Δγ). This is equivalent to multiplying both the LSST coverage area and the training sets by α and reducing all systematics by a factor of 1/\\sqrt{\\alpha }, advantages that are robust to even more extreme models of intrinsic alignment.

  7. Biodiversity research in the "big data" era: GigaScience and Pensoft work together to publish the most data-rich species description.

    PubMed

    Edmunds, Scott C; Hunter, Chris I; Smith, Vincent; Stoev, Pavel; Penev, Lyubomir

    2013-10-28

    With the publication of the first eukaryotic species description, combining transcriptomic, DNA barcoding, and micro-CT imaging data, GigaScience and Pensoft demonstrate how classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. This 'holistic' approach in taxonomic description of a new species of cave-dwelling centipede is published in the Biodiversity Data Journal (BDJ), with coordinated data release in the GigaScience GigaDB database.

  8. The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

    PubMed Central

    Wilkie, Emma M; Bishop, Melanie J; O'Connor, Wayne A

    2013-01-01

    Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non-native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density-dependent effects between the native and non-native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed

  9. Peering into peer-review at GigaScience.

    PubMed

    Edmunds, Scott C

    2013-01-24

    Fostering and promoting more open and transparent science is one of the goals of GigaScience. One of the ways we have been doing this is by throwing light on the peer-review process and carrying out open peer-review as standard. In this editorial, we provide our rationale for undertaking this policy, give examples of our positive experiences to date, and encourage others to open up the normally opaque publication process.

  10. Peering into peer-review at GigaScience

    PubMed Central

    2013-01-01

    Fostering and promoting more open and transparent science is one of the goals of GigaScience. One of the ways we have been doing this is by throwing light on the peer-review process and carrying out open peer-review as standard. In this editorial, we provide our rationale for undertaking this policy, give examples of our positive experiences to date, and encourage others to open up the normally opaque publication process. PMID:23587291

  11. Two-Step Vapor/Liquid/Solid Purification

    NASA Technical Reports Server (NTRS)

    Holland, L. R.

    1986-01-01

    Vertical distillation system combines in single operation advantages of multiple zone refining with those of distillation. Developed specifically to load Bridgman-Stockbarger (vertical-solidification) growth ampoules with ultrapure tellurium and cadmium, system, with suitable modifications, serves as material refiner. In first phase of purification process, ampoule heated to drive off absorbed volatiles. Second phase, evaporator heated to drive off volatiles in charge. Third phase, slowly descending heater causes distillation from evaporator to growing crystal in ampoule.

  12. Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

    PubMed

    Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

    2015-07-01

    An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Automated large-scale purification of a G protein-coupled receptor for neurotensin.

    PubMed

    White, Jim F; Trinh, Loc B; Shiloach, Joseph; Grisshammer, Reinhard

    2004-04-30

    Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

  14. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS[beta

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.

    2012-03-16

    MutS{beta} is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutS{alpha} (MSH2-MSH6). Although mismatch recognition by MutS{alpha} has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutS{beta}. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification ofmore » recombinant human MutS{beta} and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.« less

  15. Purification and crystallization of Kokobera virus helicase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    De Colibus, Luigi; Speroni, Silvia; Coutard, Bruno

    2007-03-01

    Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method and exhibit a diffraction limit of 2.3 Å. Kokobera virus is a mosquito-borne flavivirus belonging, like West Nile virus, to the Japanese encephalitis virus serocomplex. The flavivirus genus is characterized by a positive-sense single-stranded RNA genome. The unique open reading frame of the viral RNA is transcribed and translated as a single polyprotein which is post-translationally cleaved to yield three structural and seven nonstructural proteins, one of which ismore » the NS3 gene that encodes a C-terminal helicase domain consisting of 431 amino acids. Helicase inhibitors are potential antiviral drugs as the helicase is essential to viral replication. Crystals of the Kokobera virus helicase domain were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P3{sub 1}21 (or P3{sub 2}21), with unit-cell parameters a = 88.6, c = 138.6 Å, and exhibit a diffraction limit of 2.3 Å.« less

  16. The quorum-quenching lactonase from Geobacillus caldoxylosilyticus : purification, characterization, crystallization and crystallographic analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bergonzi, Celine; Schwab, Michael; Elias, Mikael

    Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-β-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacteriumGeobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidatemore » for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (k cat/K m) against AHLs of greater than 10 6 M $-$1 s $-$1. The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Å resolution of GcL are reported.« less

  17. Facilitated giga-seal formation with a just originated glass surface.

    PubMed

    Böhle, T; Benndorf, K

    1994-07-01

    A simple technique of tip preparation in patch pipettes is described, which facilitates giga-seal formation. The pipettes were fabricated from thick-walled borosilicate glass tubing (external diameter 2.0 mm, internal diameter 0.5 mm) and the tips could be repeatedly broken in the bath. The pipette resistance correspondingly fell in steps of 3-20 M omega from about 80 M omega to about 2 M omega (double concentrated Tyrode). Scanning electron microscopy showed that the tip obtained after breaking was fairly plain. These broken tips were especially appropriate for patch-clamping. In cardiac myocytes in 11 out of 26 patches with Na+ channel activity, giga-seals developed spontaneously, i.e. without suction. In these patches the amplitude of the mean current with depolarizing pulses to -40 mV was significantly higher in comparison with patches formed under negative pressure. It is concluded that spontaneously sealed patches are most likely of planar configuration and the Na+ channel activity exceeds that in suction-induced patches.

  18. Overexpression of cinnamate 4-hydroxylase gene enhances biosynthesis of decursinol angelate in Angelica gigas hairy roots.

    PubMed

    Park, Nam Il; Park, Jee Hee; Park, Sang Un

    2012-02-01

    Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

  19. Biodiversity research in the “big data” era: GigaScience and Pensoft work together to publish the most data-rich species description

    PubMed Central

    2013-01-01

    With the publication of the first eukaryotic species description, combining transcriptomic, DNA barcoding, and micro-CT imaging data, GigaScience and Pensoft demonstrate how classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. This 'holistic’ approach in taxonomic description of a new species of cave-dwelling centipede is published in the Biodiversity Data Journal (BDJ), with coordinated data release in the GigaScience GigaDB database. PMID:24229463

  20. Giga-z: A 100,000 OBJECT SUPERCONDUCTING SPECTROPHOTOMETER FOR LSST FOLLOW-UP

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marsden, Danica W.; Mazin, Benjamin A.; O'Brien, Kieran

    2013-09-15

    We simulate the performance of a new type of instrument, a Superconducting Multi-Object Spectrograph (SuperMOS), that uses microwave kinetic inductance detectors (MKIDs). MKIDs, a new detector technology, feature good quantum efficiency in the UVOIR, can count individual photons with microsecond timing accuracy, and, like X-ray calorimeters, determine their energy to several percent. The performance of Giga-z, a SuperMOS designed for wide field imaging follow-up observations, is evaluated using simulated observations of the COSMOS mock catalog with an array of 100,000 R{sub 423{sub nm}} = E/{Delta}E = 30 MKID pixels. We compare our results against a simultaneous simulation of LSST observations.more » In 3 yr on a dedicated 4 m class telescope, Giga-z could observe Almost-Equal-To 2 billion galaxies, yielding a low-resolution spectral energy distribution spanning 350-1350 nm for each; 1000 times the number measured with any currently proposed LSST spectroscopic follow-up, at a fraction of the cost and time. Giga-z would provide redshifts for galaxies up to z Almost-Equal-To 6 with magnitudes m{sub i} {approx}< 25, with accuracy {sigma}{sub {Delta}z/(1+z)} Almost-Equal-To 0.03 for the whole sample, and {sigma}{sub {Delta}z/(1+z)} Almost-Equal-To 0.007 for a select subset. We also find catastrophic failure rates and biases that are consistently lower than for LSST. The added constraint on dark energy parameters for WL + CMB by Giga-z using the FoMSWG default model is equivalent to multiplying the LSST Fisher matrix by a factor of {alpha} = 1.27 (w{sub p} ), 1.53 (w{sub a} ), or 1.98 ({Delta}{gamma}). This is equivalent to multiplying both the LSST coverage area and the training sets by {alpha} and reducing all systematics by a factor of 1/{radical}({alpha}), advantages that are robust to even more extreme models of intrinsic alignment.« less

  1. Comparative Transcriptome Analysis of the Pacific Oyster Crassostrea gigas Characterized by Shell Colors: Identification of Genetic Bases Potentially Involved in Pigmentation

    PubMed Central

    Feng, Dandan; Li, Qi; Yu, Hong; Zhao, Xuelin; Kong, Lingfeng

    2015-01-01

    Background Shell color polymorphisms of Mollusca have contributed to development of evolutionary biology and population genetics, while the genetic bases and molecular mechanisms underlying shell pigmentation are poorly understood. The Pacific oyster (Crassostrea gigas) is one of the most important farmed oysters worldwide. Through successive family selection, four shell color variants (white, golden, black and partially pigmented) of C. gigas have been developed. To elucidate the genetic mechanisms of shell coloration in C. gigas and facilitate the selection of elite oyster lines with desired coloration patterns, differentially expressed genes (DEGs) were identified among the four shell color variants by RNA-seq. Results Digital gene expression generated over fifteen million reads per sample, producing expression data for 28,027 genes. A total number of 2,645 DEGs were identified from pair-wise comparisons, of which 432, 91, 43 and 39 genes specially were up-regulated in white, black, golden and partially pigmented shell of C. gigas, respectively. Three genes of Abca1, Abca3 and Abcb1 which belong to the ATP-binding cassette (ABC) transporters super-families were significantly associated with white shell formation. A tyrosinase transcript (CGI_10008737) represented consistent up-regulated pattern with golden coloration. We proposed that white shell variant of C. gigas could employ “endocytosis” to down-regulate notch level and to prevent shell pigmentation. Conclusion This study discovered some potential shell coloration genes and related molecular mechanisms by the RNA-seq, which would provide foundational information to further study on shell coloration and assist in selective breeding in C. gigas. PMID:26693729

  2. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  3. Purification of HgI.sub.2 for nuclear detector fabrication

    DOEpatents

    Schieber, Michael M.

    1978-01-01

    A process for purification of mercuric iodide (HgI.sub.2) to be used as a source material for the growth of detector quality crystals. The high purity HgI.sub.2 raw material is produced by a combination of three stages: synthesis of HgI.sub.2 from Hg and I.sub.2, repeated sublimation, and zone refining.

  4. Effect of impurities and post-experimental purification in SAD phasing with serial femtosecond crystallography data.

    PubMed

    Zhang, Tao; Gu, Yuanxin; Fan, Haifu

    2016-06-01

    In serial crystallography (SX) with either an X-ray free-electron laser (XFEL) or synchrotron radiation as the light source, huge numbers of micrometre-sized crystals are used in diffraction data collection. For a SAD experiment using a derivative with introduced heavy atoms, it is difficult to completely exclude crystals of the native protein from the sample. In this paper, simulations were performed to study how the inclusion of native crystals in the derivative sample could affect the result of SAD phasing and how the post-experimental purification proposed by Zhang et al. [(2015), Acta Cryst. D71, 2513-2518] could be used to remove the impurities. A gadolinium derivative of lysozyme and the corresponding native protein were used in the test. Serial femtosecond crystallography (SFX) diffraction snapshots were generated by CrystFEL. SHELXC/D, Phaser, DM, ARP/wARP and REFMAC were used for automatic structure solution. It is shown that a small amount of impurities (snapshots from native crystals) in the set of derivative snapshots can strongly affect the SAD phasing results. On the other hand, post-experimental purification can efficiently remove the impurities, leading to results similar to those from a pure sample.

  5. Sclerochronology of Holocene oyster shells (Crassostrea gigas) from the West Coast of Bohai Sea, China

    NASA Astrophysics Data System (ADS)

    Fan, C.; Koeniger, P.; Wang, H.; Frechen, M.

    2009-04-01

    Sclerochronology, the study of periodic increments in skeletal organisms, can decipher the life history and environmental records preserved in fossil shells. Although there have been a number of studies that apply isotopic analyses to shells in open ocean and fresh water, investigations for brackish environments are rare. One of the common inhabitants in estuaries is the Crassostrea oyster. Kirby et al. (1998) demonstrated a close correspondence between the ligamental increments of convex and concave bands and yearly ^18O cycles; Andrus and Crowe (2000) found a close correspondence between translucent growth bands on the cross-section of the hinge and yearly ^18O cycles. They conclude that the morphological features on hinge and growth bands on the cross-section are formed annually and can be used to determine accurately age and growth rate in this species. However, Surge et al. (2001) could not find that these morphologic features have seasonal significance in the C. virginica shells. Therefore, these concave ridges are not reliable independent proxies of seasonality. These studies were carried out with C. virginica shells; none was studied with nature C. gigas, which was widely distributed along the Pacific coastal area. C. gigas has been introduced from its native home to all over the world, ranging from North America to Australia and Europe; it has become an important commercial harvest in many of these places. Buried Holocene oyster shells of C. gigas were sampled from a huge buried oyster reef on the West of Bohai Sea, China. One of these shells was selected for high resolution micro-sampling and stable isotope analyses testing the assumption that C. gigas ligamental increments are annual in nature. We analyzed 236 consecutive samples from the shell to show that morphologic features both on hinge and cross-section are annual by comparing them to the ^18O profiles. We tested the assumption that the morphologic features of C.gigas are delineated by convex tops

  6. Fluxing purification and its effect on magnetic properties of high-Bs FeBPSiC amorphous alloy

    NASA Astrophysics Data System (ADS)

    Pang, Jing; Wang, Anding; Yue, Shiqiang; Kong, Fengyu; Qiu, Keqiang; Chang, Chuntao; Wang, Xinmin; Liu, Chain-Tsuan

    2017-07-01

    A high-Bs amorphous alloy with the base composition Fe83B11P3Si2C1 was used to study the effects of fluxing purification on amorphous forming ability and magnetic properties of the alloy prepared with raw materials in industrialization. By using fluxing purification, the surface crystallization was suppressed and fully amorphous Fe83B11P3Si2C1 ribbons with a maximum thickness of 48 μm were successfully achieved by using an industrial process and materials. The amorphous ribbons made with industrial-purified alloys exhibit excellent magnetic properties, containing high-Bs of 1.65 T, low Hc of 2.0 A/m, and high μe of 9.7 × 103 at 1 kHz. Impurities in the melting alloys exist in three forms and have different effluences on magnetic properties. The surface crystallization was triggered by the impurities which exist as high melting point inclusions serving as nuclei. Thus, fluxing purification is a feasible way for industrialization of high-Bs FeBPSiC amorphous alloys.

  7. Biases in determining the diet of jumbo squid Dosidicus gigas (D' Orbigny 1835) (Cephalopoda: Ommastrephidae) off southern-central Chile (34°S-40°S)

    NASA Astrophysics Data System (ADS)

    Ibáñez, Christian M.; Arancibia, Hugo; Cubillos, Luis A.

    2008-12-01

    The diet of jumbo squid ( Dosidicus gigas) off southern-central Chile is described to examine potential biases in the determination of their main prey. Specimens were collected from catches using different fishing gear (jigging, trawl and purse-seine), from July 2003 to January 2004, and from December 2005 to October 2006. The stomach contents were analyzed in terms of frequency of occurrence, number, and weight of prey items and the diet composition was analyzed using Detrended Correspondence Analysis. In the industrial purse-seine fleet for jack mackerel ( Trachurus murphyi), the dominant prey of D. gigas was T. murphyi. In the industrial mid-trawl fishery for Patagonian grenadier ( Macruronus magellanicus), the dominant species in the diet of D. gigas was M. magellanicus. Similarly, Chilean hake ( Merluccius gayi) was the main prey in the diet of D. gigas obtained in the industrial trawl fishery for Chilean hake; and, in both artisanal fisheries (purse-seine for small pelagics and jigging), small pelagic fish and D. gigas were the main prey in the stomach contents of D. gigas. Cannibalism in D. gigas varied between different fleets and probably is related to stress behavior during fishing. The Detrended Correspondence Analysis ordination showed that the main prey in the diet of D. gigas is associated with the target species of the respective fishery. Consequently, biases are associated with fishing gear, leading to an overestimate in the occurrence of the target species in the diet. We recommend analyzing samples from jigging taken at the same time and place where the trawl and purse-seine fleets are operating to avoid this problem, and the application of new tools like stable isotope, heavy metal, and fatty acid signature analyses.

  8. RAPID-COMMUNICATION Genetic diversity and differentiation in natural populations of Arapaima gigas from lower Amazon revealed by microsatellites.

    PubMed

    Fazzi-Gomes, P F; Melo, N; Palheta, G; Guerreiro, S; Amador, M; Ribeiro-Dos-Santos, A K; Santos, S; Hamoy, I

    2017-02-08

    Genetic variability is one of the important criteria for species conservation decisions. This study aimed to analyze the genetic diversity and the population differentiation of two natural populations of Arapaima gigas, a species with a long history of being commercially exploited. We collected 87 samples of A. gigas from Grande Curuai Lake and Paru Lake, located in the Lower Amazon region of Amazônia, Brazil, and genotyped these samples using a multiplex panel of microsatellite markers. Our results showed that the populations of A. gigas analyzed had high levels of genetic variability, which were similar to those described in previous studies. These two populations had a significant population differentiation supported by the estimates of F ST and R ST (0.06), by Bayesian analysis (K = 2), and by population assignment tests, which revealed a moderate genetic distance.

  9. Physicochemical characterization of actomyosin-paramyosin from giant squid mantle (Dosidicus gigas).

    PubMed

    Tolano-Villaverde, Ivan J; Ocaño-Higuera, Victor; Ezquerra-Brauer, Josafat; Santos-Sauceda, Irela; Santacruz-Ortega, Hisila; Cárdenas-López, José L; Rodríguez-Olibarria, Guillermo; Márquez-Ríos, Enrique

    2018-03-01

    The giant squid (Dosidicus gigas) has been proposed as raw material to obtain myofibrillar protein concentrates. However, it has been observed that colloidal systems formed from squid proteins have limited stability. Therefore, the isolation and characterization of the actomyosin-paramyosin isolated (API) complex were performed, because they are the main proteins to which functionality has been attributed. Densitogram analysis revealed 45% of actin, 38% of myosin and 17% of paramyosin. The amino acid profile indicates a higher proportion of acidic amino acids, which gives a higher negative charge; this was supported by the zeta potential. Total sulfhydryl (TSH) content was lower compared with proteins of other aquatic species. The higher percentage of actin in relation to myosin, the presence of paramyosin, as well as the low content of sulfhydryl groups, could comprise the main causes of the low technological functional property of proteins from D. gigas mantle. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  10. A comparative study of spatially clustered distribution of jumbo flying squid ( Dosidicus gigas) offshore Peru

    NASA Astrophysics Data System (ADS)

    Feng, Yongjiu; Cui, Li; Chen, Xinjun; Liu, Yu

    2017-06-01

    We examined spatially clustered distribution of jumbo flying squid ( Dosidicus gigas) in the offshore waters of Peru bounded by 78°-86°W and 8°-20°S under 0.5°×0.5° fishing grid. The study is based on the catch-per-unit-effort (CPUE) and fishing effort from Chinese mainland squid jigging fleet in 2003-2004 and 2006-2013. The data for all years as well as the eight years (excluding El Niño events) were studied to examine the effect of climate variation on the spatial distribution of D. gigas. Five spatial clusters reflecting the spatial distribution were computed using K-means and Getis-Ord Gi* for a detailed comparative study. Our results showed that clusters identified by the two methods were quite different in terms of their spatial patterns, and K-means was not as accurate as Getis-Ord Gi*, as inferred from the agreement degree and receiver operating characteristic. There were more areas of hot and cold spots in years without the impact of El Niño, suggesting that such large-scale climate variations could reduce the clustering level of D. gigas. The catches also showed that warm El Niño conditions and high water temperature were less favorable for D. gigas offshore Peru. The results suggested that the use of K-means is preferable if the aim is to discover the spatial distribution of each sub-region (cluster) of the study area, while Getis-Ord Gi* is preferable if the aim is to identify statistically significant hot spots that may indicate the central fishing ground.

  11. Production of extreme-purity aluminum and silicon by fractional crystallization processing

    NASA Astrophysics Data System (ADS)

    Dawless, R. K.; Troup, R. L.; Meier, D. L.; Rohatgi, A.

    1988-06-01

    Large scale fractional crystallization is used commercially at Alcoa to produce extreme purity aluminum (99.999+% Al). The primary market is sputtering targets used to make interconnects for integrated circuits. For some applications the impurities uranium and thorium are reduced to less than 1 ppbw to avoid "soft errors" associated with α particle emission. The crystallization process achieves segregation coefficients which are close to theoretical at normal yields, and this, coupled with the scale of the units, allows practical production of this material. The silicon purification process involves crystallization of Si from molten aluminum alloys containing about 30% silicon. The crystallites from this process are further treated to remove residual Al and an extreme purity ingot is obtained. This material is considered suitable for single crystal or ribbon type photovoltaic cells and for certain IC applications, including highly doped substrates used for epitaxial growth. In production of both extreme purity Al and Si, impurities are rejected to the remaining melt as the crystals form and some separation is achieved by draining this downgraded melt from the unit. Purification of this downgrade by crystallization has also been demonstrated for both systems and is important for achieving high recoveries.

  12. Sex Ratio and Sex Reversal in Two-year-old Class of Oyster, Crassostrea gigas (Bivalvia: Ostreidae)

    PubMed Central

    Park, Jung Jun; Kim, Hyejin; Kang, Seung Wan; An, Cheul Min; Lee, Sung-Ho; Gye, Myung Chan; Lee, Jung Sick

    2012-01-01

    The sex ratio (F:M) in the same population of oyster, Crassostrea gigas at the commencement of the study (2007) was 1:1.0, but changed to 1:2.8 by the end of the study (2008). The sex reversal rate in two-year-old oysters was 40.2%. Specifically, female to male sex reversal rate was 66.1%, which is higher than the male to female sex reversal rate of 21.1%. The sex reversal pattern of C. gigas appears to go from male⇒female⇒male, and as such is determined to be rhythmical hermaphroditism. PMID:25949114

  13. Protective Effect of Decursin Extracted from Angelica gigas in Male Infertility via Nrf2/HO-1 Signaling Pathway.

    PubMed

    Bae, Woong Jin; Ha, U Syn; Choi, Jin Bong; Kim, Kang Sup; Kim, Su Jin; Cho, Hyuk Jin; Hong, Sung Hoo; Lee, Ji Youl; Wang, Zhiping; Hwang, Sung Yeoun; Kim, Sae Woong

    2016-01-01

    Higher testicular temperature results in altered spermatogenesis due to heat-related oxidative stress. We examined the effects of decursin extracted from Angelica gigas Nakai on antioxidant activity in vitro and in a cryptorchidism-induced infertility rat model. TM3 Leydig cell viability was measured based on oxidative stress according to treatment. Either distilled water or AG 400 mg/kg of A. gigas extract was administered orally for 4 weeks after unilateral cryptorchidism was induced. After 1, 2, and 4 weeks, six rats from the control group and six rats from treatment group were sacrificed. Testicular weight, semen quality, antioxidant activities, nuclear factor erythroid 2-related factor 2 (Nrf2) protein, and mRNA expression of Nrf2-regulated genes were analyzed. Treatment with A. gigas extract (1) protected TM3 cells against oxidative stress in a dose-dependent manner, (2) improved the mean weight of the cryptorchid testis, (3) maintained sperm counts, motility, and spermatogenic cell density, (4) decreased levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and increased levels of superoxide dismutase (SOD), (5) significantly increased Nrf2 and heme oxygenase-1 (HO-1), and (6) significantly decreased apoptosis. This study suggests that decursin extracted from A. gigas is a supplemental agent that can reduce oxidative stress by Nrf2-mediated upregulation of HO-1 in rat experimentally induced unilateral cryptorchidism and may improve cryptorchidism-induced infertility.

  14. Protein Crystallization.

    PubMed

    McPherson, Alexander

    2017-01-01

    Protein crystallization was discovered by chance nearly 200 years ago and was developed in the late nineteenth century as a powerful purification tool, and a demonstration of chemical purity. The crystallization of proteins, nucleic acids, and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule, but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by manipulation of various parameters that include temperature, ionic strength, and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors, or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch, and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years due to the advent of practical, easy-to-use screening kits, and the application of laboratory robotics.

  15. Purification, Crystallization, and Preliminary X-ray Analysis of Native Canavalin

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Dowell, Jennifer; Ng, Joseph; Gavira, Jose A.

    2003-01-01

    The protein canavalin is a 7S vicilin, from the Jack Bean, Canavalis ensfomis. Canavalin is described as a seed storage protein, an energy source for a developing seed, as no other known activity or function has been found. The protein was first isolated and crystallized by Sumner and Howell (J. Biol. Chem. 113, 607-610, 1936). Canavalin spontaneously crystallizes after proteolytic cleavage at neutral pH, which removes residues 1-46, 224-245, and 325-330 and produces peptides of approximately 25, 13, and 12 kDa. Preliminary gel filtration experiments indicated the presence of nucleic acid with the uncleaved protein. We developed a dual column procedure, ion exchange followed by hydroxy apatite chromatography, that effectively removes the nucleic acid and yields an essentially pure uncut canavalin preparation with an OD 280/260 ratio of approximately 1.9-2.0. Standard crystallization screens using this material gave a number of positive results having a common requirement for alcohols and Mg(2+) ion, with crystals typically appearing within a day or less. Optimization experiments to date have shown that we can obtain crystals from pH 6.5 to pH 8.2, using MPD from 5 to 20% and 0.05 to 0.2M Mg(2+) (sulfate or acetate). The crystals are of space group P2(sub 1)2(sub 1)2(sub 1), unit cell dimensions, and a complete data set to 1.5 Angstroms, resolution has now been collected at a synchrotron source. Most importantly, the crystals are not twinned, a persistent problem with the most commonly obtained rhombohedral form of proteolytically cleaved canavalin.

  16. Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium Bartonella henselae strain Houston-1 at 2.1 Å resolution

    DOE PAGES

    Naqvi, Kubra F.; Staker, Bart L.; Dobson, Renwick C. J.; ...

    2016-01-01

    The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of L-aspartate 4-semialdehyde and pyruvate to synthesize L-2,3-dihydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacteriumBartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mMsodium citrate tribasic pH 5.5 and were shown to diffract to ~2.10 Å resolution. They belonged to space groupP2 12 12 1, with unit-cell parametersa= 79.96,b= 106.33,c= 136.25more » Å. The finalRvalues wereR r.i.m.= 0.098,R work= 0.183,R free= 0.233.« less

  17. The Rehbinder effect in iron during giga-cycle fatigue loading

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bannikov, M. V., E-mail: mbannikov@icmm.ru; Naimark, O. B.

    The influence of the adsorptive strength reduction effect (the Rehbinder effect) on the fatigue life of pure iron under the giga-cycle loading regime was investigated. Specimens were loaded by an ultrasonic testing machine with a frequency of 20 kHz in air and in contact with eutectic alloy of gallium with tin and indium. A significant (by several orders of magnitude) worsening of the life-time of iron in contact with a molten metal as compared with tests in air was established. The liquid metal penetrates into the material to a depth of 200 μm to the center of a fatigue crack. Themore » mechanism of the fatigue crack initiation in the giga-cycle regime of loading in contact with a surfactant is differing: the crack is formed on the surface of the specimen rather than within it as is the case for air. Based on the electron and optical microscopy data for the fracture surface, it can be concluded that exactly the change in the crack initiation mechanism reduces the fatigue life of iron in contact with a liquid metal because the initiated crack propagates regardless of the surfactant.« less

  18. The Rehbinder effect in iron during giga-cycle fatigue loading

    NASA Astrophysics Data System (ADS)

    Bannikov, M. V.; Naimark, O. B.

    2015-10-01

    The influence of the adsorptive strength reduction effect (the Rehbinder effect) on the fatigue life of pure iron under the giga-cycle loading regime was investigated. Specimens were loaded by an ultrasonic testing machine with a frequency of 20 kHz in air and in contact with eutectic alloy of gallium with tin and indium. A significant (by several orders of magnitude) worsening of the life-time of iron in contact with a molten metal as compared with tests in air was established. The liquid metal penetrates into the material to a depth of 200 μm to the center of a fatigue crack. The mechanism of the fatigue crack initiation in the giga-cycle regime of loading in contact with a surfactant is differing: the crack is formed on the surface of the specimen rather than within it as is the case for air. Based on the electron and optical microscopy data for the fracture surface, it can be concluded that exactly the change in the crack initiation mechanism reduces the fatigue life of iron in contact with a liquid metal because the initiated crack propagates regardless of the surfactant.

  19. Growth Defects in Biomacromolecular Crystals

    NASA Technical Reports Server (NTRS)

    2003-01-01

    NASA's ground based program confirmed close similarity between protein and small molecules crystal growth, but also revealed essential differences. No understanding exists as to why and when crystals grown in space are, in approx. 20 percent of cases, of higher quality. More rationale is needed in flight experiments. Ferritin crystals grown in space are 2.5 times cleaner than their terrestrial counterparts. This may occur because of the existence of a zone depleted with respect to impurities around a crystal growing in stagnant solution. This zone should appear since the distribution coefficient for homologous impurities exceeds unity. This impurity depletion zone hypothesis requires verification and development. Thorough purification from homologous impurities brought about resolution improvement from 2.6 to 1.8 angstroms for ferritin and from 2.6 to 2.0 angstroms for canavalin.

  20. Transcriptomic Responses to Salinity Stress in the Pacific Oyster Crassostrea gigas

    PubMed Central

    Zhao, Xuelin; Yu, Hong; Kong, Lingfeng; Li, Qi

    2012-01-01

    Background Low salinity is one of the main factors limiting the distribution and survival of marine species. As a euryhaline species, the Pacific oyster Crassostrea gigas is considered to be tolerant to relative low salinity. The genes that regulate C. gigas responses to osmotic stress were monitored using the next-generation sequencing of whole transcriptome with samples taken from gills. By RNAseq technology, transcript catalogs of up- and down-regulated genes were generated from the oysters exposed to low and optimal salinity seawater. Methodology/Principal Findings Through Illumina sequencing, we reported 1665 up-regulated transcripts and 1815 down-regulated transcripts. A total of 45771 protein-coding contigs were identified from two groups based on sequence similarities with known proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the genes recovered diverse biological functions and processes. The genes that changed expression significantly were highly represented in cellular process and regulation of biological process, intracellular and cell, binding and protein binding according to GO annotation. The results highlighted genes related to osmoregulation, signaling and interactions of osmotic stress response, anti-apoptotic reactions as well as immune response, cell adhesion and communication, cytoskeleton and cell cycle. Conclusions/Significance Through more than 1.5 million sequence reads and the expression data of the two libraries, the study provided some useful insights into signal transduction pathways in oysters and offered a number of candidate genes as potential markers of tolerance to hypoosmotic stress for oysters. In addition, the characterization of C. gigas transcriptome will not only provide a better understanding of the molecular mechanisms about the response to osmotic stress of the oysters, but also facilitate research into biological processes to find underlying physiological adaptations to

  1. Purification of Restriction Endonuclease EcoRII and its Co-Crystallization

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Chen, L.; Meehan, E.; Pusey, M.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.

  2. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    PubMed

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Microsatellite Analysis for Identification of Individuals Using Bone from the Extinct Steller's Sea Cow (Hydrodamalis gigas).

    PubMed

    Warner, Jeffery F; Harpole, Michael G; Crerar, Lorelei D

    2017-01-01

    Microsatellite DNA can provide more detailed population genetic information than mitochondrial DNA which is normally used to research ancient bone. The methods detailed in this chapter can be utilized for any type of bone. However, for this example, four microsatellite loci were isolated from Steller's sea cow (Hydrodamalis gigas) using published primers for manatee and dugong microsatellites. The primers DduC05 (Broderick et al., Mol Ecol Notes 6:1275-1277, 2007), Tmakb60, TmaSC5 (Pause et al., Mol Ecol Notes 6: 1073-1076, 2007), and TmaE11 (Garcia-Rodriguez et al., Mol Ecol 12:2161-2163, 2000) all successfully amplified microsatellites from H. gigas. The DNA samples were from bone collected on Bering or St. Lawrence Islands. DNA was analyzed using primers with the fluorescent label FAM-6. Sequenced alleles were then used to indicate a difference in the number of repeats and thus a difference in individuals. This is the first time that H. gigas microsatellite loci have been isolated. These techniques for ancient bone microsatellite analysis allow an estimate of population size for a newly discovered St. Lawrence Island sea cow population.

  4. Genome and Transcriptome Analyses Provide Insight into the Euryhaline Adaptation Mechanism of Crassostrea gigas

    PubMed Central

    Zhang, Linlin; Li, Chunyan; Li, Li; She, Zhicai; Huang, Baoyu; Zhang, Guofan

    2013-01-01

    Background The Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster’s euryhaline adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR. Results Transcriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na+/K+ channel and aquaporin but increased calcium-activated K+ channel and Ca2+ channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster’s powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas. Conclusions Our results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as

  5. Expression, purification and crystallization of Streptococcus dysgalactiae-derived mitogen

    PubMed Central

    Papageorgiou, Anastassios C.; Saarinen, Susanna; Ramirez-Bartutis, Rosa; Kato, Hidehito; Uchiyama, Takehiko; Kirikae, Teruo; Miyoshi-Akiyama, Toru

    2006-01-01

    Superantigens are bacterial or viral toxins with potent immunostimulatory properties. Streptococcus dysgalactiae-derived mitogen, a 25 kDa protein, is a recently discovered superantigen isolated from S. dysgalactiae culture supernatant. Sequence considerations suggest that it belongs to a new superantigen family distinct from other superantigens. The protein was expressed in Escherichia coli cells and purified to homogeneity. Crystals were grown at pH 4.2–4.4 in the presence of 18–20%(w/v) PEG 3350 and 0.4 M lithium nitrate. A complete data set to 2.4 Å resolution was collected from a single crystal at liquid-nitrogen temperatures using synchrotron radiation. The crystals belong to space group P3/P31/P32, with unit-cell parameters a = b = 52.7, c = 62.4 Å, γ = 120° and one molecule in the crystallographic asymmetric unit. PMID:16511312

  6. Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc.

    PubMed

    Zhang, Na; Xu, Fei; Guo, Ximing

    2014-09-11

    Despite the prevalence of sex in animal kingdom, we have only limited understanding of how sex is determined and evolved in many taxa. The mollusc Pacific oyster Crassostrea gigas exhibits complex modes of sexual reproduction that consists of protandric dioecy, sex change, and occasional hermaphroditism. This complex system is controlled by both environmental and genetic factors through unknown molecular mechanisms. In this study, we investigated genes related to sex-determining pathways in C. gigas through transcriptome sequencing and analysis of female and male gonads. Our analysis identified or confirmed novel homologs in the oyster of key sex-determining genes (SoxH or Sry-like and FoxL2) that were thought to be vertebrate-specific. Their expression profile in C. gigas is consistent with conserved roles in sex determination, under a proposed model where a novel testis-determining CgSoxH may serve as a primary regulator, directly or indirectly interacting with a testis-promoting CgDsx and an ovary-promoting CgFoxL2. Our findings plus previous results suggest that key vertebrate sex-determining genes such as Sry and FoxL2 may not be inventions of vertebrates. The presence of such genes in a mollusc with expression profiles consistent with expected roles in sex determination suggest that sex determination may be deeply conserved in animals, despite rapid evolution of the regulatory pathways that in C. gigas may involve both genetic and environmental factors. Copyright © 2014 Zhang et al.

  7. Genomic Analysis of the Pacific Oyster (Crassostrea gigas) Reveals Possible Conservation of Vertebrate Sex Determination in a Mollusc

    PubMed Central

    Zhang, Na; Xu, Fei; Guo, Ximing

    2014-01-01

    Despite the prevalence of sex in animal kingdom, we have only limited understanding of how sex is determined and evolved in many taxa. The mollusc Pacific oyster Crassostrea gigas exhibits complex modes of sexual reproduction that consists of protandric dioecy, sex change, and occasional hermaphroditism. This complex system is controlled by both environmental and genetic factors through unknown molecular mechanisms. In this study, we investigated genes related to sex-determining pathways in C. gigas through transcriptome sequencing and analysis of female and male gonads. Our analysis identified or confirmed novel homologs in the oyster of key sex-determining genes (SoxH or Sry-like and FoxL2) that were thought to be vertebrate-specific. Their expression profile in C. gigas is consistent with conserved roles in sex determination, under a proposed model where a novel testis-determining CgSoxH may serve as a primary regulator, directly or indirectly interacting with a testis-promoting CgDsx and an ovary-promoting CgFoxL2. Our findings plus previous results suggest that key vertebrate sex-determining genes such as Sry and FoxL2 may not be inventions of vertebrates. The presence of such genes in a mollusc with expression profiles consistent with expected roles in sex determination suggest that sex determination may be deeply conserved in animals, despite rapid evolution of the regulatory pathways that in C. gigas may involve both genetic and environmental factors. PMID:25213692

  8. Protective Effect of Decursin Extracted from Angelica gigas in Male Infertility via Nrf2/HO-1 Signaling Pathway

    PubMed Central

    Bae, Woong Jin; Ha, U. Syn; Choi, Jin Bong; Kim, Kang Sup; Kim, Su Jin; Cho, Hyuk Jin; Hong, Sung Hoo; Lee, Ji Youl; Wang, Zhiping; Hwang, Sung Yeoun; Kim, Sae Woong

    2016-01-01

    Higher testicular temperature results in altered spermatogenesis due to heat-related oxidative stress. We examined the effects of decursin extracted from Angelica gigas Nakai on antioxidant activity in vitro and in a cryptorchidism-induced infertility rat model. TM3 Leydig cell viability was measured based on oxidative stress according to treatment. Either distilled water or AG 400 mg/kg of A. gigas extract was administered orally for 4 weeks after unilateral cryptorchidism was induced. After 1, 2, and 4 weeks, six rats from the control group and six rats from treatment group were sacrificed. Testicular weight, semen quality, antioxidant activities, nuclear factor erythroid 2-related factor 2 (Nrf2) protein, and mRNA expression of Nrf2-regulated genes were analyzed. Treatment with A. gigas extract (1) protected TM3 cells against oxidative stress in a dose-dependent manner, (2) improved the mean weight of the cryptorchid testis, (3) maintained sperm counts, motility, and spermatogenic cell density, (4) decreased levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) and increased levels of superoxide dismutase (SOD), (5) significantly increased Nrf2 and heme oxygenase-1 (HO-1), and (6) significantly decreased apoptosis. This study suggests that decursin extracted from A. gigas is a supplemental agent that can reduce oxidative stress by Nrf2-mediated upregulation of HO-1 in rat experimentally induced unilateral cryptorchidism and may improve cryptorchidism-induced infertility. PMID:27034737

  9. Effect of linear alkylbenzene mixtures and sanitary sewage in biochemical and molecular responses in pacific oyster Crassostrea gigas.

    PubMed

    Flores-Nunes, Fabrício; Mattos, Jacó J; Zacchi, Flávia L; Serrano, Miguel A S; Piazza, Clei E; Sasaki, Silvio T; Taniguchi, Satie; Bicego, Márcia C; Melo, Cláudio M R; Bainy, Afonso C D

    2015-11-01

    Urban effluents are rich in nutrients, organic matter, pharmaceuticals and personal care products (PPCPs), pesticides, hydrocarbons, surfactants, and others. Previous studies have shown that oysters Crassostrea gigas accumulate significant levels of linear alkylbenzenes (LABs) in sanitary sewage contaminated sites, but there is little information about its toxicological effects in marine bivalves. The aim of this study was to analyze the transcription of genes in two tissues of C. gigas exposed for 12, 24, and 36 h to LABs or sanitary sewage. Likewise, the activity of antioxidant and biotransformation enzymes was measured in oysters exposed for 36 h in all groups. Oysters exposed to LABs and oysters exposed to sanitary sewage showed different patterns of transcriptional responses. LAB-exposed oysters showed lower level of biological responses than the oysters exposed to sanitary sewage. Despite the ability of the oyster C. gigas to accumulate LABs (28-fold), the data indicate that these contaminants are not the cause for the transcriptional responses observed in oysters exposed to sanitary sewage. Possibly, the biological changes observed in the sanitary sewage-exposed oysters are associated with the presence of other contaminants, which might have caused synergistic, additive, or antagonistic effects. The results show that FABP-like and GST-ω-like messenger RNAs (mRNAs) have a rapid response in tissues of oyster C. gigas exposed to sanitary sewage, suggesting a possible protective response and a role in maintaining homeostasis of these organisms.

  10. Molecular adaptation in the world's deepest-living animal: Insights from transcriptome sequencing of the hadal amphipod Hirondellea gigas.

    PubMed

    Lan, Yi; Sun, Jin; Tian, Renmao; Bartlett, Douglas H; Li, Runsheng; Wong, Yue Him; Zhang, Weipeng; Qiu, Jian-Wen; Xu, Ting; He, Li-Sheng; Tabata, Harry G; Qian, Pei-Yuan

    2017-07-01

    The Challenger Deep in the Mariana Trench is the deepest point in the oceans of our planet. Understanding how animals adapt to this harsh environment characterized by high hydrostatic pressure, food limitation, dark and cold is of great scientific interest. Of the animals dwelling in the Challenger Deep, amphipods have been captured using baited traps. In this study, we sequenced the transcriptome of the amphipod Hirondellea gigas collected at a depth of 10,929 m from the East Pond of the Challenger Deep. Assembly of these sequences resulted in 133,041 contigs and 22,046 translated proteins. Functional annotation of these contigs was made using the go and kegg databases. Comparison of these translated proteins with those of four shallow-water amphipods revealed 10,731 gene families, of which 5659 were single-copy orthologs. Base substitution analysis on these single-copy orthologs showed that 62 genes are positively selected in H. gigas, including genes related to β-alanine biosynthesis, energy metabolism and genetic information processing. For multiple-copy orthologous genes, gene family expansion analysis revealed that cold-inducible proteins (i.e., transcription factors II A and transcription elongation factor 1) as well as zinc finger domains are expanded in H. gigas. Overall, our results indicate that genetic adaptation to the hadal environment by H. gigas may be mediated by both gene family expansion and amino acid substitutions of specific proteins. © 2017 John Wiley & Sons Ltd.

  11. Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

    PubMed

    Takahashi, Keisuke G; Izumi-Nakajima, Nakako; Mori, Katsuyoshi

    2017-11-01

    For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and

  12. Purification of Germanium Crystals by Zone Refining

    NASA Astrophysics Data System (ADS)

    Kooi, Kyler; Yang, Gang; Mei, Dongming

    2016-09-01

    Germanium zone refining is one of the most important techniques used to produce high purity germanium (HPGe) single crystals for the fabrication of nuclear radiation detectors. During zone refining the impurities are isolated to different parts of the ingot. In practice, the effective isolation of an impurity is dependent on many parameters, including molten zone travel speed, the ratio of ingot length to molten zone width, and number of passes. By studying the theory of these influential factors, perfecting our cleaning and preparation procedures, and analyzing the origin and distribution of our impurities (aluminum, boron, gallium, and phosphorous) identified using photothermal ionization spectroscopy (PTIS), we have optimized these parameters to produce HPGe. We have achieved a net impurity level of 1010 /cm3 for our zone-refined ingots, measured with van der Pauw and Hall-effect methods. Zone-refined ingots of this purity can be processed into a detector grade HPGe single crystal, which can be used to fabricate detectors for dark matter and neutrinoless double beta decay detection. This project was financially supported by DOE Grant (DE-FG02-10ER46709) and the State Governor's Research Center.

  13. Effect of essential oil and supercritical carbon dioxide extract from the root of Angelica gigas on human EEG activity.

    PubMed

    Sowndhararajan, Kandhasamy; Seo, Min; Kim, Minju; Kim, Heeyeon; Kim, Songmun

    2017-08-01

    The present study aimed to investigate the effect of inhalation of essential oil (EO) and supercritical carbon dioxide extract (SC-CO 2 ) from the root of A. gigas on human electroencephalographic (EEG) activity. For this purpose, the EO was obtained from the root of A. gigas by steam distillation and SC-CO 2 was obtained at 50 °C and 400 bar for 1 h. The EEG readings were recorded using the QEEG-8 system from 8 electrode sites according to the International 10-20 system. In the EEG study, the absolute low beta (left temporal and left parietal) activity significantly increased during the inhalation of EO. In the case of SC-CO 2 inhalation, there was no significant change in absolute waves. The results revealed that the EO of A. gigas root produced significant changes in the absolute low beta activity and these changes may enhance the language learning abilities of human brain. Copyright © 2017. Published by Elsevier Ltd.

  14. Control of crystal growth in water purification by directional freeze crystallization

    NASA Technical Reports Server (NTRS)

    Conlon, William M. (Inventor)

    1996-01-01

    A Directional Freeze Crystallization system employs an indirect contact heat exchanger to freeze a fraction of liquid to be purified. The unfrozen fraction is drained away and the purified frozen fraction is melted. The heat exchanger must be designed in accordance with a Growth Habit Index to achieve efficient separation of contaminants. If gases are dissolved in the liquid, the system must be pressurized.

  15. Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents.

    PubMed

    Piletska, Elena V; Karim, Kal; Cutler, Malcolm; Piletsky, Sergey A

    2013-01-01

    A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g(-1) ), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC-MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL(-1) . Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step-purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high-purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. GaN Initiative for Grid Applications (GIGA)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Turner, George

    2015-07-03

    For nearly 4 ½ years, MIT Lincoln Laboratory (MIT/LL) led a very successful, DoE-funded team effort to develop GaN-on-Si materials and devices, targeting high-voltage (>1 kV), high-power, cost-effective electronics for grid applications. This effort, called the GaN Initiative for Grid Applications (GIGA) program, was initially made up of MIT/LL, the MIT campus group of Prof. Tomas Palacios (MIT), and the industrial partner M/A Com Technology Solutions (MTS). Later in the program a 4th team member was added (IQE MA) to provide commercial-scale GaN-on-Si epitaxial materials. A basic premise of the GIGA program was that power electronics, for ubiquitous utilization -evenmore » for grid applications - should be closer in cost structure to more conventional Si-based power electronics. For a number of reasons, more established GaN-on-SiC or even SiC-based power electronics are not likely to reach theses cost structures, even in higher manufacturing volumes. An additional premise of the GIGA program was that the technical focus would be on materials and devices suitable for operating at voltages > 1 kV, even though there is also significant commercial interest in developing lower voltage (< 1 kV), cost effective GaN-on-Si devices for higher volume applications, like consumer products. Remarkable technical progress was made during the course of this program. Advances in materials included the growth of high-quality, crack-free epitaxial GaN layers on large-diameter Si substrates with thicknesses up to ~5 μm, overcoming significant challenges in lattice mismatch and thermal expansion differences between Si and GaN in the actual epitaxial growth process. Such thick epilayers are crucial for high voltage operation of lateral geometry devices such as Schottky barrier (SB) diodes and high electron mobility transistors (HEMTs). New “Normally-Off” device architectures were demonstrated – for safe operation of power electronics circuits. The trade-offs between lateral

  17. Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2.

    PubMed

    Wei, Lei; Wang, Qing; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin; Cao, Ruiwen; Wu, Huifeng; Cong, Ming; Li, Fei; Ji, Chenglong; Zhao, Jianmin

    2015-03-01

    Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Populations of Pacific oysters Crassostrea gigas respond variably to elevated CO2 and predation by Morula marginalba.

    PubMed

    Wright, John M; Parker, Laura M; O'Connor, Wayne A; Williams, Mark; Kube, Peter; Ross, Pauline M

    2014-06-01

    Ocean acidification is anticipated to decrease calcification and increase dissolution of shelled molluscs. Molluscs with thinner and weaker shells may be more susceptible to predation, but not all studies have measured negative responses of molluscs to elevated pCO2. Recent studies measuring the response of molluscs have found greater variability at the population level than first expected. Here we investigate the impact of acidification on the predatory whelk Morula marginalba and genetically distinct subpopulations of the Pacific oyster Crassostrea gigas. Whelks and eight family lines of C. gigas were separately exposed to ambient (385 ppm) and elevated (1000 ppm) pCO2 for 6 weeks. Following this period, individuals of M. marginalba were transferred into tanks with oysters at ambient and elevated pCO2 for 17 days. The increase in shell height of the oysters was on average 63% less at elevated compared to ambient pCO2. There were differences in shell compression strength, thickness, and mass among family lines of C. gigas, with sometimes an interaction between pCO2 and family line. Against expectations, this study found increased shell strength in the prey and reduced shell strength in the predator at elevated compared to ambient pCO2. After 10 days, the whelks consumed significantly more oysters regardless of whether C. gigas had been exposed to ambient or elevated CO2, but this was not dependent on the family line and the effect was not significant after 17 days. Our study found an increase in predation after exposure of the predator to predicted near-future levels of estuarine pCO2. © 2014 Marine Biological Laboratory.

  19. Aqueous sulfate separation by crystallization of sulfate–water clusters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Custelcean, Radu; Williams, Neil J.; Seipp, Charles A.

    An effective approach to separating sulfates from aqueous solutions is based on the crystallization of extended [SO 4(H 2O) 5 2-] n sulfate–water clusters with a bis(guanidinium) ligand. The ligand was generated in situ by hydrazone condensation in water, thus avoiding elaborate syntheses, tedious purifications, and organic solvents. Crystallization of sulfate–water clusters represents an alternative to the now established sulfate separation strategies that involve encapsulating the “naked” anion.

  20. Aqueous sulfate separation by crystallization of sulfate–water clusters

    DOE PAGES

    Custelcean, Radu; Williams, Neil J.; Seipp, Charles A.

    2015-08-07

    An effective approach to separating sulfates from aqueous solutions is based on the crystallization of extended [SO 4(H 2O) 5 2-] n sulfate–water clusters with a bis(guanidinium) ligand. The ligand was generated in situ by hydrazone condensation in water, thus avoiding elaborate syntheses, tedious purifications, and organic solvents. Crystallization of sulfate–water clusters represents an alternative to the now established sulfate separation strategies that involve encapsulating the “naked” anion.

  1. Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space

    NASA Technical Reports Server (NTRS)

    Lee, Chun P.; Chernov, Alexander A.

    2004-01-01

    At least some protein crystals were found to preferentially trap microheterogeneous impurities. The latter are, for example, dimmer molecules of the crystallizing proteines (e.g. ferritin, lysozyme), or the regular molecules on which surfaces small molecules or ions are adsorbed (e.g. acetilated lysozyme) and modi@ molecular charge. Impurities may induce lattice defects and deteriorate structural resolution. Distribution of impurities between mother solution and gorwing crystal is defined by two interrelated distribution coefficients: kappa = rho(sup c, sub 2) and K = (rho(sup c, sub 2)/rho(sup c, sub 1)/rho(sub 2)/rho(sub 1). Here, rho(sub 2), rho(sub 1) and rho(sup c, sub 2) are densities of impurity (2) and regular protein (1) in solution at the growing interface and within the crystal ("c"). For the microheterogeneous impurities studied, K approx. = 2 - 4, so that kappa approx. - 10(exp 2) - 10(exp 3), since K = kappa (rho(sub 1)/rho(sup c, sub 1) and protein solubility ratio rho(sub 1)/rho(sub=p c, sub 2) much less than 1. Therefore, a crystal growing in absence of convection purifies mother solution around itself, grows cleaner and, probably, more perfect. If convection is present, the solution flow permanently brings new impurities to the crystal. This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities.

  2. Examining spatiotemporal distribution and CPUE-environment relationships for the jumbo flying squid Dosidicus gigas offshore Peru based on spatial autoregressive model

    NASA Astrophysics Data System (ADS)

    Feng, Yongjiu; Chen, Xinjun; Liu, Yang

    2017-09-01

    The spatiotemporal distribution and relationship between nominal catch-per-unit-effort (CPUE) and environment for the jumbo flying squid (Dosidicus gigas) were examined in offshore Peruvian waters during 2009-2013. Three typical oceanographic factors affecting the squid habitat were investigated in this research, including sea surface temperature (SST), sea surface salinity (SSS) and sea surface height (SSH). We studied the CPUE-environment relationships for D. gigas using a spatially-lagged version of spatial autoregressive (SAR) model and a generalized additive model (GAM), with the latter for auxiliary and comparative purposes. The annual fishery centroids were distributed broadly in an area bounded by 79.5°-82.7°W and 11.9°-17.1°S, while the monthly fishery centroids were spatially close and lay in a smaller area bounded by 81.0°-81.2°W and 14.3°-15.4°S. Our results show that the preferred environmental ranges for D. gigas offshore Peru were 20.9°-21.9°C for SST, 35.16-35.32 for SSS and 27.2-31.5 cm for SSH in the areas bounded by 78°-80°W/82-84°W and 15°-18°S. Monthly spatial distributions during October to December were predicted using the calibrated GAM and SAR models and general similarities were found between the observed and predicted patterns for the nominal CPUE of D. gigas. The overall accuracies for the hotspots generated by the SAR model were much higher than those produced by the GAM model for all three months. Our results contribute to a better understanding of the spatiotemporal distributions of D. gigas offshore Peru, and offer a new SAR modeling method for advancing fishery science.

  3. GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii

    PubMed Central

    Shuman, Howard A.

    2017-01-01

    ABSTRACT A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as Acinetobacter baumannii, a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of A. baumannii is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying A. baumannii pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTSNtr), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in A. baumannii, and coupling this pathway with the PTSNtr allows A. baumannii to integrate cellular metabolic status with external environmental cues. IMPORTANCE Opportunistic pathogens, including Acinetobacter baumannii, encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in A. baumannii has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we

  4. GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii.

    PubMed

    Gebhardt, Michael J; Shuman, Howard A

    2017-05-15

    A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as Acinetobacter baumannii , a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of A. baumannii is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying A. baumannii pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTS Ntr ), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in A. baumannii , and coupling this pathway with the PTS Ntr allows A. baumannii to integrate cellular metabolic status with external environmental cues. IMPORTANCE Opportunistic pathogens, including Acinetobacter baumannii , encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in A. baumannii has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we

  5. Approaches to automated protein crystal harvesting

    PubMed Central

    Deller, Marc C.; Rupp, Bernhard

    2014-01-01

    The harvesting of protein crystals is almost always a necessary step in the determination of a protein structure using X-ray crystallographic techniques. However, protein crystals are usually fragile and susceptible to damage during the harvesting process. For this reason, protein crystal harvesting is the single step that remains entirely dependent on skilled human intervention. Automation has been implemented in the majority of other stages of the structure-determination pipeline, including cloning, expression, purification, crystallization and data collection. The gap in automation between crystallization and data collection results in a bottleneck in throughput and presents unfortunate opportunities for crystal damage. Several automated protein crystal harvesting systems have been developed, including systems utilizing microcapillaries, microtools, microgrippers, acoustic droplet ejection and optical traps. However, these systems have yet to be commonly deployed in the majority of crystallography laboratories owing to a variety of technical and cost-related issues. Automation of protein crystal harvesting remains essential for harnessing the full benefits of fourth-generation synchrotrons, free-electron lasers and microfocus beamlines. Furthermore, automation of protein crystal harvesting offers several benefits when compared with traditional manual approaches, including the ability to harvest microcrystals, improved flash-cooling procedures and increased throughput. PMID:24637746

  6. DNA barcoding reveals that the common cupped oyster in Taiwan is the Portuguese oyster Crassostrea angulata (Ostreoida; Ostreidae), not C. gigas

    PubMed Central

    Hsiao, Sheng-Tai; Chuang, Shin-Chang; Chen, Kao-Sung; Ho, Ping-Ho; Wu, Chi-Lun; Chen, Chaolun Allen

    2016-01-01

    The Pacific cupped oyster, Crassostrea gigas, is one of the major aquacultural shellfish species that has been introduced to Europe and America from its native source in the West Pacific. In Taiwan, the cultivated cupped oysters along the west coast have been identified as C. gigas for over centuries; however, several molecular phylogenetic studies have cast doubt upon the existence of this species in Taiwan and adjacent waters. Indeed, our analyses of mitochondrial cytochrome oxidase I (COI) sequences from 313 Crassostrea collected from 12 locations along Taiwanese and southern Chinese coastlines confirm that all samples were the Portuguese oyster, C. angulata, rather than C. gigas. Multiple lines of evidence, including haplotypic and nucleotide diversity of the COI gene, demographic history, and population genetics, suggest that Taiwanese C. angulata is unique, probably experienced a sudden population expansion after the Last Glacial Maxima around 20,000 years ago, and has a significantly limited genetic connectivity across the Taiwan Strait. Our study applies an extended sampling and DNA barcoding to confirm the absence of C. gigas in natural and cultivated populations in Taiwan and southern China, where we only found C. angulata. We highlight the importance of conserving the gene pool of the C. angulata population in Taiwan, particularly considering the current threats by large-scale environmental disturbances such as marine pollution, habitat destruction, and climate change. PMID:27666088

  7. DNA barcoding reveals that the common cupped oyster in Taiwan is the Portuguese oyster Crassostrea angulata (Ostreoida; Ostreidae), not C. gigas.

    PubMed

    Hsiao, Sheng-Tai; Chuang, Shin-Chang; Chen, Kao-Sung; Ho, Ping-Ho; Wu, Chi-Lun; Chen, Chaolun Allen

    2016-09-26

    The Pacific cupped oyster, Crassostrea gigas, is one of the major aquacultural shellfish species that has been introduced to Europe and America from its native source in the West Pacific. In Taiwan, the cultivated cupped oysters along the west coast have been identified as C. gigas for over centuries; however, several molecular phylogenetic studies have cast doubt upon the existence of this species in Taiwan and adjacent waters. Indeed, our analyses of mitochondrial cytochrome oxidase I (COI) sequences from 313 Crassostrea collected from 12 locations along Taiwanese and southern Chinese coastlines confirm that all samples were the Portuguese oyster, C. angulata, rather than C. gigas. Multiple lines of evidence, including haplotypic and nucleotide diversity of the COI gene, demographic history, and population genetics, suggest that Taiwanese C. angulata is unique, probably experienced a sudden population expansion after the Last Glacial Maxima around 20,000 years ago, and has a significantly limited genetic connectivity across the Taiwan Strait. Our study applies an extended sampling and DNA barcoding to confirm the absence of C. gigas in natural and cultivated populations in Taiwan and southern China, where we only found C. angulata. We highlight the importance of conserving the gene pool of the C. angulata population in Taiwan, particularly considering the current threats by large-scale environmental disturbances such as marine pollution, habitat destruction, and climate change.

  8. DNA barcoding reveals that the common cupped oyster in Taiwan is the Portuguese oyster Crassostrea angulata (Ostreoida; Ostreidae), not C. gigas

    NASA Astrophysics Data System (ADS)

    Hsiao, Sheng-Tai; Chuang, Shin-Chang; Chen, Kao-Sung; Ho, Ping-Ho; Wu, Chi-Lun; Chen, Chaolun Allen

    2016-09-01

    The Pacific cupped oyster, Crassostrea gigas, is one of the major aquacultural shellfish species that has been introduced to Europe and America from its native source in the West Pacific. In Taiwan, the cultivated cupped oysters along the west coast have been identified as C. gigas for over centuries; however, several molecular phylogenetic studies have cast doubt upon the existence of this species in Taiwan and adjacent waters. Indeed, our analyses of mitochondrial cytochrome oxidase I (COI) sequences from 313 Crassostrea collected from 12 locations along Taiwanese and southern Chinese coastlines confirm that all samples were the Portuguese oyster, C. angulata, rather than C. gigas. Multiple lines of evidence, including haplotypic and nucleotide diversity of the COI gene, demographic history, and population genetics, suggest that Taiwanese C. angulata is unique, probably experienced a sudden population expansion after the Last Glacial Maxima around 20,000 years ago, and has a significantly limited genetic connectivity across the Taiwan Strait. Our study applies an extended sampling and DNA barcoding to confirm the absence of C. gigas in natural and cultivated populations in Taiwan and southern China, where we only found C. angulata. We highlight the importance of conserving the gene pool of the C. angulata population in Taiwan, particularly considering the current threats by large-scale environmental disturbances such as marine pollution, habitat destruction, and climate change.

  9. Relationship between single nucleotide polymorphism of glycogen synthase gene of Pacific oyster Crassostrea gigas and its glycogen content

    NASA Astrophysics Data System (ADS)

    Liu, Siwei; Li, Qi; Yu, Hong; Kong, Lingfeng

    2017-02-01

    Glycogen is important not only for the energy supplementary of oysters, but also for human consumption. High glycogen content can improve the stress survival of oyster. A key enzyme in glycogenesis is glycogen synthase that is encoded by glycogen synthase gene GYS. In this study, the relationship between single nucleotide polymorphisms (SNPs) in coding regions of Crassostrea gigas GYS (Cg-GYS) and individual glycogen content was investigated with 321 individuals from five full-sib families. Single-strand conformation polymorphism (SSCP) procedure was combined with sequencing to confirm individual SNP genotypes of Cg-GYS. Least-square analysis of variance was performed to assess the relationship of variation in glycogen content of C. gigas with single SNP genotype and SNP haplotype. As a consequence, six SNPs were found in coding regions to be significantly associated with glycogen content ( P < 0.01), from which we constructed four main haplotypes due to linkage disequilibrium. Furthermore, the most effective haplotype H2 (GAGGAT) had extremely significant relationship with high glycogen content ( P < 0.0001). These findings revealed the potential influence of Cg-GYS polymorphism on the glycogen content and provided molecular biological information for the selective breeding of good quality traits of C. gigas.

  10. Counting the dead to determine the source and transmission of the marine herpesvirus OsHV-1 in Crassostrea gigas.

    PubMed

    Whittington, Richard J; Paul-Pont, Ika; Evans, Olivia; Hick, Paul; Dhand, Navneet K

    2018-04-10

    Marine herpesviruses are responsible for epizootics in economically, ecologically and culturally significant taxa. The recent emergence of microvariants of Ostreid herpesvirus 1 (OsHV-1) in Pacific oysters Crassostrea gigas has resulted in socioeconomic losses in Europe, New Zealand and Australia however, there is no information on their origin or mode of transmission. These factors need to be understood because they influence the way the disease may be prevented and controlled. Mortality data obtained from experimental populations of C. gigas during natural epizootics of OsHV-1 disease in Australia were analysed qualitatively. In addition we compared actual mortality data with those from a Reed-Frost model of direct transmission and analysed incubation periods using Sartwell's method to test for the type of epizootic, point source or propagating. We concluded that outbreaks were initiated from an unknown environmental source which is unlikely to be farmed oysters in the same estuary. While direct oyster-to-oyster transmission may occur in larger oysters if they are in close proximity (< 40 cm), it did not explain the observed epizootics, point source exposure and indirect transmission being more common and important. A conceptual model is proposed for OsHV-1 index case source and transmission, leading to endemicity with recurrent seasonal outbreaks. The findings suggest that prevention and control of OsHV-1 in C. gigas will require multiple interventions. OsHV-1 in C. gigas, which is a sedentary animal once beyond the larval stage, is an informative model when considering marine host-herpesvirus relationships.

  11. SNP mining in Crassostrea gigas EST data: transferability to four other Crassostrea species, phylogenetic inferences and outlier SNPs under selection.

    PubMed

    Zhong, Xiaoxiao; Li, Qi; Yu, Hong; Kong, Lingfeng

    2014-01-01

    Oysters, with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. Our study is intended to generate new EST-SNP markers and to evaluate their potential for cross-species utilization in phylogenetic study of the genus Crassostrea. In the study, 57 novel SNPs were developed from an EST database of C. gigas by the HRM (high-resolution melting) method. Transferability of 377 SNPs developed for C. gigas was examined on four other Crassostrea species: C. sikamea, C. angulata, C. hongkongensis and C. ariakensis. Among the 377 primer pairs tested, 311 (82.5%) primers showed amplification in C. sikamea, 353 (93.6%) in C. angulata, 254 (67.4%) in C. hongkongensis and 253 (67.1%) in C. ariakensis. A total of 214 SNPs were found to be transferable to all four species. Phylogenetic analyses showed that C. hongkongensis was a sister species of C. ariakensis and that this clade was sister to the clade containing C. sikamea, C. angulata and C. gigas. Within this clade, C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister group. In addition, we detected eight SNPs as potentially being under selection by two outlier tests (fdist and hierarchical methods). The SNPs studied here should be useful for genetic diversity, comparative mapping and phylogenetic studies across species in Crassostrea and the candidate outlier SNPs are worth exploring in more detail regarding association genetics and functional studies.

  12. On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7

    PubMed Central

    Boer, D. Roeland; Müller, Axel; Fetzner, Susanne; Lowe, David J.; Romão, Maria João

    2005-01-01

    Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map. PMID:16508115

  13. Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

    NASA Technical Reports Server (NTRS)

    Feigelson, R. S. (Editor)

    1986-01-01

    Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

  14. Genetics of the Steller's Sea Cow (Hydrodamalis gigas): A Study of Ancient Bone Material

    NASA Astrophysics Data System (ADS)

    Crerar, Lorelei D.

    Georg Wilhelm Steller was born 100 years before Darwin in 1709 and he was part of a vast exploration fifty years before Lewis and Clark explored America. Steller was important to the study of marine mammals because he was the only naturalist to see and describe the great northern sea cow ( Hydrodamalis gigas). Knowledge of an extinct population can be used to aid the conservation of a current population. Extraction of DNA from this extinct animal was performed in order to determine the population structure of the Steller's sea cow. A test was also developed that can definitively state whether or not a random bone sample came from H. gigas. This test could be used by the Fish and Wildlife Service (FWS) and the National Oceanic and Atmospheric Administration (NOAA) to examine material distributed in the North Pacific to determine whether samples are legally traded extinct Steller's sea cow or illegally traded extant marine mammal species protected under the Marine Mammal Protection Act (MMPA).

  15. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin.

    PubMed

    Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J; McHugh, Tara H; Zhang, Yu-Zhu

    2014-08-01

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369-792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121.

  16. Expression, purification and crystallization of pecan (Carya illinoinensis) vicilin

    PubMed Central

    Lee, BoRam; Zhang, Renhao; Du, Wen-Xian; Grauke, Larry J.; McHugh, Tara H.; Zhang, Yu-Zhu

    2014-01-01

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein vicilin has been identified as a food allergen in many kinds of tree nuts. The vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369–792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 Å resolution in space group P212121. PMID:25084379

  17. PredPPCrys: Accurate Prediction of Sequence Cloning, Protein Production, Purification and Crystallization Propensity from Protein Sequences Using Multi-Step Heterogeneous Feature Fusion and Selection

    PubMed Central

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed ‘PredPPCrys’ using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of

  18. PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

    PubMed

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed 'PredPPCrys' using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of currently

  19. Proteomic and metabolomic responses of Pacific oyster Crassostrea gigas to elevated pCO2 exposure.

    PubMed

    Wei, Lei; Wang, Qing; Wu, Huifeng; Ji, Chenglong; Zhao, Jianmin

    2015-01-01

    The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being well understood. In this work, the effects of exposure to elevated pCO2 were characterized in gills and hepatopancreas of Crassostrea gigas using integrated proteomic and metabolomic approaches. Metabolic responses indicated that high CO2 exposure mainly caused disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose, glycogen, amino acids and organic osmolytes in oysters, and the depletions of ATP in gills and the accumulations of ATP, glucose and glycogen in hepatopancreas accounted for the difference in energy distribution between these two tissues. Proteomic responses suggested that OA could not only affect energy and primary metabolisms, stress responses and calcium homeostasis in both tissues, but also influence the nucleotide metabolism in gills and cytoskeleton structure in hepatopancreas. This study demonstrated that the combination of proteomics and metabolomics could provide an insightful view into the effects of OA on oyster C. gigas. The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being understood. To our knowledge, few studies have focused on the responses induced by pCO2 at both protein and metabolite levels. The pacific oyster C. gigas, widely distributed

  20. Studies on the "Aerobic" Acetyl-Coenzyme A Synthetase of Saccharomyces Cerevisiae: Purification, Crystallization, and Physical Properties of the Enzyme

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Klein, Harold P.

    1976-01-01

    A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.

  1. Enhancement of pheochromocytoma nerve cell growth by consecutive fractionization of Angelica gigas Nakai extracts

    PubMed Central

    Jeong, Myoung Hoon; Kim, Ji Seon; Zou, Yunyun; Yoon, Chang Soon; Lim, Hye Won; Ahn, Juhee

    2010-01-01

    This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 μm in adding 50 μg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study. PMID:20936343

  2. The "New Polyethylene Glycol Dilemma": Polyethylene Glycol Impurities and Their Paradox Role in mAb Crystallization.

    PubMed

    Hildebrandt, Christian; Joos, Lea; Saedler, Rainer; Winter, Gerhard

    2015-06-01

    Polyethylene glycols (PEG) represent the most successful and frequently applied class of excipients used for protein crystallization. PEG auto-oxidation and formation of impurities such as peroxides and formaldehydes that foster protein drug degradation is known. However, their effect on mAb crystallization has not been studied in detail before. During the present study, a model IgG1 antibody (mAb1) was crystallized in PEG solutions. Aggregate formation was observed during crystallization and storage that was ascribed to PEG degradation products. Reduction of peroxide and formaldehyde levels prior to crystallization by vacuum and freeze-drying was investigated for its effect on protein degradation. Vacuum drying was superior in removal of peroxides but inferior in reducing formaldehyde residues. Consequently, double purification allowed extensive removal of both impurities. Applying of purified PEG led to 50% lower aggregate fractions. Surprisingly, PEG double purification or addition of methionine prior to crystallization prevented crystal formation. With increased PEG concentration or spiking with peroxides and formaldehydes, crystal formation could be recovered again. With these results, we demonstrate that minimum amounts of oxidizing impurities and thus in consequence chemically altered proteins are vital to initiate mAb1 crystallization. The present study calls PEG as good precipitant for therapeutic biopharmaceuticals into question. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  3. Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meining, Winfried, E-mail: wim@csb.ki.se; Scheuring, Johannes; Fischer, Markus

    2006-06-01

    SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å,more » α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.« less

  4. Genetic variation assessed with microsatellites in mass selection lines of the Pacific oyster ( Crassostrea gigas) in China

    NASA Astrophysics Data System (ADS)

    Wang, Xubo; Li, Qi; Yu, Hong; Kong, Lingfeng

    2016-12-01

    Four successive mass selection lines of the Pacific oyster, Crassostrea gigas, selected for faster growth in breeding programs in China were examined at ten polymorphic microsatellite loci to assess the level of allelic diversity and estimate the effective population size. These data were compared with those of their base population. The results showed that the genetic variation of the four generations were maintained at high levels with an average allelic richness of 18.8-20.6, and a mean expected heterozygosity of 0.902-0.921. They were not reduced compared with those of their base population. Estimated effective population sizes based on temporal variances in microsatellite frequencies were smaller to that of sex ratio-corrected broodstock count estimates. Using a relatively large number of broodstock and keeping an equal sex ratio in the broodstock each generation may have contributed to retaining the original genetic diversity and maintaining relatively large effective population size. The results obtained in this study showed that the genetic variation was not affected greatly by mass selection progress and high genetic variation still existed in the mass selection lines, suggesting that there is still potential for increasing the gains in future generations of C. gigas. The present study provided important information for future genetic improvement by selective breeding, and for the design of suitable management guidelines for genetic breeding of C. gigas.

  5. The sense of hearing in the Pacific oyster, Magallana gigas.

    PubMed

    Charifi, Mohcine; Sow, Mohamedou; Ciret, Pierre; Benomar, Soumaya; Massabuau, Jean-Charles

    2017-01-01

    There is an increasing concern that anthropogenic noise could have a significant impact on the marine environment, but there is still insufficient data for most invertebrates. What do they perceive? We investigated this question in oysters Magallana gigas (Crassostrea gigas) using pure tone exposures, accelerometer fixed on the oyster shell and hydrophone in the water column. Groups of 16 oysters were exposed to quantifiable waterborne sinusoidal sounds in the range of 10 Hz to 20 kHz at various acoustic energies. The experiment was conducted in running seawater using an experimental flume equipped with suspended loudspeakers. The sensitivity of the oysters was measured by recording their valve movements by high-frequency noninvasive valvometry. The tests were 3 min tone exposures including a 70 sec fade-in period. Three endpoints were analysed: the ratio of responding individuals in the group, the resulting changes of valve opening amplitude and the response latency. At high enough acoustic energy, oysters transiently closed their valves in response to frequencies in the range of 10 to <1000 Hz, with maximum sensitivity from 10 to 200 Hz. The minimum acoustic energy required to elicit a response was 0.02 m∙s-2 at 122 dBrms re 1 μPa for frequencies ranging from 10 to 80 Hz. As a partial valve closure cannot be differentiated from a nociceptive response, it is very likely that oysters detect sounds at lower acoustic energy. The mechanism involved in sound detection and the ecological consequences are discussed.

  6. Trace impurities analysis determined by neutron activation in the PbI 2 crystal semiconductor

    NASA Astrophysics Data System (ADS)

    Hamada, M. M.; Oliveira, I. B.; Armelin, M. J.; Mesquita, C. H.

    2003-06-01

    In this work, a methodology for impurity analysis of PbI 2 was studied to investigate the effectiveness of the purification. Commercial salts were purified by the multi passes zone refining and grown by the Bridgman method. To evaluate the purification efficiency, samples from the bottom, middle and upper sections of the ZR ingot were analyzed after 200, 300 and 500 purification passes, by measurements of the impurity concentrations, using the neutron activation analysis (NAA) technique. There was a significant reduction of the impurities according to the purification numbers. The reduction efficiency was different for each element, namely: Au>Mn>Co˜Ag>K˜Br. The impurity concentration of the crystals grown after 200, 300 and 500 passes and the PbI 2 starting material were analyzed by NAA and plasma optical emission spectroscopy.

  7. Production, Purification and Preliminary X-ray Crystallographic Studies of Adeno-Associated Virus Serotype 9

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mitchell, M.; Nam, H; Carter, A

    2009-01-01

    Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetricmore » unit capsid have been determined by molecular-replacement methods and structure determination is in progress.« less

  8. Development of a Medium Density Combined-Species SNP Array for Pacific and European Oysters (Crassostrea gigas and Ostrea edulis).

    PubMed

    Gutierrez, Alejandro P; Turner, Frances; Gharbi, Karim; Talbot, Richard; Lowe, Natalie R; Peñaloza, Carolina; McCullough, Mark; Prodöhl, Paulo A; Bean, Tim P; Houston, Ross D

    2017-07-05

    SNP arrays are enabling tools for high-resolution studies of the genetic basis of complex traits in farmed and wild animals. Oysters are of critical importance in many regions from both an ecological and economic perspective, and oyster aquaculture forms a key component of global food security. The aim of our study was to design a combined-species, medium density SNP array for Pacific oyster ( Crassostrea gigas ) and European flat oyster ( Ostrea edulis ), and to test the performance of this array on farmed and wild populations from multiple locations, with a focus on European populations. SNP discovery was carried out by whole-genome sequencing (WGS) of pooled genomic DNA samples from eight C. gigas populations, and restriction site-associated DNA sequencing (RAD-Seq) of 11 geographically diverse O. edulis populations. Nearly 12 million candidate SNPs were discovered and filtered based on several criteria, including preference for SNPs segregating in multiple populations and SNPs with monomorphic flanking regions. An Affymetrix Axiom Custom Array was created and tested on a diverse set of samples ( n = 219) showing ∼27 K high quality SNPs for C. gigas and ∼11 K high quality SNPs for O. edulis segregating in these populations. A high proportion of SNPs were segregating in each of the populations, and the array was used to detect population structure and levels of linkage disequilibrium (LD). Further testing of the array on three C. gigas nuclear families ( n = 165) revealed that the array can be used to clearly distinguish between both families based on identity-by-state (IBS) clustering parental assignment software. This medium density, combined-species array will be publicly available through Affymetrix, and will be applied for genome-wide association and evolutionary genetic studies, and for genomic selection in oyster breeding programs. Copyright © 2017 Gutierrez et al.

  9. Metabolic Cost of Protein Synthesis in Larvae of the Pacific Oyster (Crassostrea gigas) Is Fixed Across Genotype, Phenotype, and Environmental Temperature.

    PubMed

    Lee, Jimmy W; Applebaum, Scott L; Manahan, Donal T

    2016-06-01

    The energy made available through catabolism of specific biochemical reserves is constant using standard thermodynamic conversion equivalents (e.g., 24.0 J mg protein(-1)). In contrast, measurements reported for the energy cost of synthesis of specific biochemical constituents are highly variable. In this study, we measured the metabolic cost of protein synthesis and determined whether this cost was influenced by genotype, phenotype, or environment. We focused on larval stages of the Pacific oyster Crassostrea gigas, a species that offers several experimental advantages: availability of genetically pedigreed lines, manipulation of ploidy, and tractability of larval forms for in vivo studies of physiological processes. The cost of protein synthesis was measured in larvae of C. gigas for 1) multiple genotypes, 2) phenotypes with different growth rates, and 3) different environmental temperatures. For all treatments, the cost of protein synthesis was within a narrow range--near the theoretical minimum--with a fixed cost (mean ± one standard error, n = 21) of 2.1 ± 0.2 J (mg protein synthesized)(-1) We conclude that there is no genetic variation in the metabolic cost of protein synthesis, thereby simplifying bioenergetic models. Protein synthesis is a major component of larval metabolism in C. gigas, accounting for more than half the metabolic rate in diploid (59%) and triploid larvae (54%). These results provide measurements of metabolic cost of protein synthesis in larvae of C. gigas, an indicator species for impacts of ocean change, and provide a quantitative basis for evaluating the cost of resilience. © 2016 Marine Biological Laboratory.

  10. [Resolution of chiral molecules of pharmaceutical interest by means of preferential crystallization].

    PubMed

    Coquerel, G

    2009-07-01

    Various aspects of the chiral discrimination in the solid state are examined. The interests of the conglomerate are illustrated by two applications: the preparative enantiomeric purification and the preferential crystallization. The latter process is described by a careful examination of the heterogeneous equilibria that govern the crystallization and its selectivity. Two variants of the preferential crystallization are detailed. A "good" example illustrates the productivity at the laboratory scale. The ratio between homochiral interaction energies and heterochiral interaction energies at different (hkl) interfaces are involved in the "difficult" cases where the entrainment effect is limited.

  11. Reversible stress softening of collagen based networks from the jumbo squid mantle (Dosidicus gigas).

    PubMed

    Torres, F G; Troncoso, O P; Rivas, E R; Gomez, C G; Lopez, D

    2014-04-01

    Dosidicus gigas is the largest and one of the most abundant jumbo squids in the eastern Pacific Ocean. In this paper we have studied the muscle of the mantle of D. gigas (DGM). Morphological, thermal and rheological properties were assessed by means of atomic force microscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy, differential scanning calorimetry, thermogravimetry and oscillatory rheometry. This study allowed us to assess the morphological and rheological properties of a collagen based network occurring in nature. The results showed that the DGM network displays a nonlinear effect called reversible stress softening (RSS) that has been previously described for other types of biological structures such as naturally occurring cellulose networks and actin networks. We propose that the RSS could play a key role on the way jumbo squids withstand hydrostatic pressure. The results presented here confirm that this phenomenon occurs in a wider number of materials than previously thought, all of them exhibiting different size scales as well as physical conformation. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Anti-Inflammatory Effect of Angelica gigas via Heme Oxygenase (HO)-1 Expression.

    PubMed

    Cho, Joon Hyeong; Kwon, Jung Eun; Cho, Youngmi; Kim, Inhye; Kang, Se Chan

    2015-06-15

    Angelica gigas (AG) is effective against various medical conditions such as bacterial infection, inflammation, and cancer. It contains a number of coumarin compounds and the group of interest is the pyranocoumarin, which comprises decursin and decursinol angelate. This group has an effect on controlling inflammation, which is caused by excessive nitric oxide (NO) production. Heme oxygenases (HOs), particularly HO-1, play a role in regulating the production of NO. Thus, this study aimed to investigate the anti-inflammatory effects of AG by measuring HO-1 expression. Treatments with CH2Cl2 layer and Angelica gigas extract (AGE) showed the highest NO inhibition effects. Decursin, decursinol angelate, and nodakenin were isolated from the CH2Cl2 layer of AGE. Decursin also demonstrated the highest anti-oxidative effect among the coumarins. Although decursin had the best NO inhibition and anti-oxidative effects, the effects of AGE treatment far surpassed that of decursin. This is owing to the combination effect of the coumarins present within AGE, which is a solvent extract of AG. The expression of HO-1 is an effective indicator of the anti-inflammatory effects of AG. Based on the results of the coumarin compounds, HO-1 expression was found to be dose dependent and specific to decursin.

  13. Anti-Inflammatory Effect of Angelica gigas via Heme Oxygenase (HO)-1 Expression

    PubMed Central

    Cho, Joon Hyeong; Kwon, Jung Eun; Cho, Youngmi; Kim, Inhye; Kang, Se Chan

    2015-01-01

    Angelica gigas (AG) is effective against various medical conditions such as bacterial infection, inflammation, and cancer. It contains a number of coumarin compounds and the group of interest is the pyranocoumarin, which comprises decursin and decursinol angelate. This group has an effect on controlling inflammation, which is caused by excessive nitric oxide (NO) production. Heme oxygenases (HOs), particularly HO-1, play a role in regulating the production of NO. Thus, this study aimed to investigate the anti-inflammatory effects of AG by measuring HO-1 expression. Treatments with CH2Cl2 layer and Angelica gigas extract (AGE) showed the highest NO inhibition effects. Decursin, decursinol angelate, and nodakenin were isolated from the CH2Cl2 layer of AGE. Decursin also demonstrated the highest anti-oxidative effect among the coumarins. Although decursin had the best NO inhibition and anti-oxidative effects, the effects of AGE treatment far surpassed that of decursin. This is owing to the combination effect of the coumarins present within AGE, which is a solvent extract of AG. The expression of HO-1 is an effective indicator of the anti-inflammatory effects of AG. Based on the results of the coumarin compounds, HO-1 expression was found to be dose dependent and specific to decursin. PMID:26083119

  14. Introduction to protein crystallization

    PubMed Central

    McPherson, Alexander; Gavira, Jose A.

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid–liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies. PMID:24419610

  15. Introduction to protein crystallization.

    PubMed

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  16. Results and insights from the NCSU Insect Museum GigaPan project

    PubMed Central

    Bertone, Matthew A.; Blinn, Robert L.; Dew1, Tanner M. StanfieldKelly J.; Seltmann, Katja C.; Deans, Andrew R.

    2012-01-01

    Abstract Pinned insect specimens stored in museum collections are a fragile and valuable resource for entomological research. As such, they are usually kept away from viewing by the public and hard to access by experts. Here we present a method for mass imaging insect specimens, using GigaPan technology to achieve highly explorable, many-megapixel panoramas of insect museum drawers. We discuss the advantages and limitations of the system, and describe future avenues of collections research using this technology. PMID:22859883

  17. Succinonitrile Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Succinonitrile (SCN) Purification Facility provides succinonitrile and succinonitrile alloys to several NRA selected investigations for flight and ground research at various levels of purity. The purification process employed includes both distillation and zone refining. Once the appropriate purification process is completed, samples are characterized to determine the liquidus and/or solidus temperature, which is then related to sample purity. The lab has various methods for measuring these temperatures with accuracies in the milliKelvin to tenths of milliKelvin range. The ultra-pure SCN produced in our facility is indistinguishable from the standard material provided by NIST to well within the stated +/- 1.5mK of the NIST triple point cells. In addition to delivering material to various investigations, our current activities include process improvement, characterization of impurities and triple point cell design and development. The purification process is being evaluated for each of the four vendors to determine the efficacy of each purification step. We are also collecting samples of the remainder from distillation and zone refining for analysis of the constituent impurities. The large triple point cells developed will contain SCN with a melting point of 58.0642 C +/- 1.5mK for use as a calibration standard for Standard Platinum Resistance Thermometers (SPRTs).

  18. Comprehensive model of Jumbo squid Dosidicus gigas trophic ecology in the Northern Humboldt current system.

    PubMed

    Alegre, Ana; Ménard, Frédéric; Tafur, Ricardo; Espinoza, Pepe; Argüelles, Juan; Maehara, Víctor; Flores, Oswaldo; Simier, Monique; Bertrand, Arnaud

    2014-01-01

    The jumbo squid Dosidicus gigas plays an important role in marine food webs both as predator and prey. We investigated the ontogenetic and spatiotemporal variability of the diet composition of jumbo squid in the northern Humboldt Current system. For that purpose we applied several statistical methods to an extensive dataset of 3,618 jumbo squid non empty stomachs collected off Peru from 2004 to 2011. A total of 55 prey taxa was identified that we aggregated into eleven groups. Our results evidenced a large variability in prey composition as already observed in other systems. However, our data do not support the hypothesis that jumbo squids select the most abundant or energetic taxon in a prey assemblage, neglecting the other available prey. Indeed, multinomial model predictions showed that stomach fullness increased with the number of prey taxa, while most stomachs with low contents contained one or two prey taxa only. Our results therefore question the common hypothesis that predators seek locally dense aggregations of monospecific prey. In addition D. gigas consumes very few anchovy Engraulis ringens in Peru, whereas a tremendous biomass of anchovy is potentially available. It seems that D. gigas cannot reach the oxygen unsaturated waters very close to the coast, where the bulk of anchovy occurs. Indeed, even if jumbo squid can forage in hypoxic deep waters during the day, surface normoxic waters are then required to recover its maintenance respiration (or energy?). Oxygen concentration could thus limit the co-occurrence of both species and then preclude predator-prey interactions. Finally we propose a conceptual model illustrating the opportunistic foraging behaviour of jumbo squid impacted by ontogenetic migration and potentially constrained by oxygen saturation in surface waters.

  19. A novel family of cyclic oligopeptides derived from ribosomal peptide synthesis of an in planta-induced gene, gigA, in Epichloë endophytes of grasses.

    PubMed

    Johnson, Richard D; Lane, Geoffrey A; Koulman, Albert; Cao, Mingshu; Fraser, Karl; Fleetwood, Damien J; Voisey, Christine R; Dyer, Jolon M; Pratt, Jennifer; Christensen, Michael; Simpson, Wayne R; Bryan, Gregory T; Johnson, Linda J

    2015-12-01

    Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Exploitable Lipids and Fatty Acids in the Invasive Oyster Crassostrea gigas on the French Atlantic Coast

    PubMed Central

    Dagorn, Flore; Couzinet-Mossion, Aurélie; Kendel, Melha; Beninger, Peter G.; Rabesaotra, Vony; Barnathan, Gilles; Wielgosz-Collin, Gaëtane

    2016-01-01

    Economic exploitation is one means to offset the cost of controlling invasive species, such as the introduced Pacific oyster (Crassostrea gigas Thunberg) on the French Atlantic coast. Total lipid and phospholipid (PL) fatty acids (FAs) and sterols were examined in an invasive population of C. gigas in Bourgneuf Bay, France, over four successive seasons, with a view to identify possible sources of exploitable substances. The total lipid level (% dry weight) varied from 7.1% (winter) to 8.6% (spring). Of this, PLs accounted for 28.1% (spring) to 50.4% (winter). Phosphatidylcholine was the dominant PL throughout the year (up to 74% of total PLs in winter). Plasmalogens were identified throughout the year as a series of eleven dimethylacetals (DMAs) with chain lengths between C16 and C20 (up to 14.5% of PL FAs + DMAs in winter). Thirty-seven FAs were identified in the PL FAs. Eicosapentaenoic acid (20:5n-3 EPA/7.53% to 14.5%) and docosahexaenoic acid (22:6n-3 DHA/5.51% to 9.5%) were the dominant polyunsaturated FAs in all seasons. Two non-methylene-interrupted dienoic (NMID) FAs were identified in all seasons: 7,13-docosadienoic and 7,15-docosadienoic acids, the latter being present at relatively high levels (up to 9.6% in winter). Twenty free sterols were identified, including cholesterol at 29.9% of the sterol mixture and about 33% of phytosterols. C. gigas tissues thus contained exploitable lipids for health benefits or as a potential source of high-quality commercial lecithin. PMID:27231919

  1. Crystallization of Membrane Proteins by Vapor Diffusion

    PubMed Central

    Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

  2. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1995-01-01

    During the fifth semi-annual period under this grant we have pursued the following activities: (1) Characterization of the purity and further purification of lysozyme solutions, these efforts are summarized in Section 2; (2) Crystal growth morphology and kinetics studies with tetragonal lysozyme, our observation on the dependence of lysozyme growth kinetics on step sources and impurities has been summarized in a manuscript which was accepted for publication in the Journal of Crystal Growth; (3) Numerical modelling of the interaction between bulk transport and interface kinetics, for a detailed summary of this work see the manuscript which was accepted for publication in the Journal of Crystal Growth; and (4) Light scattering studies, this work has been summarized in a manuscript that has been submitted for publication to the Journal of Chemical Physics.

  3. Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

    PubMed

    Jandaruang, Jinda; Siritapetawee, Jaruwan; Songsiriritthigul, Chomphunuch; Preecharram, Sutthidech; Azuma, Taoka; Dhiravisit, Apisak; Fukumori, Yoshihiro; Thammasirirak, Sompong

    2014-08-01

    Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.

  4. Liquid Crystals of Dendron-Like Pt Complexes Processable Into Nanofilms Dendrimers. Phase 2. Cholesteric Liquid Crystal Glass Platinum Acetylides

    DTIC Science & Technology

    2014-08-01

    Std. Z39.18 Final Report Liquid Crystals of Dendron-Like Pt Complexes Processable Into Nanofilms. Dendrimers Eduardo Arias...to pack and also the presence of a polar group. Figure 4. Summary of phase behavior. DENDRIMERS New Denrimers. The synthesis...purification and some spectral characteristics of the new dendrimers shown in Fig 5 were reported in AFOSR FA9550-11-1-0169, May, 2013. Further

  5. The sense of hearing in the Pacific oyster, Magallana gigas

    PubMed Central

    Charifi, Mohcine; Sow, Mohamedou; Ciret, Pierre; Benomar, Soumaya

    2017-01-01

    There is an increasing concern that anthropogenic noise could have a significant impact on the marine environment, but there is still insufficient data for most invertebrates. What do they perceive? We investigated this question in oysters Magallana gigas (Crassostrea gigas) using pure tone exposures, accelerometer fixed on the oyster shell and hydrophone in the water column. Groups of 16 oysters were exposed to quantifiable waterborne sinusoidal sounds in the range of 10 Hz to 20 kHz at various acoustic energies. The experiment was conducted in running seawater using an experimental flume equipped with suspended loudspeakers. The sensitivity of the oysters was measured by recording their valve movements by high-frequency noninvasive valvometry. The tests were 3 min tone exposures including a 70 sec fade-in period. Three endpoints were analysed: the ratio of responding individuals in the group, the resulting changes of valve opening amplitude and the response latency. At high enough acoustic energy, oysters transiently closed their valves in response to frequencies in the range of 10 to <1000 Hz, with maximum sensitivity from 10 to 200 Hz. The minimum acoustic energy required to elicit a response was 0.02 m∙s-2 at 122 dBrms re 1 μPa for frequencies ranging from 10 to 80 Hz. As a partial valve closure cannot be differentiated from a nociceptive response, it is very likely that oysters detect sounds at lower acoustic energy. The mechanism involved in sound detection and the ecological consequences are discussed. PMID:29069092

  6. New Insight for the Genetic Evaluation of Resistance to Ostreid Herpesvirus Infection, a Worldwide Disease, in Crassostrea gigas.

    PubMed

    Dégremont, Lionel; Lamy, Jean-Baptiste; Pépin, Jean-François; Travers, Marie-Agnès; Renault, Tristan

    2015-01-01

    The Pacific oyster, Crassostrea gigas, is the most important commercial oyster species cultivated in the world. Meanwhile, the ostreid herpesvirus 1 (OsHV-1) is one of the major pathogens affecting the Pacific oyster, and numerous mortality outbreaks related to this pathogen are now reported worldwide. To assess the genetic basis of resistance to OsHV-1 infection in spat C. gigas and to facilitate breeding programs for such a trait, if any exist, we compared the mortality of half- and full-sib families using three field methods and a controlled challenge by OsHV-1 in the laboratory. In the field, three methods were tested: (A) one family per bag; (B) one family per small soft mesh bag and all families inside one bag; (C) same as the previous methods but the oysters were individually labelled and then mixed. The mean mortality ranged from 80 to 82% and was related to OsHV-1 based on viral DNA detection. The narrow-sense heritability for mortality, and thus OsHV-1 resistance, ranged from 0.49 to 0.60. The high positive genetic correlations across the field methods suggested no genotype by environment interaction. Ideally, selective breeding could use method B, which is less time- and space-consuming. The narrow sense heritability for mortality under OsHV-1 challenge was 0.61, and genetic correlation between the field and the laboratory was ranged from 0.68 to 0.75, suggesting a weak genotype by environment interaction. Thus, most of families showing the highest survival performed well in field and laboratory conditions, and a similar trend was also observed for families with the lowest survival. In conclusion, this is the first study demonstrating a large additive genetic variation for resistance to OsHV-1 infection in C. gigas, regardless of the methods used, which should help in selective breeding to improve resistance to viral infection in C. gigas.

  7. Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

    PubMed Central

    Gibbons, Don L.; Reilly, Brigid; Ahn, Anna; Vaney, Marie-Christine; Vigouroux, Armelle; Rey, Felix A.; Kielian, Margaret

    2004-01-01

    The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein. PMID:15016874

  8. Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate air-breathing teleost from the Amazon.

    PubMed

    Brauner, C J; Matey, V; Wilson, J M; Bernier, N J; Val, A L

    2004-04-01

    The transition from aquatic to aerial respiration is associated with dramatic physiological changes in relation to gas exchange, ion regulation, acid-base balance and nitrogenous waste excretion. Arapaima gigas is one of the most obligate extant air-breathing fishes, representing a remarkable model system to investigate (1) how the transition from aquatic to aerial respiration affects gill design and (2) the relocation of physiological processes from the gills to the kidney during the evolution of air-breathing. Arapaima gigas undergoes a transition from water- to air-breathing during development, resulting in striking changes in gill morphology. In small fish (10 g), the gills are qualitatively similar in appearance to another closely related water-breathing fish (Osteoglossum bicirrhosum); however, as fish grow (100-1000 g), the inter-lamellar spaces become filled with cells, including mitochondria-rich (MR) cells, leaving only column-shaped filaments. At this stage, there is a high density of MR cells and strong immunolocalization of Na(+)/K(+)-ATPase along the outer cell layer of the gill filament. Despite the greatly reduced overall gill surface area, which is typical of obligate air-breathing fish, the gills may remain an important site for ionoregulation and acid-base regulation. The kidney is greatly enlarged in A. gigas relative to that in O. bicirrhosum and may comprise a significant pathway for nitrogenous waste excretion. Quantification of the physiological role of the gill and the kidney in A. gigas during development and in adults will yield important insights into developmental physiology and the evolution of air-breathing.

  9. A comparison of fishery biology of jumbo flying squid, Dosidicus gigas outside three Exclusive Economic Zones in the Eastern Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Liu, Bilin; Chen, Xinjun; Yi, Qian

    2013-05-01

    Although many studies on the fishery biology of jumbo flying squid, Dosidicus gigas, have been conducted in the coastal areas within Exclusive Economic Zones (EEZs) of various countries due to its commercial and ecological importance, limited biological information is available from waters outside these EEZs. In this paper, we examined D. gigas fishery biology from waters outside Chilean, Peruvian and Costa Rican EEZs, based on the fishery data collected by Chinese jigging vessels during 2006 to 2010. The dominant mantle lengths of D. gigas were 350-450 mm, 250-400 mm and 250-350 mm outside Chilean, Peruvian and Costa Rican EEZs, respectively. Size structure analysis show that a medium-sized group existed mostly in the waters outside the Chilean and Peruvian EEZs, whereas a small-sized group occurred mainly in the waters outside the Costa Rican EEZ. The longevity of the squid outside the Costa Rican EEZ was less than 10 months, while most of those outside Chilean and Peruvian EEZs were about 1-1.5 years and very few large individuals were 1.5-2 years old. A higher percentage of mature individuals existed outside Costa Rican EEZ implying the region as a potential spawning ground, while lower proportions of mature squid outside the Peruvian and Chilean EEZs indicated that spawning may be occurring outside our study area. Spatial differences in sizes at maturity of the squid are thought to be result from different environmental factors especially different temperature and nutrition among the three areas. Stomach-content analysis showed that cannibalism was important in the diet of D. gigas. Stress generated by jigging may increase the incidence of cannibalism.

  10. Isolation, purification, crystallization, and preliminary X-ray diffraction study of the crystals of HU protein from M. gallisepticum

    NASA Astrophysics Data System (ADS)

    Nikolaeva, A. Yu.; Timofeev, V. I.; Boiko, K. M.; Korzhenevskii, D. A.; Rakitina, T. V.; Dorovatovskii, P. V.; Lipkin, A. V.

    2015-11-01

    HU proteins are involved in bacterial DNA and RNA repair. Since these proteins are absent in cells of higher organisms, inhibitors of HU proteins can be used as effective and safe antibiotics. The crystallization conditions for the M. gallisepticum HU protein were found and optimized by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.91 Å resolution from the crystals grown by the vapor-diffusion method on a synchrotron source. The crystals of the HU protein belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 97.94 Å, c = 77.92 Å, α = β = γ = 90°.

  11. Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bacik, John-Paul; Brigley, Angela M.; Channon, Lisa D.

    2005-06-01

    Ectromelia virus glutaredoxin has been crystallized in the presence of the reducing agent DTT. A diffraction data set has been collected and processed to 1.8 Å resolution. Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized.more » EVM053 crystallizes in space group C222{sub 1}, with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 Å. Diffraction data have been processed to 1.8 Å resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.« less

  12. "Two-dimensional" molecularly imprinted solid-phase extraction coupled with crystallization and high performance liquid chromatography for fast semi-preparative purification of tannins from pomegranate husk extract.

    PubMed

    Sun, Guangying; Liu, Yanfang; Ahat, Hasanjan; Shen, Aijin; Liang, Xinmiao; Xue, Xingya; Luo, Yuqin; Yang, Jian; Liu, Zhao-Sheng; Aisa, Haji Akber

    2017-07-07

    In this study, "two dimensional" molecularly imprinted solid-phase extraction (2D-MIP-SPE) of semi-preparative grade was constructed to fast purify ellagitannins in pomegranate husk extract with the help of crystallization and reverse-phase liquid chromatgoraphy (RPLC). Ellagic acid and punicalagin imprinted polymers were synthesized in batch mode and two semi-preparative MIP-SPE columns were individually packed. After investigaing "functional complementation", 2D-MIP-SPE was constructed using ellagic acid MIP and punicalagin MIP-SPE as the first and second dimension, respectively. Then, pomegranate husk extract was fast divided into four fractions individually enriching in ellagic acid, granatin A, punicalagin and ellagic acid glucoside by 2D-MIP-SPE. With the aid of crystallization and RPLC, ellagic acid (13.5mg) and punicalagin (53.4mg) were fast obtained in 30min. Ellagic acid glucoside was purified to the purity near 100% with a recovery of 86.1%. Granatin A (92%) was directly obtained by 2D-MIP-SPE with the recovery of 81.8%. All above indicated that 2D-MIP-SPE was highly efficient in natural product purification. The concept of "functional complementation" was expected to be a useful tool in the construction of 2D-MIP-SPE. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Identification and Functional Characterization of the Glycogen Synthesis Related Gene Glycogenin in Pacific Oysters (Crassostrea gigas).

    PubMed

    Li, Busu; Meng, Jie; Li, Li; Liu, Sheng; Wang, Ting; Zhang, Guofan

    2017-09-06

    High glycogen levels in the Pacific oyster (Crassostrea gigas) contribute to its flavor, quality, and hardiness. Glycogenin (CgGN) is the priming glucosyltransferase that initiates glycogen biosynthesis. We characterized the full sequence and function of C. gigas CgGN. Three CgGN isoforms (CgGN-α, β, and γ) containing alternative exon regions were isolated. CgGN expression varied seasonally in the adductor muscle and gonadal area and was the highest in the adductor muscle. Autoglycosylation of CgGN can interact with glycogen synthase (CgGS) to complete glycogen synthesis. Subcellular localization analysis showed that CgGN isoforms and CgGS were located in the cytoplasm. Additionally, a site-directed mutagenesis experiment revealed that the Tyr200Phe and Tyr202Phe mutations could affect CgGN autoglycosylation. This is the first study of glycogenin function in marine bivalves. These findings will improve our understanding of glycogen synthesis and accumulation mechanisms in mollusks. The data are potentially useful for breeding high-glycogen oysters.

  14. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Åmore » resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.« less

  15. Giga-pixel lensfree holographic microscopy and tomography using color image sensors.

    PubMed

    Isikman, Serhan O; Greenbaum, Alon; Luo, Wei; Coskun, Ahmet F; Ozcan, Aydogan

    2012-01-01

    We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2). This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ± 50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3) across a sample volume of ~5 mm(3), which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

  16. Formation of crystal-like structures and branched networks from nonionic spherical micelles

    NASA Astrophysics Data System (ADS)

    Cardiel, Joshua J.; Furusho, Hirotoshi; Skoglund, Ulf; Shen, Amy Q.

    2015-12-01

    Crystal-like structures at nano and micron scales have promise for purification and confined reactions, and as starting points for fabricating highly ordered crystals for protein engineering and drug discovery applications. However, developing controlled crystallization techniques from batch processes remain challenging. We show that neutrally charged nanoscale spherical micelles from biocompatible nonionic surfactant solutions can evolve into nano- and micro-sized branched networks and crystal-like structures. This occurs under simple combinations of temperature and flow conditions. Our findings not only suggest new opportunities for developing controlled universal crystallization and encapsulation procedures that are sensitive to ionic environments and high temperatures, but also open up new pathways for accelerating drug discovery processes, which are of tremendous interest to pharmaceutical and biotechnological industries.

  17. Polymorph selection: the role of nucleation, crystal growth and molecular modeling.

    PubMed

    Erdemir, Deniz; Lee, Alfred Y; Myerson, Allan S

    2007-11-01

    Solution crystallization is an important separation and purification process used in the chemical, pharmaceutical and food industries. The quality of a crystalline product is generally judged by four main criteria: purity, crystal habit, particle size and solid form. Consistent production of the desired polymorph is crucial as the unanticipated emergence of a different crystal form may have severe consequences. Thus, the selection of a solid-state form for a crystalline product is vital and is ultimately based on knowledge of the properties of the other polymorphs. This review discusses the role of nucleation, crystal growth and molecular modeling on polymorphism in molecular crystals. Examples are presented demonstrating how the first two factors can govern the appearance of a particular crystalline form, and how the latter factor can be used as a tool for understanding polymorphism.

  18. The Influence of Surface Morphology and Diffraction Resolution of Canavalin Crystals

    NASA Technical Reports Server (NTRS)

    Plomp, M.; Thomas, B. R.; Day, J. S.; McPherson, A.; Chernov, A. A.; Malkin, A.

    2003-01-01

    Canavalin crystals grown from material purified and not purified by High Performance Liquid Chromatography were studied by atomic force microscopy and x-ray diffraction. After purification, resolution was improved from 2.55Angstroms to 2.22Angstroms and jagged isotropic spiral steps transformed into regular, well polygonized steps.

  19. Comprehensive Model of Jumbo Squid Dosidicus gigas Trophic Ecology in the Northern Humboldt Current System

    PubMed Central

    Alegre, Ana; Ménard, Frédéric; Tafur, Ricardo; Espinoza, Pepe; Argüelles, Juan; Maehara, Víctor; Flores, Oswaldo; Simier, Monique; Bertrand, Arnaud

    2014-01-01

    The jumbo squid Dosidicus gigas plays an important role in marine food webs both as predator and prey. We investigated the ontogenetic and spatiotemporal variability of the diet composition of jumbo squid in the northern Humboldt Current system. For that purpose we applied several statistical methods to an extensive dataset of 3,618 jumbo squid non empty stomachs collected off Peru from 2004 to 2011. A total of 55 prey taxa was identified that we aggregated into eleven groups. Our results evidenced a large variability in prey composition as already observed in other systems. However, our data do not support the hypothesis that jumbo squids select the most abundant or energetic taxon in a prey assemblage, neglecting the other available prey. Indeed, multinomial model predictions showed that stomach fullness increased with the number of prey taxa, while most stomachs with low contents contained one or two prey taxa only. Our results therefore question the common hypothesis that predators seek locally dense aggregations of monospecific prey. In addition D. gigas consumes very few anchovy Engraulis ringens in Peru, whereas a tremendous biomass of anchovy is potentially available. It seems that D. gigas cannot reach the oxygen unsaturated waters very close to the coast, where the bulk of anchovy occurs. Indeed, even if jumbo squid can forage in hypoxic deep waters during the day, surface normoxic waters are then required to recover its maintenance respiration (or energy?). Oxygen concentration could thus limit the co-occurrence of both species and then preclude predator-prey interactions. Finally we propose a conceptual model illustrating the opportunistic foraging behaviour of jumbo squid impacted by ontogenetic migration and potentially constrained by oxygen saturation in surface waters. PMID:24465788

  20. The jumbo squid, Dosidicus gigas (Ommastrephidae), living in oxygen minimum zones I: Oxygen consumption rates and critical oxygen partial pressures

    NASA Astrophysics Data System (ADS)

    Trueblood, Lloyd A.; Seibel, Brad A.

    2013-10-01

    Dosidicus gigas is a large, metabolically active, epipelagic squid known to undertake diel vertical migrations across a large temperature and oxygen gradient in the Eastern Pacific. Hypoxia is known to cause metabolic suppression in D. gigas. However, the precise oxygen level at which metabolic suppression sets in is unknown. Here we describe a novel ship-board swim tunnel respirometer that was used to measure metabolic rates and critical oxygen partial pressures (Pcrit) for adult squids (2-7kg). Metabolic rate measurements were validated by comparison to the activity of the Krebs cycle enzyme, citrate synthase, in mantle muscle tissue (2-17kg). We recorded a mean routine metabolic rate of 5.91μmolg-1h-1 at 10°C and 12.62μmolg-1h-1 at 20°C. A temperature coefficient, Q10, of 2.1 was calculated. D. gigas had Pcrits of 1.6 and 3.8kPa at 10 and 20°C, respectively. Oxygen consumption rate (MO2) varied with body mass (M) according to MO2=11.57M-0.12±0.03 at 10°C. Citrate synthase activity varied with body mass according to Y=9.32M-0.19±0.02.

  1. The bubble method of water purification

    NASA Astrophysics Data System (ADS)

    Smirnov, B. M.; Babaeva, N. Yu.; Naidis, G. V.; Panov, V. A.; Saveliev, A. S.; Son, E. E.; Tereshonok, D. V.

    2018-02-01

    The processes of water purification from admixture molecules are analyzed. The purification rate is limited due to a low diffusion coefficient of the admixture molecules in water. At non-small concentrations of the admixture molecules, the water purication can proceed through association of molecules in condensed nanoparticles which fall on the bottom of the water volume. The rate of association may be increased in an external electric field, but in reality this cannot change significantly the rate of the purification process. The bubble method of water purification is considered, where air bubbles formed at the bottom of the water volume, transfer admixture molecules to the interface. This method allows one to clean small water volumes fast. This mechanism of water purification is realized experimentally and exhibits the promises of the bubble purification method.

  2. Gonad Transcriptome Analysis of the Pacific Oyster Crassostrea gigas Identifies Potential Genes Regulating the Sex Determination and Differentiation Process.

    PubMed

    Yue, Chenyang; Li, Qi; Yu, Hong

    2018-04-01

    The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide. C. gigas has a fascinating sexual reproduction system consisting of dioecism, sex change, and occasional hermaphroditism, while knowledge of the molecular mechanisms of sex determination and differentiation is still limited. In this study, the transcriptomes of male and female gonads at different gametogenesis stages were characterized by RNA-seq. Hierarchical clustering based on genes differentially expressed revealed that 1269 genes were expressed specifically in female gonads and 817 genes were expressed increasingly over the course of spermatogenesis. Besides, we identified two and one gene modules related to female and male gonad development, respectively, using weighted gene correlation network analysis (WGCNA). Interestingly, GO and KEGG enrichment analysis showed that neurotransmitter-related terms were significantly enriched in genes related to ovary development, suggesting that the neurotransmitters were likely to regulate female sex differentiation. In addition, two hub genes related to testis development, lncRNA LOC105321313 and Cg-Sh3kbp1, and one hub gene related to ovary development, Cg-Malrd1-like, were firstly investigated. This study points out the role of neurotransmitter and non-coding RNA regulation during gonad development and produces lists of novel relevant candidate genes for further studies. All of these provided valuable information to understand the molecular mechanisms of C. gigas sex determination and differentiation.

  3. Semiconductor Grade, Solar Silicon Purification Project. [photovoltaic solar energy conversion

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Rosler, R. S.; Thompson, S. W.; Chaney, R. E.

    1979-01-01

    A low cost by-product, SiF4, is reacted with mg silicon to form SiF2 gas which is polymerized. The (SiF2)x polymer is heated forming volatile SixFy homologues which disproportionate on a silicon particle bed forming silicon and SiF4. The silicon analysis procedure relied heavily on mass spectroscopic and emission spectroscopic analysis. These analyses demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). However, electrical analysis via crystal growth reveal that the product contains compensated phosphorus and boron.

  4. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    PubMed Central

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

    2016-01-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

  5. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    DOE PAGES

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

    2016-03-01

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

  6. In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin

    The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

  7. Bioaccumulation of trace metals in farmed pacific oysters Crassostrea gigas from SW Gulf of California coast, Mexico.

    PubMed

    Jonathan, M P; Muñoz-Sevilla, N P; Góngora-Gómez, Andrés Martin; Luna Varela, Raquel Gabriela; Sujitha, S B; Escobedo-Urías, D C; Rodríguez-Espinosa, P F; Campos Villegas, Lorena Elizabeth

    2017-11-01

    The aim of the study was to evaluate the bioavailability of trace metals (Chromium, Copper, Nickel, Lead, Zinc, Cadmium, Arsenic, and Mercury) in the commercially consumed Crassostrea gigas oysters collected over a 12-month growth period (2011-12) from an experimental cultivation farm in La Pitahaya, Sinaloa State, Mexico. Sediment and water samples were also collected from four different zones adjacent to the cultivation area to identify the concentration patterns of metals. The results revealed that sewage disposals, fertilizers used for agricultural practices and shrimp culture are the major sources for the enrichment of certain toxic metals. The metal concentrations in oysters presented a decreasing order of abundance (all values in mg Kg -1 ): Zn (278.91 ± 93.03) > Cu (63.13 ± 31.72) > Cr (22.29 ± 30.23) > Cd (14.54 ± 4.28) > Ni (9.41 ± 11.33) > Pb (2.22 ± 1.33) > As (0.58 ± 0.91) > Hg (0.04 ± 0.06). Bioconcentration Factor (BCF) and Biota Sediment Accumulation Factor (BSAF) exhibited that C. gigas in the region are strong accumulators for Zn and Cd respectively. Thus, the present study proves to fulfill the gap in understanding the rate of bioaccumulation of metals in C. gigas which is regarded as the most sought after oyster species globally. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae▿

    PubMed Central

    Hasegawa, Hiroaki; Lind, Erin J.; Boin, Markus A.; Häse, Claudia C.

    2008-01-01

    Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae. PMID:18456850

  9. Roles of HynAB and Ech, the Only Two Hydrogenases Found in the Model Sulfate Reducer Desulfovibrio gigas

    PubMed Central

    Morais-Silva, Fabio O.; Santos, Catia I.; Rodrigues, Rute

    2013-01-01

    Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism. PMID:23974026

  10. Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.; Lee, C. P.

    2002-01-01

    This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities. Onset of convection was considered analytically and numerically. Crystal growth was characterized by slow surface incorporation kinetics, i.e. growth kinetic coefficient beta (cm/s) small as compared to the typical bulk diffusion rate, D(sub 1)/h, where D(sub 1) is diffusivity of major crystallizing protein and h is the crystal size. Scaling type analysis predicted two laws on how the convection rate, v, essentially the Peclet number, Pe exactly equal to vh/D(sub 1), depends on dimensionless kinetic coefficient a exactly equal to beta h/D(sub 1). Namely: Pe = C(sub 2/5)(aRa(sup 2/5)) and Pe = C(sub 1) aRa. Here, Reynolds number Ra = rho(sub 1)(sup 0)gh(sup 3)(rho(sub p) - rho(sub w))/rho(sup p)rho(sub 1)vD(sub 1), v being solution viscosity. The constants C(sub 2/5), exactly equal to 0.28 and C(sub 1) exactly equal to 10(exp -2) found from the full scale computer simulation for a cylindrical crystal inside big cylindrical vessel. The linear boundary conditions connecting protein and impurity concentration at the interface with the flux to/from the interface was applied. No-slip condition for Navier-Shocker equations was employed. With these conditions, flow and concentration distributions were calculated. Validity of the Pe(Ra) dependencies follows for wide range of parameters for which numerical calculations have been accomplished and presented by various points.

  11. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum.

    PubMed

    Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J; Ren, Bin

    2013-04-01

    Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell parameters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.

  12. The purification, crystallization and preliminary diffraction of a glycerophosphodiesterase from Enterobacter aerogenes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung

    2006-07-01

    The metallo-glycerophosphodiesterase from E. aerogenes (GpdQ) has been cloned, expressed in E. coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals. The metallo-glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) has been cloned, expressed in Escherichia coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme andmore » subsequent optimization of these conditions led to crystals that diffracted to 2.9 Å and belonged to space group P2{sub 1}3, with unit-cell parameter a = 164.1 Å. Self-rotation function analysis and V{sub M} calculations indicated that the asymmetric unit contains two copies of the monomeric enzyme, corresponding to a solvent content of 79%. It is intended to determine the structure of this protein utilizing SAD phasing from transition metals or molecular replacement.« less

  13. Growth of Solid Solution Crystals

    NASA Technical Reports Server (NTRS)

    Lehoczky, S. L.; Szofran, F. R.; Holland, L. R.

    1985-01-01

    The major objective of this program is to determine the conditions under which single crystals of solid solutions can be grown from the melt in a Bridgman configuration with a high degree of chemical homogeneity. The central aim is to assess the role of gravity in the growth process and to explore the possible advantages for growth in the absence of gravity. The alloy system being investigated is the solid solution semiconductor with x-values appropriate for infrared detector applications in Hg sub (1-x) Cd sub x Te the 8 to 14 micro m wavelength region. Both melt and Te-solvent growth are being considered. The study consists of an extensive ground-based experimental and theoretical research effort followed by flight experimentation where appropriate. Experimental facilities have been established for the purification, casting, and crystal growth of the alloy system. Facilities have been also established for the metallurgical, compositional, electric and optical characterization of the alloys. Crystals are being grown by the Bridgman-Stockbarger method and are analyzed by various experimental techniques to evaluate the effects of growth conditions on the longitudinal and radial compositional variations and defect densities in the crystals.

  14. Gigas Meets Ulysses

    NASA Technical Reports Server (NTRS)

    2003-01-01

    [figure removed for brevity, see original site]

    Released 9 July 2003

    Roughly halfway between the great volcanoes of Olympus Mons and Pavonis Mons, the graben (troughs) of Ulysses Fossae intersect with the furrows of Gigas (gigantic) Sulci. A clear time sequence is evident: first came the formation of the sulci terrain (to the left), which then was fractured by graben radial to Olympus Mons, followed by flooding of lava. All but the deepest graben are filled by lava in the topographic low between the two volcanic rises.

    Image information: VIS instrument. Latitude 11.8, Longitude 234.3 East (125.7 West). 19 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  15. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum

    PubMed Central

    Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J.; Ren, Bin

    2013-01-01

    Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell paramters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution. PMID:23545659

  16. Inclusion behavior of Cs, Sr, and Ba impurities in LiCl crystal formed by layer-melt crystallization: Combined first-principles calculation and experimental study

    NASA Astrophysics Data System (ADS)

    Choi, Jung-Hoon; Cho, Yung-Zun; Lee, Tae-Kyo; Eun, Hee-Chul; Kim, Jun-Hong; Kim, In-Tae; Park, Geun-Il; Kang, Jeung-Ku

    2013-05-01

    The pyroprocessing which uses a dry method to recycle spent oxide fuel generates a waste LiCl salt containing radioactive elements. To reuse LiCl salt, the radioactive impurities has to be separated by the purification process such as layer-melt crystallization. To enhance impurity separation efficiency, it is important to understand the inclusion mechanism of impurities within the LiCl crystal. Herein, we report the inclusion properties of impurities in LiCl crystals. First of all, the substitution enthalpies of Cs+, Sr2+, and Ba2+ impurities with 0-6 at% in LiCl crystal were evaluated via first-principles calculations. Also, the molten LiCl containing 1 mol of Cs+, Sr2+, and Ba2+ impurities was crystallized through the experimental layer-melt crystallization method. These substitution enthalpy and experiment clarify that a high substitution enthalpy should result in the high separation efficiency for an impurity. Furthermore, we find that the electron density map gives a clue to the mechanism for inclusion of impurities into LiCl crystal.

  17. Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

    PubMed Central

    Wysoczynski, Christina L.; Roemer, Sarah C.; Dostal, Vishantie; Barkley, Robert M.; Churchill, Mair E. A.; Malarkey, Christopher S.

    2013-01-01

    Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications. PMID:24013567

  18. Modification of the crystal habit of celecoxib for improved processability.

    PubMed

    Banga, Sheere; Chawla, Garima; Varandani, Deepak; Mehta, B R; Bansal, Arvind K

    2007-01-01

    Crystallization is often used in the pharmaceutical industry for purification and isolation of drugs, and also as a means of generating polymorphs or isomorphs. The aim of this study was to investigate the role of extrinsic crystallization parameters on the crystallized product, with special emphasis on improving the mechanical properties of acicular celecoxib. Celecoxib isomorphs were prepared using different techniques (solvent crystallization and vapour diffusion) and crystallization conditions (solvents, stirring, degree of supersaturation, crystallization temperature and seeding). Powder X-ray diffractometry, spectroscopic and thermal methods were used to investigate physical characteristics of crystals. Growth kinetics and aggregation dynamics of crystallization in polar and non-polar solvents were simulated using a dynamic light scattering method. The quick appearance of broad peaks over the range of 10-8000 nm in chloroform during crystallization simulation studies indicated faster aggregation in non-polar solvents. Aspect ratio, flow, compressibility and surface area of recrystallized products were also determined. Surface topography was determined by atomic force microscopy and the lath-shaped crystals (aspect ratio of 2-4) exhibited a roughness index of 1.79 in comparison with 2.92 for needles. Overall, the lath-shaped isomorphs exhibited improved flow and better compressibility.

  19. In situ distribution and characterization of the organic content of the oyster shell Crassostrea gigas (Mollusca, Bivalvia).

    PubMed

    Dauphin, Yannicke; Ball, Alexander D; Castillo-Michel, Hiram; Chevallard, Corinne; Cuif, Jean-Pierre; Farre, Bastien; Pouvreau, Stéphane; Salomé, Murielle

    2013-01-01

    Cultivation of commercial oysters is now facing the possible influence of global change in sea water composition, commonly referred to as "ocean acidification". In order to test the potential consequence of the predicted environmental changes, a cultivation experiment was carried out. The left and right valves of the oyster shell Crassostrea gigas differ in their structure; moreover, lenses of non compact layers are irregular. The shell layers of juvenile C. gigas are studied using a variety of highly spatially resolved techniques to establish their composition and structure. Our results confirm the presence of three different calcitic structural types. The role of the lenses of chalky layers is not yet deciplered. Despite a common mineralogy, the elemental composition of the layers differs. The sulphur aminoacids and sulphated polysaccharide contents of the intracrystalline and intercrystalline matrices differ, as well as those of the structural types. The possible different sensitivity of these structures to environmental changes is still unknown. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Peculiarities of Crystallization of the Restriction Endonuclease EcoRII

    NASA Technical Reports Server (NTRS)

    Karpove, Elizaveta; Pusey, M.arc L.

    1998-01-01

    Nucleases interfere with most standard molecular biology procedures. We have purified and crystallized the restriction endonuclease EcoRII, which belongs to the type II of restriction- modification enzyme, to study the protein crystallization process using a "non standard" macromolecule. A procedure for the purification of EcoRII was developed and 99% pure protein as determined by SDS PAGE electrophoresis obtained. Light scattering experiments were performed to assist in screening protein suitable crystallization conditions. The second virial coefficient was determined as a function of precipitating salt concentration, using sodium chloride, ammonium sulfate, and sodium sulfate. Small (maximum size approximately 0.2 mm) well shaped crystals have been obtained. Larger poorly formed crystals (ca 0.5 mm) have also been obtained, but we have been unable to mount them for diff-raction analysis due to their extreme fragility. Crystallization experiments with PEG have shown that using this precipitant, the best crystals are obtained from slightly over-saturated solutions. Use of higher precipitant concentration leads to dendritic crystal formation. EcoRII is difficult to solubilize and meticulous attention must be paid to the presence of reducing agents.

  1. Comparative sensitivity of Crassostrea angulata and Crassostrea gigas embryo-larval development to As under varying salinity and temperature.

    PubMed

    Moreira, Anthony; Figueira, Etelvina; Libralato, Giovanni; Soares, Amadeu M V M; Guida, Marco; Freitas, Rosa

    2018-06-07

    Oysters are a diverse group of marine bivalves that inhabit coastal systems of the world's oceans, providing a variety of ecosystem services, and represent a major socioeconomic resource. However, oyster reefs have become inevitably impacted from habitat destruction, overfishing, pollution and disease outbreaks that have pushed these structures to the break of extinction. In addition, the increased frequency of climate change related events promise to further challenge oyster species survival worldwide. Oysters' early embryonic development is likely the most vulnerable stage to climate change related stressors (e.g. salinity and temperature shifts) as well as to pollutants (e.g. arsenic), and therefore can represent the most important bottleneck that define populations' survival in a changing environment. In light of this, the present study aimed to assess two important oyster species, Crassostrea angulata and Crassostrea gigas embryo-larval development, under combinations of salinity (20, 26 and 33), temperature (20, 24 and 28 °C) and arsenic (As) exposure (0, 30, 60, 120, 240, 480, 960 and 1920 μg. As L -1 ), to infer on different oyster species capacity to cope with these environmental stressors under the eminent threat of climate change and increase of pollution worldwide. Results showed differences in each species range of salinity and temperature for successful embryonic development. For C angulata, embryo-larval development was successful at a narrower range of both salinity and temperature, compared to C. gigas. Overall, As induced higher toxicity to C. angulata embryos, with calculated EC50 values at least an order of magnitude lower than those calculated for C. gigas. The toxicity of As (measured as median effective concentration, EC50) showed to be influenced by both salinity and temperature in both species. Nonetheless, salinity had a greater influence on embryos' sensitivity to As. This pattern was mostly noticed for C. gigas, with lower salinity

  2. Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

    NASA Astrophysics Data System (ADS)

    Liu, Gang; Huan, Pin; Liu, Baozhong

    2014-11-01

    Increasing evidence indicates that transforming growth factor β (TGF-β) signaling pathways play many important roles in the early development of mollusks. However, limited information is known concerning their detailed mechanisms. Here, we describe the identification, cloning and characterization of two Smad genes, the key components of TGF-β signaling pathways, from the Pacific oyster Crassostrea gigas. Sequence analysis of the two genes, designated as cgi-smad1/ 5/ 8 and cgi-smad4, revealed conserved functional characteristics. The two genes were widely expressed in embryos and larvae, suggesting multiple roles in the early development of C. gigas. The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula, indicating that TGF-β signaling pathways may be involved in dorsoventral patterning in C. gigas. Furthermore, high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-β signaling pathways in particular phases of shell development, including the formation of the initial shell field and the biomineralization of larval shells. The results of this study provide fundamental support for elucidating how TGF-β signaling pathways participate in the early development of bivalve mollusks, and suggest that further work is warranted to this end.

  3. Strep-Tagged Protein Purification.

    PubMed

    Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

    2015-01-01

    The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

  4. Physicochemical characterization and toxicity of decursin and their derivatives from Angelica gigas.

    PubMed

    Mahat, Bimit; Chae, Jung-Woo; Baek, In-Hwan; Song, Gyu-Yong; Song, Jin-Sook; Cho, Seong-Kwon; Kwon, Kwang-Il

    2012-01-01

    Angelica gigas NAKAI is used to treat dysmenorrhea, amenorrhea, menopause, abdominal pain, injuries, migraine, and arthritis. The present study provided a physicochemical and toxicological characterization of compounds in A. gigas NAKAI (decursin, decursinol angelate, diketone decursin, ether decursin, epoxide decursin and oxim decursin). Diketone decursin (173.16 μg/mL) and epoxide decursin (122.12 μg/mL) exhibited >100 μg/mL kinetic solubility after applying nephelometry, suggesting a highly soluble compound. The Student’s t-test revealed significant differences in the pKa ranges of the compounds by automatic titration from capillary electrophoresis (p<0.05). Diketone decursin, epoxide decursin and oxim decursin might be formulated into an oral dosage form (log P: 0-3) by an automatic titration analysis. A parallel artificial membrane permeability assay demonstrated permeability coefficients of <10 x 10⁻⁶ cm/s for all of the compounds, suggesting poor permeability. Ether decursin exhibited a toxic effect after being applied to mouse (NIH 3T3, EC₅₀: 57.9 μM) and human (HT-29, EC₅₀: 36.1 μM; Hep-G2, EC₅₀: 4.92 μM) cells. Additionally, epoxide and oxim decursin were toxic through acute oral toxicity (four and three deaths of Institute of Cancer Research (ICR) mice) and mutation toxicity testing by applying Salmonella typhimurium cells with and without S9. Although diketone decursin exhibited less permeability, it is potentially valuable pharmacological compound that should be investigated.

  5. Purification, crystallization and preliminary crystallographic analysis of a 6-pyruvoyltetrahydropterin synthase homologue from Esherichia coli.

    PubMed

    Seo, Kyung Hye; Supangat; Kim, Hye Lim; Park, Young Shik; Jeon, Che Ok; Lee, Kon Ho

    2008-02-01

    6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.

  6. Purification, crystallization and preliminary crystallographic analysis of a 6-pyruvoyltetrahydropterin synthase homologue from Esherichia coli

    PubMed Central

    Seo, Kyung Hye; Supangat; Kim, Hye Lim; Park, Young Shik; Jeon, Che Ok; Lee, Kon Ho

    2008-01-01

    6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 Å resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 Å, and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 Å, and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement. PMID:18271114

  7. Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster ( Crassostrea gigas)

    NASA Astrophysics Data System (ADS)

    Liu, Ting; Li, Qi; Song, Junlin; Yu, Hong

    2017-02-01

    There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

  8. Fluorescent Applications to Crystallization

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Forsythe, Elizabeth; Achari, Aniruddha

    2006-01-01

    By covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. Preliminary tests, using model proteins, indicates that we can use high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to test this approach using a wider range, of proteins. The trace fluorescently labeled crystals will also

  9. [Phylogenetic analysis of tyrosinase gene family in the Pacific oyster (Crassostrea gigas Thunberg)].

    PubMed

    Yu, Xue; Yu, Hong; Kong, Lingfeng; Li, Qi

    2014-02-01

    The deduced amino acid sequence characteristics, classification and phylogeny of tyrosinase gene family in the Pacific oyster (Crassostrea gigas Thunberg) were analyzed using bioinformatics methods. The results showed that gene duplication was the major cause of tyrosinase gene expansion in the Pacific oyster. The tyrosinase gene family in the Pacific oyster can be further classified into three types: secreted form (Type A), cytosolic form (Type B) and membrane-bound form (Type C). Based on the topology of the phylogenetic tree of the Pacific oyster tyrosinases, among Type A isoforms, tyr18 seemed divergent from other Type A tyrosinases early, while tyr2 and tyr9 appeared divergent early in Type B. In Type C tyrosinses, tyr8 was divergent early. The cluster of the Pacific oyster tyrosinasesis determined by their classifications and positions in the scaffolds. Further analysis suggested that Type A tyrosinases of C. gigas clustered with those from cephalopods and then with nematodes and cnidarians. Type B tyrosinases were generally clustered with the same type of tyrosinases from molluscas and nematodes, and then with those from platyhelminths, cnidarians and chordates. Type A tyrosinases in the Pacific oyster and the Pearl oyster expanded independently and were divergent from membrane-bound form of tyrosinases in chordata, platyhelminthes and annelida. These observations suggested that Type C tyrosinases in the bivalve had a distinct evolution direction.

  10. Behavioral ecology of jumbo squid (Dosidicus gigas) in relation to oxygen minimum zones

    NASA Astrophysics Data System (ADS)

    Stewart, Julia S.; Field, John C.; Markaida, Unai; Gilly, William F.

    2013-10-01

    Habitat utilization, behavior and food habits of the jumbo or Humboldt squid, Dosidicus gigas, were compared between an area recently inhabited in the northern California Current System (CCS) and a historically established area of residence in the Gulf of California (GOC). Low dissolved oxygen concentrations at midwater depths define the oxygen minimum zone (OMZ), an important environmental feature in both areas. We analyzed vertical diving behavior and diet of D. gigas and hydrographic properties of the water column to ascertain the extent to which squid utilized the OMZ in the two areas. The upper boundary of the OMZ has been shoaling in recent decades in the CCS, and this phenomenon has been proposed to vertically compress the pelagic environment inhabited by aerobic predators. A shoaling OMZ will also bring mesopelagic communities into a depth range with more illumination during daytime, making these organisms more vulnerable to predation by visual predators (i.e. jumbo squid). Because the OMZ in the GOC is considerably shallower than in the CCS, our study provides insight into the behavioral plasticity of jumbo squid and how they may respond to a shoaling OMZ in the CCS. We propose that shoaling OMZs are likely to be favorable to jumbo squid and could be a key indirect factor behind the recent range expansion of this highly migratory predator.

  11. Implications for osmorespiratory compromise by anatomical remodeling in the gills of Arapaima gigas.

    PubMed

    Ramos, Cleverson Agner; Fernandes, Marisa Narciso; da Costa, Oscar Tadeu Ferreira; Duncan, Wallice Paxiuba

    2013-10-01

    The gill structure of the Amazonian fish Arapaima gigas, an obligatory air breather, was investigated during its transition from water breathing to the obligatory air breathing modes of respiration. The gill structure of A. gigas larvae is similar to that of most teleost fish; however, the morphology of the gills changes as the fish grow. The main morphological changes in the gill structure of a growing fish include the following: (1) intense cell proliferation in the filaments and lamellae, resulting in increasing epithelial thickness and decreasing interlamellar distance; (2) pillar cell system atrophy, which reduces the blood circulation through the lamellae; (3) the generation of long cytoplasmic processes from the epithelial cells into the intercellular space, resulting in continuous and sinuous paracellular channels between the epithelial cells of the filament and lamella that may be involved in gas, ion, and nutrient transport to epithelial cells; and (4) intense mitochondria-rich cell (MRC) proliferation in the lamellar epithelium. All of these morphological changes in the gills contribute to a low increase of the respiratory surface area for gas exchange and an increase in the water-blood diffusion distance increasing their dependence on air-breathing as fish developed. The increased proliferation of MRCs may contribute to increased ion uptake, which favors the regulation of ion content and pH equilibrium. Copyright © 2013 Wiley Periodicals, Inc.

  12. Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use

    PubMed Central

    Šiman, Pavel; Filipová, Alžběta; Tichá, Alena; Niang, Mohamed; Bezrouk, Aleš; Havelek, Radim

    2016-01-01

    A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use. PMID:27152419

  13. Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

    PubMed Central

    Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M.; Bartlam, Mark

    2016-01-01

    Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate. PMID:26919520

  14. Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

    PubMed

    Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M; Bartlam, Mark

    2016-03-01

    Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

  15. Membrane-Based Technologies in the Pharmaceutical Industry and Continuous Production of Polymer-Coated Crystals/Particles.

    PubMed

    Chen, Dengyue; Sirkar, Kamalesh K; Jin, Chi; Singh, Dhananjay; Pfeffer, Robert

    2017-01-01

    Membrane technologies are of increasing importance in a variety of separation and purification applications involving liquid phases and gaseous mixtures. Although the most widely used applications at this time are in water treatment including desalination, there are many applications in chemical, food, healthcare, paper and petrochemical industries. This brief review is concerned with existing and emerging applications of various membrane technologies in the pharmaceutical and biopharmaceutical industry. The goal of this review article is to identify important membrane processes and techniques which are being used or proposed to be used in the pharmaceutical and biopharmaceutical operations. How novel membrane processes can be useful for delivery of crystalline/particulate drugs is also of interest. Membrane separation technologies are extensively used in downstream processes for bio-pharmaceutical separation and purification operations via microfiltration, ultrafiltration and diafiltration. Also the new technique of membrane chromatography allows efficient purification of monoclonal antibodies. Membrane filtration techniques of reverse osmosis and nanofiltration are being combined with bioreactors and advanced oxidation processes to treat wastewaters from pharmaceutical plants. Nanofiltration with organic solvent-stable membranes can implement solvent exchange and catalyst recovery during organic solvent-based drug synthesis of pharmaceutical compounds/intermediates. Membranes in the form of hollow fibers can be conveniently used to implement crystallization of pharmaceutical compounds. The novel crystallization methods of solid hollow fiber cooling crystallizer (SHFCC) and porous hollow fiber anti-solvent crystallization (PHFAC) are being developed to provide efficient methods for continuous production of polymer-coated drug crystals in the area of drug delivery. This brief review provides a general introduction to various applications of membrane technologies in

  16. Towards decoding the conifer giga-genome.

    PubMed

    Mackay, John; Dean, Jeffrey F D; Plomion, Christophe; Peterson, Daniel G; Cánovas, Francisco M; Pavy, Nathalie; Ingvarsson, Pär K; Savolainen, Outi; Guevara, M Ángeles; Fluch, Silvia; Vinceti, Barbara; Abarca, Dolores; Díaz-Sala, Carmen; Cervera, María-Teresa

    2012-12-01

    Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world's conifer forests.

  17. Cloning, expression, purification and crystallization of saccharopine reductase from Magnaporthe grisea.

    PubMed

    Johansson, E; Steffens, J J; Emptage, M; Lindqvist, Y; Schneider, G

    2000-05-01

    The gene coding for saccharopine reductase (E.C. 1.5.1.10), an enzyme of the alpha-aminoadipic pathway of lysine biosynthesis in the pathogenic fungus Magnaporthe grisea, was cloned and expressed in Escherichia coli. The purified enzyme was crystallized in space groups C2 and C222(1) using ammonium sulfate pH 4.8 or PEG 6000 pH 4. 1 as precipitants. The unit-cell parameters are a = 115.0, b = 56.6, c = 74.3 A, beta = 111.1 degrees for space group C2, and a = 89.3, b = 119.0, c = 195.9 A for space group C222(1). The crystals diffract to resolutions of 2.0 A (C2) and 2.4 A (C222(1)) at synchrotron sources.

  18. Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin.

    PubMed

    Broco, Manuela; Soares, Cláudio M; Oliveira, Solange; Mayhew, Stephen G; Rodrigues-Pousada, Claudina

    2007-09-18

    Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5'-phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of K(d) values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.

  19. Adult Pacific Oyster (Crassostrea gigas) May Have Light Sensitivity

    PubMed Central

    Yang, Yanjian; Li, Zhuang; Guo, Ting; Li, Yongchuan; Wang, Xiaotong

    2015-01-01

    Light-sensitivity is an important aspect of mollusk survival as it plays a vital role in reproduction and predator avoidance. In the Pacific oyster Crassostrea gigas light sensitivity has been demonstrated in the larval stage but has not yet been conclusively demonstrated in adult oysters. In this paper we describe an experiment which was undertaken to determine if adult Pacific oysters were sensitive to light. One LED flashlight was used to shine light onto adult oysters while they were filtering seawater through their shell openings. We found that the degree of opening increased gradually during the light period but rapidly decreased when the flashlight was turned off in the treated group but not in the control group. These results suggest that adult Pacific oyster may be sensitive to light. PMID:26474058

  20. Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Harris, Paul T.; Raghunathan, Kannan; Spurbeck, Rachel R.

    2010-09-02

    Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 {angstrom} resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 {angstrom}. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.8 {angstrom}{sup 3} Da{sup -1}, corresponding to 55.2% solvent content.

  1. Use of nanoindentation technique for a better understanding of the fracture toughness of Strombus gigas conch shell

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Romana, L., E-mail: laurence.romana@univ-ag.fr; Thomas, P.; Bilas, P.

    2013-02-15

    In this work the nanochemical properties of the composite organomineral biomaterial constituting Strombus gigas conch shell are studied by means of dynamic mechanical analyses associated to nanoidentation technique. The measurements are performed on shell samples presenting different surface orientations relative to the growth axis of the conch shell. The influence of the organic component of the biomaterial on its nanomechanical properties is also investigated by studying fresh and dried S. gigas conch shells. Monocrystalline aragonite is used as a reference. For the understanding of nanochemical behaviour, special attention is paid to the pop in events observed on the load/displacement curvesmore » which results from nanofractures' initiation and propagation occuring during the load process. In order to better understand the mechanical properties systematic studies of the structure and morphology are performed using scanning electron microscopy, atomic force microscopy and X-ray diffractometry. The hardness and Young's modulus values measured on bio aragonite samples are close to those of the aragonite mineral standard. This surprising result shows that, H and E values are not related to the bio composition and lamellar structure of the bio aragonite. However, it was found that the organic layer and the micro architecture strongly influence the nanofracture initiation and propagation processes in the samples. Statistic study of the pop-in events can help to predict the macroscopic mechanical behaviour of the material. - Highlights: ► Nanomechanical properties of Strombus gigas conch shell ► Low influence of the crossed lamellar structure on H and E values at the nano scale ► Strong influence of the crossed lamellar on nanocracks initiation ► Correlation between mechanical behaviors at the macro and nano scales.« less

  2. Purification, crystallization and preliminary crystallographic study of low oxygen-affinity haemoglobin from cat (Felis silvestris catus) in two different crystal forms.

    PubMed

    Balasubramanian, M; Moorthy, Pon Sathya; Neelagandan, K; Ponnuswamy, M N

    2009-03-01

    Haemoglobin is a metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. The present work reports the preliminary crystallographic study of low oxygen-affinity haemoglobin from cat in different crystal forms. Cat blood was collected, purified by anion-exchange chromatography and crystallized in two different conditions by the hanging-drop vapour-diffusion method under unbuffered low-salt and buffered high-salt concentrations using PEG 3350 as a precipitant. Intensity data were collected using MAR345 and MAR345dtb image-plate detector systems. Cat haemoglobin crystallizes in monoclinic and orthorhombic crystal forms with one and two whole biological molecules (alpha(2)beta(2)), respectively, in the asymmetric unit.

  3. (NZ)CH...O contacts assist crystallization of a ParB-like nuclease.

    PubMed

    Shaw, Neil; Cheng, Chongyun; Tempel, Wolfram; Chang, Jessie; Ng, Joseph; Wang, Xin-Yu; Perrett, Sarah; Rose, John; Rao, Zihe; Wang, Bi-Cheng; Liu, Zhi-Jie

    2007-07-07

    The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2-3.7 A) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 A resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.

  4. Rising water temperatures, reproduction and recruitment of an invasive oyster, Crassostrea gigas, on the French Atlantic coast.

    PubMed

    Dutertre, Mickaël; Beninger, Peter G; Barillé, Laurent; Papin, Mathias; Haure, Joël

    2010-02-01

    The recent appearance and invasion of feral oysters (Crassostrea gigas) along the northern European Atlantic coast, underscores the necessity to investigate the relationship between environmental variables, reproductive physiology, larval development and recruitment. We studied these relationships at both high (HT) and intermediate (IT) - turbidity sites, through historical data on water temperatures, multi-parameter environmental probes, histological analyses, and field collections of planktonic larvae and settled post-larvae in 2005 and 2006. A progressive warming trend was observed, especially since 1995, when oyster proliferation first became severe. Threshold temperatures for oocyte growth, larval development and settlement were achieved in both 2005 and 2006. The HT site showed greater numbers of larvae and post-larvae than the IT site for both years, with the highest numbers of post-larvae observed at both sites during the warmer summer of 2006. These results suggest that increased temperatures in northern European waters allow successful reproduction, larval development, and recruitment of C. gigas. High turbidity conditions further enhance this success. 2009 Elsevier Ltd. All rights reserved.

  5. Evidence of neutralizing activity against T3 coliphage in oyster Crassostrea gigas hemolymph.

    PubMed

    Bachère, E; Hervio, D; Mialhe, E; Grizel, H

    1990-01-01

    To investigate defense reactions of bivalve molluscs against viruses, experimental in vitro assays have been developed using T3 coliphage as a test virus. A native neutralizing factor in oyster Crassostrea gigas serum showed high individual variability and was enhanced significantly by repeated sampling of hemolymph from the same oysters. The responsible factor is apparently thermolabile and sensitive to EDTA treatment. Because of an inhibitory effect by the enzymatic inhibitor, phenylmethylsulphonyl fluoride (PMSF), the T3-neutralizing factor may be related to serine protease.

  6. Purification and Crystal Growth of Lead Iodide by Physical Vapor Transport Method

    NASA Technical Reports Server (NTRS)

    Wright, G. W.; Cole, M.; Chen, Y.-F.; Chen, K.-T.; Chen, H.; Chattopadhyay, K.; Burger, A.

    1998-01-01

    Lead iodide (PbI2) is a layered compound semiconductor being developed as room temperature x- and gamma-ray detector. Compared to the more studied material, mercuric iodide, PbI2 has a higher melting temperature and no phase transition until liquid phase which are indications of better mechanical properties. In this study, the source material was purified by the zone-refining process, and the purest section was extracted from center of the the zone-refined ingot to be grown by physical vapor transport (PVT) method. The zone-refined material and as-grown crystals were characterized by optical microscopy and differential scanning calorimetry (DSC) to reveal the surface morphology, purity and stoichiometry. The results shows that both materials are near-stoichiometric composition, with the purity of the as-grown crystals higher than zone-refined materials. The resistivity of the as-grown crystal (10" Omega-cm) was derived from current-voltage (I-V) measurement, and is 10 times higher than the zone-refined materials. Detail results will be presented and discussed.

  7. Growth of Bulk Wide Bandgap Semiconductor Crystals and Their Potential Applications

    NASA Technical Reports Server (NTRS)

    Chen, Kuo-Tong; Shi, Detang; Morgan, S. H.; Collins, W. Eugene; Burger, Arnold

    1997-01-01

    Developments in bulk crystal growth research for electro-optical devices in the Center for Photonic Materials and Devices since its establishment have been reviewed. Purification processes and single crystal growth systems employing physical vapor transport and Bridgman methods were assembled and used to produce high purity and superior quality wide bandgap materials such as heavy metal halides and II-VI compound semiconductors. Comprehensive material characterization techniques have been employed to reveal the optical, electrical and thermodynamic properties of crystals, and the results were used to establish improved material processing procedures. Postgrowth treatments such as passivation, oxidation, chemical etching and metal contacting during the X-ray and gamma-ray device fabrication process have also been investigated and low noise threshold with improved energy resolution has been achieved.

  8. Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gaur, Vineet; Sethi, Dhruv K.; Salunke, Dinakar M., E-mail: dinakar@nii.res.in

    The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported. Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presencemore » of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 150.7, c = 164.9 Å.« less

  9. Purification, crystallization and preliminary X-ray diffraction of human S100A15

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boeshans, Karen M.; Wolf, Ronald; Voscopoulos, Christopher

    2006-05-01

    S100 proteins are differentially expressed during epithelial cell maturation, tumorigenesis and inflammation. The novel human S100A15 protein has been cloned, expressed, purified and crystallized in two crystal forms, a triclinic and a monoclinic form, which diffract to 1.7 and 2.0 Å, respectively. Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that itmore » contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 Å, α = 71.2, β = 68.1, γ = 67.8° and an estimated two molecules in the asymmetric unit, and diffract to 1.7 Å resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 Å, β = 128.2° and an estimated one molecule in the asymmetric unit, and diffract to 2.0 Å resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.« less

  10. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Balasubramanian, Anuradha; Ponnuraj, Karthe, E-mail: pkarthe@hotmail.com

    Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized andmore » the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å.« less

  11. Mineral and water content of A. gigas scales determine local micromechanical properties and energy dissipation mechanisms

    NASA Astrophysics Data System (ADS)

    Troncoso, Omar P.; Gigos, Florian; Torres, Fernando G.

    2017-11-01

    Arapaima gigas scales are natural laminated composite materials made of individual layers with different degrees of mineralization, accompanied of varying mechanical properties. This natural design provides scales with hardness and flexibility, and can serve as a source of inspiration for the development of new layered composites with a hard surface and flexible base. In this paper, we have carried out cyclic micro-indentation tests on both; the internal and the highly mineralized external surface of air dried and wet scales, in order to assess the variation of their local micromechanical properties with regard to the mineral and water content. The load-penetration (P-h) curves showed that creep takes place throughout the application of a constant force during the micro-indentation tests, confirming the time dependent response of A. gigas scales. A model that accounted for the elastic, plastic and viscous responses of the samples was used to fit the experimental results. The penetration depth during loading and creep, as well as the energy dissipated are dependent on the water content. The used model suggests that the viscous response of the internal layer increases with the water content.

  12. Unusual varieties and duplication of Rig-I like receptors encoded in the marine mollusk, Crassostrea gigas

    NASA Astrophysics Data System (ADS)

    Tian, Z. H.; Jiao, C. Z.

    2017-07-01

    RIG-I like receptors (RLRs) play key roles in sensing non-self nucleic acids in cytoplasm and trigger antiviral innate immune response in vertebrates and human body. Here we carried out in silico analysis to identify and investigate the putative RLRs encoded in the genome of marine mollusk, Crassostrea gigas (cgRLRs), an invertebrate species. We found the unusual duplication and varieties on domain architecture of putative cgRLRs encoded in the genome of C. gigas. Three putative cgRLRs (accessions numbers are EKC24603, EKC31344.1 and EKC38304.1 on GenBank), have the similar domain architecture with that of human RIG-I or MDA5, and one protein (EKC34573.1) with that of human LGP2; The fifth putative cgRLRs (EKC38303.1) is somewhat similar with human RIG-I/MDA5 except that it has only one caspase activation and recruitment domain (CARD) in its N-terminal. Other nine proteins were identified to be partialy similar with RLRs while with the incomplete sequences, which maybe reflect the events of partial duplication of cgRLRs genes occurred in the oyster genome.

  13. Characterization of microbiota in Arapaima gigas intestine and isolation of potential probiotic bacteria.

    PubMed

    do Vale Pereira, G; da Cunha, D G; Pedreira Mourino, J L; Rodiles, A; Jaramillo-Torres, A; Merrifield, D L

    2017-11-01

    The aim of this study was to determine the intestinal microbiota of pirarucu (Arapaima gigas) in different growth stages (adult and fingerlings) and to isolate and identify potential probiotic bacteria. High-throughput sequencing analysis of the intestinal contents revealed that the majority of sequences belonged to the Proteobacteria, Fusobacteria and Firmicutes phyla. At the genus level, the greatest number of sequences belonged to Bradyrhizobium in adult fish, while Cetobacterium was the most abundant in juvenile fish. Twenty-three lactic-acid bacteria (LABs) were isolated on MRS agar from healthy juvenile fish. The isolates were tested in vitro for probiotic properties. Two isolates (identified as strains of Lactococcus lactis subsp. lactis and Enterococcus faecium) displayed antagonism against all 10 pathogens tested, were nonhaemolytic and maintained good viability for at least 3 weeks when supplemented to fish diets. The presence of a number of antibiotic resistance genes (ARGs), conferring resistance to erythromycin, tetracycline and chloramphenicol, was investigated by PCR. The absence of ARGs investigated the potential to antagonize pathogens, and favourable growth and survival characteristics indicate that these autochthonous isolates have the potential to be considered probiotics, which will be studied in future in vivo experiments. This study has demonstrated, for the first time, the normal microbiota in the A. gigas intestine during different life stages and the presence of LAB strains. It also demonstrated LAB antibiotic resistance and antagonistic behaviour against pathogens isolated from the same fish. © 2017 The Society for Applied Microbiology.

  14. First-pass metabolism of decursin, a bioactive compound of Angelica gigas, in rats.

    PubMed

    Park, Hyun Seo; Kim, Byunghyun; Oh, Ju-Hee; Kim, Young Choong; Lee, Young-Joo

    2012-06-01

    Decursin is considered the major bioactive compound of Angelica gigas roots, a popular Oriental herb and dietary supplement. In this study, the pharmacokinetics of decursin and its active metabolite, decursinol, were evaluated after the administration of decursin in rats. The plasma concentration of decursin decreased rapidly, with an initial half-life of 0.05 h. It was not detectable at 1 h after intravenous administration at an area under the plasma concentration-time curve of 1.20 µg · mL-1·h, whereas the concentration of decursinol increased rapidly reaching a maximum concentration of 2.48 µg · mL-1 at the time to maximum plasma concentration of 0.25 h and an area under the plasma concentration-time curve of 5.23 µg · mL-1·h. Interestingly, after oral administration of decursin, only decursinol was present in plasma, suggesting an extensive hepatic first-pass metabolism of decursin. The extremely low bioavailability of decursin after its administration via the hepatic portal vein (the fraction of dose escaping first-pass elimination in the liver, FH = 0.11) is indicative of extensive hepatic first-pass metabolism of decursin, which was confirmed by a tissue distribution study. These findings suggest that decursin is not directly associated with the bioactivity of A. gigas and that it may work as a type of natural prodrug of decursinol. Georg Thieme Verlag KG Stuttgart · New York.

  15. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens β2-toxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gurjar, Abhijit A.; Yennawar, Neela H.; Yennawar, Hemant P.

    2007-06-01

    The cloning, expression, purification and crystallization of recombinant Clostridium perfringens β2-toxin is described. The crystals diffracted to 2.9 Å resolution. Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. β2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of β2-toxin using themore » batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 Å resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 Å, α = β = 90, γ = 120° using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles ω = 90.0, ϕ = 210.3°.« less

  16. Oxygen Sag and Stream Purification.

    ERIC Educational Resources Information Center

    Neal, Larry; Herwig, Roy

    1978-01-01

    Presents a literature review of water quality related to oxygen sag and stream purification, covering publications of 1976-77. This review includes: (1) self-purification models; (2) oxygen demand; and (3) reaeration and oxygen transfer. A list of 60 references is also presented. (HM)

  17. The Trilogy is Complete - GigaGalaxy Zoom Phase 3

    NASA Astrophysics Data System (ADS)

    2009-09-01

    The third image of ESO's GigaGalaxy Zoom project has just been released online, completing this eye-opening dive into our galactic home in outstanding fashion. The latest image follows on from views, released over the last two weeks, of the sky as seen with the unaided eye and through an amateur telescope. This third instalment provides another breathtaking vista of an astronomical object, this time a 370-million-pixel view of the Lagoon Nebula of the quality and depth needed by professional astronomers in their quest to understand our Universe. The newly released image extends across a field of view of more than one and a half square degree - an area eight times larger than that of the full Moon - and was obtained with the Wide Field Imager attached to the MPG/ESO 2.2-metre telescope at the La Silla Observatory in Chile. This 67-million-pixel camera has already created several of ESO's iconic pictures. The intriguing object depicted here - the Lagoon Nebula - is located four to five thousand light-years away towards the constellation of Sagittarius (the Archer). The nebula is a giant interstellar cloud, 100 light-years across, where stars are forming. The scattered dark patches seen all over the nebula are huge clouds of gas and dust that are collapsing under their own weight and which will soon give birth to clusters of young, glowing stars. Some of the smallest clouds are known as "globules" and the most prominent ones have been catalogued by the astronomer Edward Emerson Barnard. The Lagoon Nebula hosts the young open stellar cluster known as NGC 6530. This is home for 50 to 100 stars and twinkles in the lower left portion of the nebula. Observations suggest that the cluster is slightly in front of the nebula itself, though still enshrouded by dust, as revealed by reddening of the starlight, an effect that occurs when small dust particles scatter light. The name of the Lagoon Nebula derives from the wide lagoon-shaped dark lane located in the middle of the

  18. Size increment of jumbo flying squid Dosidicus gigas mature females in Peruvian waters, 1989-2004

    NASA Astrophysics Data System (ADS)

    Argüelles, Juan; Tafur, Ricardo; Taipe, Anatolio; Villegas, Piero; Keyl, Friedeman; Dominguez, Noel; Salazar, Martín

    2008-10-01

    Changes in population structure of the jumbo flying squid Dosidicus gigas in Peruvian waters were studied based on size-at-maturity from 1989 to 2004. From 1989 to 1999, mature squid belonging to the medium-sized group prevailed, but from 2001 on, mature squids were larger. This change is not related to the changes in sea surface temperature and we hypothesized that it was caused by the population increase of mesopelagic fishes as prey.

  19. Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunicliffe, Alan; Mitchell, Timothy J.

    2006-07-01

    The choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γmore » = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V{sub M} = 2.77 Å{sup 3} Da{sup −1}) and shows a diffraction limit of 3.5 Å.« less

  20. Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

    PubMed

    Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

    2014-02-01

    In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Patterning technology for solution-processed organic crystal field-effect transistors

    PubMed Central

    Li, Yun; Sun, Huabin; Shi, Yi; Tsukagoshi, Kazuhito

    2014-01-01

    Organic field-effect transistors (OFETs) are fundamental building blocks for various state-of-the-art electronic devices. Solution-processed organic crystals are appreciable materials for these applications because they facilitate large-scale, low-cost fabrication of devices with high performance. Patterning organic crystal transistors into well-defined geometric features is necessary to develop these crystals into practical semiconductors. This review provides an update on recentdevelopment in patterning technology for solution-processed organic crystals and their applications in field-effect transistors. Typical demonstrations are discussed and examined. In particular, our latest research progress on the spin-coating technique from mixture solutions is presented as a promising method to efficiently produce large organic semiconducting crystals on various substrates for high-performance OFETs. This solution-based process also has other excellent advantages, such as phase separation for self-assembled interfaces via one-step spin-coating, self-flattening of rough interfaces, and in situ purification that eliminates the impurity influences. Furthermore, recommendations for future perspectives are presented, and key issues for further development are discussed. PMID:27877656

  2. The final spawning ground of Tachypleus gigas (Müller, 1785) on the east Peninsular Malaysia is at risk: a call for action

    PubMed Central

    Moh, Julia Hwei Zhong; Ikhwanuddin, Mhd; Chatterji, Anil; Shaharom, Faizah

    2016-01-01

    Tanjung Selongor and Pantai Balok (State Pahang) are the only two places known for spawning activity of the Malaysian horseshoe crab - Tachypleus gigas (Müller, 1785) on the east coast of Peninsular Malaysia. While the former beach has been disturbed by several anthropogenic activities that ultimately brought an end to the spawning activity of T. gigas, the status of the latter remains uncertain. In the present study, the spawning behavior of T. gigas at Pantai Balok (Sites I-III) was observed over a period of thirty six months, in three phases, between 2009 and 2013. Every year, the crab’s nesting activity was found to be high during Southwest monsoon (May–September) followed by Northeast (November–March) and Inter monsoon (April and October) periods. In the meantime, the number of female T. gigas in 2009–2010 (Phase-1) was higher (38 crabs) than in 2010–2011 (Phase-2: 7 crabs) and 2012–2013 (Phase-3: 9 crabs) for which both increased overexploitation (for edible and fishmeal preparations) as well as anthropogenic disturbances in the vicinity (sand mining since 2009, land reclamation for wave breaker/parking lot constructions in 2011 and fishing jetty construction in 2013) are responsible. In this context, the physical infrastructure developments have altered the sediment close to nesting sites to be dominated by fine sand (2.5Xφ ) with moderately-well sorted (0.6–0.7σφ), very-coarse skewed (−2.4SKφ), and extremely leptokurtic (12.6Kφ) properties. Also, increased concentrations of Cadmium (from 4.2 to 13.6 mg kg−1) and Selenium (from 11.5 to 23.3 mg kg−1) in the sediment, and Sulphide (from 21 to 28 µg l−1) in the water were observed. In relation to the monsoonal changes affecting sheltered beach topography and sediment flux, the spawning crabs have shown a seasonal nest shifting behaviour in-between Sites I-III during 2009–2011. However, in 2012–2013, the crabs were mostly restricted to the areas (i.e., Sites I and II) with high

  3. The final spawning ground of Tachypleus gigas (Müller, 1785) on the east Peninsular Malaysia is at risk: a call for action.

    PubMed

    Nelson, Bryan Raveen; Satyanarayana, Behara; Moh, Julia Hwei Zhong; Ikhwanuddin, Mhd; Chatterji, Anil; Shaharom, Faizah

    2016-01-01

    Tanjung Selongor and Pantai Balok (State Pahang) are the only two places known for spawning activity of the Malaysian horseshoe crab - Tachypleus gigas (Müller, 1785) on the east coast of Peninsular Malaysia. While the former beach has been disturbed by several anthropogenic activities that ultimately brought an end to the spawning activity of T. gigas, the status of the latter remains uncertain. In the present study, the spawning behavior of T. gigas at Pantai Balok (Sites I-III) was observed over a period of thirty six months, in three phases, between 2009 and 2013. Every year, the crab's nesting activity was found to be high during Southwest monsoon (May-September) followed by Northeast (November-March) and Inter monsoon (April and October) periods. In the meantime, the number of female T. gigas in 2009-2010 (Phase-1) was higher (38 crabs) than in 2010-2011 (Phase-2: 7 crabs) and 2012-2013 (Phase-3: 9 crabs) for which both increased overexploitation (for edible and fishmeal preparations) as well as anthropogenic disturbances in the vicinity (sand mining since 2009, land reclamation for wave breaker/parking lot constructions in 2011 and fishing jetty construction in 2013) are responsible. In this context, the physical infrastructure developments have altered the sediment close to nesting sites to be dominated by fine sand (2.5Xφ ) with moderately-well sorted (0.6-0.7σφ), very-coarse skewed (-2.4SKφ), and extremely leptokurtic (12.6Kφ) properties. Also, increased concentrations of Cadmium (from 4.2 to 13.6 mg kg(-1)) and Selenium (from 11.5 to 23.3 mg kg(-1)) in the sediment, and Sulphide (from 21 to 28 µg l(-1)) in the water were observed. In relation to the monsoonal changes affecting sheltered beach topography and sediment flux, the spawning crabs have shown a seasonal nest shifting behaviour in-between Sites I-III during 2009-2011. However, in 2012-2013, the crabs were mostly restricted to the areas (i.e., Sites I and II) with high oxygen (5.5-8.0 mg l

  4. Locomotion and behavior of Humboldt squid, Dosidicus gigas, in relation to natural hypoxia in the Gulf of California, Mexico.

    PubMed

    Gilly, William F; Zeidberg, Louis D; Booth, J Ashley T; Stewart, Julia S; Marshall, Greg; Abernathy, Kyler; Bell, Lauren E

    2012-09-15

    We studied the locomotion and behavior of Dosidicus gigas using pop-up archival transmitting (PAT) tags to record environmental parameters (depth, temperature and light) and an animal-borne video package (AVP) to log these parameters plus acceleration along three axes and record forward-directed video under natural lighting. A basic cycle of locomotor behavior in D. gigas involves an active climb of a few meters followed by a passive (with respect to jetting) downward glide carried out in a fins-first direction. Temporal summation of such climb-and-glide events underlies a rich assortment of vertical movements that can reach vertical velocities of 3 m s(-1). In contrast to such rapid movements, D. gigas spends more than 80% of total time gliding at a vertical velocity of essentially zero (53% at 0±0.05 m s(-1)) or sinking very slowly (28% at -0.05 to -0.15 m s(-1)). The vertical distribution of squid was compared with physical features of the local water column (temperature, oxygen and light). Oxygen concentrations of ≤20 μmol kg(-1), characteristic of the midwater oxygen minimum zone (OMZ), can influence the daytime depth of squid, but this depends on location and season, and squid can 'decouple' from this environmental feature. Light is also an important factor in determining daytime depth, and temperature can limit nighttime depth. Vertical velocities were compared over specific depth ranges characterized by large differences in dissolved oxygen. Velocities were generally reduced under OMZ conditions, with faster jetting being most strongly affected. These data are discussed in terms of increased efficiency of climb-and-glide swimming and the potential for foraging at hypoxic depths.

  5. Purification, crystallization and preliminary X-ray studies of human augmenter of liver regeneration.

    PubMed

    Ji, Chao-Neng; Cai, Zai-Long; Cao, Gen-Tao; Yin, Gang; Jiao, Bing-Hua; Jiang, Tao; Shu, Guang; Mao, Ji-Fang; Xie, Yi; Mao, Yu-Min

    2002-12-01

    Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).

  6. Nucleation and Convection Effects in Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1997-01-01

    Work during the second year under this grant (NAG8-1161) resulted in several major achievements. We have characterized protein impurities as well as microheterogeneities in the proteins hen egg white lysozyme and horse spleen apoferritin, and demonstrated the effects of these impurities on nucleation and crystallization. In particular, the purification of apoferritin resulted in crystals with an X-ray diffraction resolution of better than 1.8 A, i.e. a 1 A improvement over earlier work on the cubic form. Furthermore, we have shown, in association with studies of liquid-liquid phase separation, that depending on the growth conditions, lysozyme can produce all growth morphologies that have been observed with other proteins. Finally, in connection with our experimental and simulation work on growth step bunching, we have developed a system-dependent criterion for advantages and disadvantages of crystallization from solution under reduced gravity. In the following, these efforts are described in some detail.

  7. Purification and thermal analysis of perfluoro-n-alkanoic acids.

    PubMed

    Tsuji, Minami; Inoue, Tohru; Shibata, Osamu

    2008-01-15

    Purification of perfluoro-n-alkanoic acids (C(n)F(2n+1)COOH, n=7, 9, 11, 13, 15 and 17) was made by repeated recrystallizations from n-hexane/acetone mixed solvent, and their purity was found to be more than 99.5% by GC-MS, NMR, and elemental analysis. The thermal behaviors such as melting point and enthalpy change of fusion were investigated using differential scanning calorimetry (DSC). The melting point monotonously increased with increasing carbon number (n) of the acids, while the enthalpy change showed irregularity at n=14. The crystal structure of these acids was found to be dependent upon solvent used for recrystallization; that is, the acids recrystallized from the above solvent becomes more stable energetically, indicating their higher enthalpy change of fusion than that of the solidified acids from fused ones. The solid state was also found to vary depending upon the thermal history, indicating that a few crystal structures of the solid state are quite similar energetically. The melting points (T(m)) of perfluoro-n-alkanoic acids are higher than those of corresponding n-alkanoic acids, and the difference in T(m) increases with increasing carbon number in the acids.

  8. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Linke, Christian, E-mail: clin180@ec.auckland.ac.nz; Caradoc-Davies, Tom T.; Australian Synchrotron, Clayton, Victoria 3168

    2008-02-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to themore » monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.« less

  9. Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yin, Lei; Zhu, De-Yu; Yang, Na

    2005-04-01

    The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 Å. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.

  10. Protein Crystals as Novel Catalytic Materials.

    PubMed

    Margolin, Alexey L.; Navia, Manuel A.

    2001-06-18

    In this era of molecular biology, protein crystallization is often considered to be a necessary first step in obtaining structural information through X-ray diffraction analysis. In a different light, protein crystals can also be thought of as materials, whose chemical and physical properties make them broadly attractive and useful across a larger spectrum of disciplines. The full potential of these protein crystalline materials has been severely restricted in practice, however, both by their inherent fragility, and by strongly held skepticism concerning their routine and predictable growth, formulation, and practical application. Fortunately, these problems have turned out to be solvable. A systematic exploration of the biophysics and biochemistry of protein crystallization has shown that one can dependably create new protein crystalline materials more or less at will. In turn, these crystals can be readily strengthened, both chemically and mechanically, to make them suitable for practical commercialization. Today, these novel materials are used as industrial catalysts on a commercial scale, in bioremediation and "green chemistry" applications, and in enantioselective chromatography of pharmaceuticals and fine chemicals. In the near future, their utility will expand, to include the purification of protein drugs, formulation of direct protein therapeutics, and development of adjuvant-less vaccines.

  11. Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

    PubMed

    Virador, Victoria M; Reyes Grajeda, Juan P; Blanco-Labra, Alejandro; Mendiola-Olaya, Elizabeth; Smith, Gary M; Moreno, Abel; Whitaker, John R

    2010-01-27

    The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

  12. Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bäuerle, Bettina; Sandalova, Tatyana; Schneider, Gunter

    2006-08-01

    This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space groupmore » P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.« less

  13. Expression, purification and crystallization of a dye-decolourizing peroxidase from Dictyostelium discoideum.

    PubMed

    Rai, Amrita; Fedorov, Roman; Manstein, Dietmar J

    2014-02-01

    Dye-decolourizing peroxidases are haem-containing peroxidases with broad substrate specificity. Using H2O2 as an electron acceptor, they efficiently decolourize various dyes that are of industrial and environmental relevance, such as anthraquninone- and azo-based dyes. In this study, the dye-decolourizing peroxidase DdDyP from Dictyostelium discoideum was overexpressed in Escherichia coli strain Rosetta(DE3)pLysS, purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.65 Å resolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 141.03, c = 95.56 Å, α = β = γ = 90°. The asymmetric unit contains two molecules.

  14. Process for purification of silicon

    NASA Technical Reports Server (NTRS)

    Rath, H. J.; Sirtl, E.; Pfeiffer, W.

    1981-01-01

    The purification of metallurgically pure silicon having a silicon content of more than 95% by weight is accomplished by leaching with an acidic solution which substantially does not attack silicon. A mechanical treatment leading to continuous particle size reduction of the granulated silicon to be purified is combined with the chemical purification step.

  15. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Synechocystis sp. PCC 6803.

    PubMed

    Liu, Yinghui; Zhang, Yanming; Cao, Xupeng; Xue, Song

    2013-11-01

    Malonyl-coenzymeA:acyl-carrier protein transacylase (MCAT), which catalyzes the transfer of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP), is an essential enzyme in type II fatty-acid synthesis. The enzyme MCAT from Synechocystis sp. PCC 6803 (spMCAT), the first MCAT counterpart from a cyanobacterium, was cloned, purified and crystallized in order to determine its three-dimensional crystal structure. A higher-quality crystal with better diffraction was obtained by crystallization optimization. The crystal diffracted to 1.8 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 43.22, b = 149.21, c = 40.59 Å. Matthews coefficient calculations indicated that the crystal contained one spMCAT molecule in the asymmetric unit with a Matthews coefficient of 2.18 Å(3) Da(-1) and a solvent content of 43.65%.

  16. Impact and fracture analysis of fish scales from Arapaima gigas.

    PubMed

    Torres, F G; Malásquez, M; Troncoso, O P

    2015-06-01

    Fish scales from the Amazonian fish Arapaima gigas have been characterised to study their impact and fracture behaviour at three different environmental conditions. Scales were cut in two different directions to analyse the influence of the orientation of collagen layers. The energy absorbed during impact tests was measured for each sample and SEM images were taken after each test in order to analyse the failure mechanisms. The results showed that scales tested at cryogenic temperatures display fragile behaviour, while scales tested at room temperature did not fracture. Different failure mechanisms have been identified, analysed and compared with the failure modes that occur in bone. The impact energy obtained for fish scales was two to three times higher than the values reported for bone in the literature. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamermore » is the biological active form of E. coli. purine nucleoside phosphorylase.« less

  18. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan).

    PubMed

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2008-07-01

    Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 A resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 A.

  19. Entanglement of purification: from spin chains to holography

    NASA Astrophysics Data System (ADS)

    Nguyen, Phuc; Devakul, Trithep; Halbasch, Matthew G.; Zaletel, Michael P.; Swingle, Brian

    2018-01-01

    Purification is a powerful technique in quantum physics whereby a mixed quantum state is extended to a pure state on a larger system. This process is not unique, and in systems composed of many degrees of freedom, one natural purification is the one with minimal entanglement. Here we study the entropy of the minimally entangled purification, called the entanglement of purification, in three model systems: an Ising spin chain, conformal field theories holographically dual to Einstein gravity, and random stabilizer tensor networks. We conjecture values for the entanglement of purification in all these models, and we support our conjectures with a variety of numerical and analytical results. We find that such minimally entangled purifications have a number of applications, from enhancing entanglement-based tensor network methods for describing mixed states to elucidating novel aspects of the emergence of geometry from entanglement in the AdS/CFT correspondence.

  20. Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Skálová, Tereza, E-mail: skalova@imc.cas.cz; Dohnálek, Jan; Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnicka 10, 162 53 Praha 6

    2007-12-01

    The expression, purification and crystallization of the small laccase from S. coelicolor are reported. Diffraction data were collected to 3 Å resolution. The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 Å. The crystals belong to either space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases willmore » be determined from the anomalous signal of the natively present copper ions.« less

  1. Estimates of Heritability for Growth and Shell Color Traits and Their Genetic Correlations in the Black Shell Strain of Pacific Oyster Crassostrea gigas.

    PubMed

    Xu, Lan; Li, Qi; Yu, Hong; Kong, Lingfeng

    2017-10-01

    The Pacific oyster Crassostrea gigas has been introduced widely and massively and became an economically important aquaculture species on a global scale. We estimated heritabilities of growth and shell color traits and their genetic correlations in black shell strain of C. gigas. Analyses were performed on 22 full-sib families in a nested mating design including 410 individuals at harvest (24 months of age). The parentage assignment was inferred based on four panels of multiplex PCR markers including 10 microsatellite loci and 94.9% of the offspring were unambiguously assigned to single parent pairs. The Spearman correlation test (r = - 0.992, P < 0.001) demonstrated the high consistency of the shell pigmentation (SP) and L* and their same efficacy in shell color measurements. The narrow-sense heritability estimated under the animal model analysis was 0.18 ± 0.12 for shell height, 0.25 ± 0.16 for shell length, 0.10 ± 0.09 for shell width, 0.42 ± 0.20 for total weight, 0.32 ± 0.18 for shell weight, and 0.68 ± 0.16 for L*, 0.69 ± 0.16 for shell pigmentation, respectively. The considerable additive genetic variation in growth and shell color traits will make it feasible to produce genetic improvements for these traits in selective breeding program. High genetic and phenotypic correlations were found among growth traits and among shell color traits. To optimize a selection strategy for both fast growth and pure dark shell strain of C. gigas, it is proposed to take both total weight and black shell as joint objective traits in selective breeding program. Our study offers an important reference in the process of selective breeding in black shell color stain of C. gigas and will facilitate to develop favorable breeding strategies of genetic improvements for this economically important strain.

  2. Crystallization of sodium chloride from a concentrated calcium chloride-potassium chloride-sodium chloride solution in a CMSMPR crystallizer: Observation of crystal size distribution and model validation

    NASA Astrophysics Data System (ADS)

    Choi, Byung Sang

    conditions by studying the detailed crystallization processes, such as nucleation, growth, and breakage, as well as agglomeration. The purification of CaCl2 solution involving the crystallization of NaCl from the solution mixture of CaCl2, KCl, and NaCl as shipped from Dow Chemical, Ludington, in a CMSMPR crystallizer was studied as our model system because of its nucleation and crystal growth tendencies with less agglomeration. This project also generated a significant body of experimental data that are available at URL that is http://www.che.utah.edu/˜ring/CrystallizationWeb.

  3. Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine.

    PubMed

    Loren, Bradley P; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang; Nagy, Zoltan K; Thompson, David H; Cooks, R Graham

    2017-06-01

    A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis.

  4. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

    PubMed Central

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2008-01-01

    Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å. PMID:18607103

  5. Oocyte maturation and origin of the germline as revealed by the expression of Nanos-like in the Pacific oyster Crassostrea gigas.

    PubMed

    Xu, Rui; Li, Qi; Yu, Hong; Kong, Lingfeng

    2018-04-13

    Nanos gene plays an important role in germline development in animals. However, the molecular mechanisms involved in germline development in Mollusca, the second largest animal phylum, are still poorly understood. Here we identified the Nanos orthologue from the Pacific oyster Crassostrea gigas (Cg-Nanos-like), and investigated the expression patterns of Nanos during gametogenesis and embryogenesis in C. gigas. Tissue expression analysis showed that Cg-Nanos-like was specifically expressed in female gonads. During the reproductive cycle, the expression of Cg-Nanos-like mRNA increased matching the seasonal development of the ovarian tissues in diploids, while the expression levels were significantly lower in the ovaries of sterile triploids compared to diploids. High expression of Cg-Nanos-like transcripts were detected in early embryonic stages, while the expression significantly dropped at gastrulation and was barely detectable in veliger stages. In situ hybridization showed that Cg-Nanos-like was expressed at different stages of developing oocytes, whereas positive signals were detected only in spermatogonia during the spermatogenic cycle. These findings indicated that Cg-Nanos-like was involved in the development of germ cells, and maintenance of oocyte maturation. In early embryogenesis, the transcripts were broadly expressed; following gastrulation, the expression was restricted to two cell clumps, which might be the putative primordial germ cells (PGCs) or their precursors. Based on the results, the formation of the PGCs in C. gigas was consistent with the model of transition from epigenesis to preformation. Copyright © 2017. Published by Elsevier B.V.

  6. Myogenic activity and serotonergic inhibition in the chromatophore network of the squid Dosidicus gigas (family Ommastrephidae) and Doryteuthis opalescens (family Loliginidae).

    PubMed

    Rosen, Hannah E; Gilly, William F

    2017-12-15

    Seemingly chaotic waves of spontaneous chromatophore activity occur in the ommastrephid squid D osidicus gigas in the living state and immediately after surgical disruption of all known inputs from the central nervous system. Similar activity is apparent in the loliginid Doryteuthis opalescens , but only after chronic denervation of chromatophores for 5-7 days. Electrically stimulated, neurally driven activity in intact individuals of both species is blocked by tetrodotoxin (TTX), but TTX has no effect on spontaneous wave activity in either D. gigas or denervated D. opalescens Spontaneous TTX-resistant activity of this sort is therefore likely myogenic, and such activity is eliminated in both preparations by serotonin (5-HT), a known inhibitor of chromatophore activity. Immunohistochemical techniques reveal that individual axons containing L-glutamate or 5-HT (and possibly both in a minority of processes) are associated with radial muscle fibers of chromatophores in intact individuals of both species, although the area of contact between both types of axons and muscle fibers is much smaller in D. gigas Glutamatergic and serotonergic axons degenerate completely following denervation in D. opalescens Spontaneous waves of chromatophore activity in both species are thus associated with reduced (or no) serotonergic input in comparison to the situation in intact D. opalescens Such differences in the level of serotonergic inhibition are consistent with natural chromogenic behaviors in these species. Our findings also suggest that such activity might propagate via the branching distal ends of radial muscle fibers. © 2017. Published by The Company of Biologists Ltd.

  7. Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

    PubMed Central

    Zhang, Xi; Miller, Keith W.

    2015-01-01

    The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-β-d-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses—without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. PMID:25473089

  8. Photocatalytic materials and technologies for air purification.

    PubMed

    Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher

    2017-03-05

    Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Nucleation and convection effects in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz (Principal Investigator)

    1996-01-01

    The following activities are reported on: repartitioning of NaCl and protein impurities in lysozyme crystallization; dependence of lysozyme growth kinetics on step sources and impurities; facet morphology response to nonuniformities in nutrient and impurity supply; interactions in undersaturated and supersaturated lysozyme solutions; heterogeneity determination and purification of commercial hen egg white lysozyme; nonlinear response of layer growth dynamics in the mixed kinetics-bulk transport regime; development of a simultaneous multiangle light scattering technique; and x-ray topography of tetragonal lysozyme grown by the temperature-control technique.

  10. Purification, crystallization and preliminary x-ray crystallographic studies of RAIDD Death-Domain (DD).

    PubMed

    Jang, Tae-ho; Park, Hyun Ho

    2009-06-03

    Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

  11. The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, C.A.; Cohen, A.E.

    2009-05-26

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screenedmore » in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.« less

  12. Hamiltonian purification

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purificationmore » and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.« less

  13. Purification, Crystallization, and Preliminary Crystallographic Analysis of Deoxyuridine Triphosphate Nucleotidohydrolase from Arabidopsis Thaliana

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bajaj,M.; Moriyama, H.

    2007-01-01

    The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Angstroms resolution using Cu K{alpha} radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Angstroms, c = 75.55 Angstroms . Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a VM of 1.8 Angstroms 3 Da-1.

  14. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

    PubMed

    Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

    2008-06-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

  15. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

    PubMed Central

    Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

    2008-01-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P42212, with unit-cell parameters a = b = 93.665, c = 131.95. PMID:18540065

  16. STS-30 MS Cleave uses camcorder to record FEA-2 crystal growth

    NASA Image and Video Library

    1989-05-08

    STS030-10-002 (8 May 1989) --- STS-30 Mission Specialist Mary L. Cleave operates 8mm video camcorder at Fluids Experiment Apparatus 2 (FEA-2) (SK73-000102) unit located in aft middeck locker onboard Atlantis, Orbiter Vehicle (OV) 103. Two 8mm video camcorders are positioned above FEA-2 unit to record experiment titled "Floating Zone Crystal Growth and Purification". Rockwell International (RI) through its Space Transportation Systems Division, Downey, California, is engaged in a joint endeavor agreement (JEA) with NASA's Office of Commercial Programs in the field for floating zone crystal growth research. Utah State University Aggies decal appears on aft bulkhead above FEA-2 unit.

  17. A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reduction

    PubMed Central

    Moon, Andrea F; Mueller, Geoffrey A; Zhong, Xuejun; Pedersen, Lars C

    2010-01-01

    Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. PMID:20196072

  18. Moissanite anvil cell design for Giga-Pascal nuclear magnetic resonance.

    PubMed

    Meier, Thomas; Herzig, Tobias; Haase, Jürgen

    2014-04-01

    A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with external-force vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. (1)H, (23)Na, (27)Al, (69)Ga, and (71)Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.

  19. Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    D’Arcy, Allan, E-mail: allan.darcy@novartis.com; Chaillet, Maxime; Schiering, Nikolaus

    Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but maymore » also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.« less

  20. Water purification in Borexino

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Giammarchi, M.; Balata, M.; Ioannucci, L.

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  1. Affinity chromatography: A versatile technique for antibody purification.

    PubMed

    Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

    2017-03-01

    Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Protein purification in multicompartment electrolyzers for crystal growth of r-DNA products in microgravity

    NASA Technical Reports Server (NTRS)

    Righetti, Pier Giorgio; Casale, Elena; Carter, Daniel; Snyder, Robert S.; Wenisch, Elisabeth; Faupel, Michel

    1990-01-01

    Recombinant-DNA (deoxyribonucleic acid) (r-DNA) proteins, produced in large quantities for human consumption, are now available in sufficient amounts for crystal growth. Crystallographic analysis is the only method now available for defining the atomic arrangements within complex biological molecules and decoding, e.g., the structure of the active site. Growing protein crystals in microgravity has become an important aspect of biology in space, since crystals that are large enough and of sufficient quality to permit complete structure determinations are usually obtained. However even small amounts of impurities in a protein preparation are anathema for the growth of a regular crystal lattice. A multicompartment electrolyzer with isoelectric, immobiline membranes, able to purify large quantities of r-DNA proteins is described. The electrolyzer consists of a stack of flow cells, delimited by membranes of very precise isoelectric point (pI, consisting of polyacrylamide supported by glass fiber filters containing Immobiline buffers and titrants to uniquely define a pI value) and very high buffering power, able to titrate all proteins tangent or crossing such membranes. By properly selecting the pI values of two membranes delimiting a flow chamber, a single protein can be kept isoelectric in a single flow chamber and thus, be purified to homogeneity (by the most stringent criterion, charge homogeneity).

  3. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

    PubMed

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-12-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, β=95.2°. Diffraction images were processed to a resolution of 1.74 Å with an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

  4. Expression, purification and crystallization of the C-terminal LRR domain of Streptococcus pyogenes protein 0843.

    PubMed

    Haikarainen, Teemu; Loimaranta, Vuokko; Prieto-Lopez, Carlos; Battula, Pradeep; Finne, Jukka; Papageorgiou, Anastassios C

    2013-05-01

    Streptococcus pyogenes protein 0843 (Spy0843) is a recently identified protein with a potential adhesin function. Sequence analysis has shown that Spy0843 contains two leucine-rich repeat (LRR) domains that mediate interactions with the gp340 receptor. Here, the C-terminal LRR domain was overexpressed in Escherichia coli, purified and crystallized in the presence of 1.7-1.8 M ammonium sulfate pH 7.4 as precipitant. Data were collected from a single crystal to 1.59 Å resolution at 100 K at a synchrotron-radiation source. The crystal was found to belong to space group I41, with unit-cell parameters a = b = 121.4, c = 51.5 Å and one molecule in the asymmetric unit. Elucidation of the crystal structure will provide insights into the interactions of Spy0843 with the gp340 receptor and a better understanding of the role of Spy0843 in streptococcal infections.

  5. Expression, purification and crystallization of the C-terminal LRR domain of Streptococcus pyogenes protein 0843

    PubMed Central

    Haikarainen, Teemu; Loimaranta, Vuokko; Prieto-Lopez, Carlos; Battula, Pradeep; Finne, Jukka; Papageorgiou, Anastassios C.

    2013-01-01

    Streptococcus pyogenes protein 0843 (Spy0843) is a recently identified protein with a potential adhesin function. Sequence analysis has shown that Spy0843 contains two leucine-rich repeat (LRR) domains that mediate interactions with the gp340 receptor. Here, the C-terminal LRR domain was overexpressed in Escherichia coli, purified and crystallized in the presence of 1.7–1.8 M ammonium sulfate pH 7.4 as precipitant. Data were collected from a single crystal to 1.59 Å resolution at 100 K at a synchrotron-radiation source. The crystal was found to belong to space group I41, with unit-cell parameters a = b = 121.4, c = 51.5 Å and one molecule in the asymmetric unit. Elucidation of the crystal structure will provide insights into the interactions of Spy0843 with the gp340 receptor and a better understanding of the role of Spy0843 in streptococcal infections. PMID:23695577

  6. Horizontal movements, vertical-habitat utilization and diet of the jumbo squid ( Dosidicus gigas) in the Pacific Ocean off Baja California Sur, Mexico

    NASA Astrophysics Data System (ADS)

    Bazzino, Gastón; Gilly, William F.; Markaida, Unai; Salinas-Zavala, César A.; Ramos-Castillejos, Jorge

    2010-07-01

    We deployed four pop-up archival-transmitting (PAT) tags on jumbo squid ( Dosidicus gigas) collected in the Pacific Ocean off the main entrance to Magdalena Bay on the Baja California peninsula in June 2005. This is the first successful deployment of PAT tags on jumbo squid in an area outside the Gulf of California. Summary data were obtained through the ARGOS satellite system for three of the tags; the fourth tag was physically recovered. All of the tagged squid tended to remain on the shallow continental shelf for several days after tagging and then moved offshore into deeper water. Three of the four squid appeared to migrate in a general southerly direction while the fourth remained offshore of Magdalena Bay. All of the squid spent most daylight hours at depths that were associated with the hypoxic oxygen minimum layer, and at night they spent a majority of time in the upper 50 m of the water column. Stomach content analysis and tag temperature-depth data during the first days after tagging revealed that the squid were feeding on pelagic red crabs ( Pleuroncodes planipes) and several larger, neritic fishes over the continental shelf off Magdalena Bay during a seasonal nearshore upwelling. Comparison of our results with those previously collected in the Gulf of California reveal that Dosidicus gigas can vary its behavior and diet to suit local environmental conditions. This adaptability is likely to be an important factor in the ability of D. gigas to invade and colonize new areas.

  7. Effect of Amidated Low-Methoxyl Pectin on Physicochemical Characteristics of Jumbo Squid (Dosidicus gigas) Mantle Muscle Gels.

    PubMed

    Ramirez-Suarez, Juan C; Álvarez-Armenta, Andrés; García-Sánchez, Guillermina; Pacheco-Aguilar, Ramón; Scheuren-Acevedo, Susana M; Mazorra-Manzano, Miguel A; Rascón-Chu, Agustín

    2017-09-01

    Jumbo squid ( Dosidicus gigas ) muscle proteins show low functionality with limited use in gel products. This work aims to assess the influence of adding the natural and commercially available fibre, amidated low-methoxyl pectin (at 0.5, 1.0, 1.5, 2.0 and 3.0%), on the physicochemical and functional characteristics of jumbo squid ( Dosidicus gigas ) mantle muscle gels. The addition of 0.5% fibre showed an immediate effect on the gel texture profile analysis, improving hardness (p<0.05) from (3.4±0.7) N of the control (no added fibre) to (5.2±0.9) N, and increasing elasticity (p≥0.05). Shear force was significant only at 3.0% fibre addition. Water holding capacity also improved (p<0.05) with fibre addition (from 75% in the control to 90-95% after the treatments). Whiteness was affected (p<0.05) when 3.0% fibre was added. Differential scanning calorimetry showed two endothermic transition peaks in the gels. The second peak (actin) increased (p<0.05) by 1-2 °C with fibre addition. Therefore, the present study demonstrates that amidated low-methoxyl pectin (0.5-3.0%) is an excellent ingredient to improve jumbo squid mantle muscle protein functionality, increasing the gel texture and water retention characteristics.

  8. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

    PubMed Central

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-01-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase. PMID:21139221

  9. Overexpression, purification, crystallization and preliminary structural studies of catabolic ornithine transcarbamylase from Lactobacillus hilgardii

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rivas, Blanca de las; Rodríguez, Héctor; Angulo, Iván

    2007-07-01

    The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His{sub 6} tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystalsmore » obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å{sup 3} Da{sup −1}, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model.« less

  10. Antibiotics from Pseudomonas reptilivora II. Isolation, Purification, and Properties1

    PubMed Central

    Del Rio, Luís A.; Gorgé, J. López; Olivares, J.; Mayor, F.

    1972-01-01

    Under well-established culture conditions, Pseudomonas reptilivora produced several antibiotics that have been purified by solvent extraction, chromatography in Sephadex G-25, electrophoresis, and paper chromatography in different solvent systems. Activity has been monitored at the different steps of isolation and purification by measurement of the inhibition of the growth of Staphylococcus aureus by the cylinder-plate method, as well as by bioautography of chromatograms and electropherograms. Three antibiotics have been isolated and named A, B1, and B2. The B1 and B2 activities were studied in greater detail than A. The B1 substance was crystallized, and its chemical properties were found to coincide with those of YC 73 or fluopsin C described by Egawa et al. and Itoh et al., respectively. Images PMID:4790558

  11. Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2)

    PubMed Central

    Mello, Danielle F.; de Oliveira, Eliza S.; Vieira, Renato C.; Simoes, Erik; Trevisan, Rafael; Dafre, Alcir Luiz; Barracco, Margherita Anna

    2012-01-01

    Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes. PMID:22611355

  12. Demography of the ecosystem engineer Crassostrea gigas, related to vertical reef accretion and reef persistence

    NASA Astrophysics Data System (ADS)

    Walles, Brenda; Mann, Roger; Ysebaert, Tom; Troost, Karin; Herman, Peter M. J.; Smaal, Aad C.

    2015-03-01

    Marine species characterized as structure building, autogenic ecosystem engineers are recognized worldwide as potential tools for coastal adaptation efforts in the face of sea level rise. Successful employment of ecosystem engineers in coastal protection largely depends on long-term persistence of their structure, which is in turn dependent on the population dynamics of the individual species. Oysters, such as the Pacific oyster (Crassostrea gigas), are recognized as ecosystem engineers with potential for use in coastal protection. Persistence of oyster reefs is strongly determined by recruitment and shell production (growth), processes facilitated by gregarious settlement on extant shell substrate. Although the Pacific oyster has been introduced world-wide, and has formed dense reefs in the receiving coastal waters, the population biology of live oysters and the quantitative mechanisms maintaining these reefs has rarely been studied, hence the aim of the present work. This study had two objectives: (1) to describe the demographics of extant C. gigas reefs, and (2) to estimate vertical reef accretion rates and carbonate production in these oyster reefs. Three long-living oyster reefs (>30 years old), which have not been exploited since their first occurrence, were examined in the Oosterschelde estuary in the Netherlands. A positive reef accretion rate (7.0-16.9 mm year-1 shell material) was observed, consistent with self-maintenance and persistent structure. We provide a framework to predict reef accretion and population persistence under varying recruitment, growth and mortality scenarios.

  13. How annual course of photoperiod shapes seasonal behavior of diploid and triploid oysters, Crassostrea gigas

    PubMed Central

    Payton, Laura; Sow, Mohamedou; Massabuau, Jean-Charles; Ciret, Pierre

    2017-01-01

    In this work, we study if ploidy (i.e. number of copies of chromosomes) in the oyster Crassostrea gigas may introduce differences in behavior and in its synchronization by the annual photoperiod. To answer to the question about the effect of the seasonal course of the photoperiod on the behavior of C. gigas according to its ploidy, we quantified valve activity by HFNI valvometry in situ for 1 year in both diploid and triploid oysters. Chronobiological analyses of daily, tidal and lunar rhythms were performed according the annual change of the photoperiod. In parallel, growth and gametogenesis status were measured and spawning events were detected by valvometry. The results showed that triploids had reduced gametogenesis, without spawning events, and approximately three times more growth than diploids. These differences in physiological efforts could explain the result that photoperiod (daylength and/or direction of daylength) differentially drives and modulates seasonal behavior of diploid and triploid oysters. Most differences were observed during long days (spring and summer), where triploids showed longer valve opening duration but lower opening amplitude, stronger daily rhythm and weaker tidal rhythm. During this period, diploids did major gametogenesis and spawning whereas triploids did maximal growth. Differences were also observed in terms of moonlight rhythmicity and neap-spring tidal cycle rhythmicity. We suggest that the seasonal change of photoperiod differentially synchronizes oyster behavior and biological rhythms according to physiological needs based on ploidy. PMID:29020114

  14. How annual course of photoperiod shapes seasonal behavior of diploid and triploid oysters, Crassostrea gigas.

    PubMed

    Payton, Laura; Sow, Mohamedou; Massabuau, Jean-Charles; Ciret, Pierre; Tran, Damien

    2017-01-01

    In this work, we study if ploidy (i.e. number of copies of chromosomes) in the oyster Crassostrea gigas may introduce differences in behavior and in its synchronization by the annual photoperiod. To answer to the question about the effect of the seasonal course of the photoperiod on the behavior of C. gigas according to its ploidy, we quantified valve activity by HFNI valvometry in situ for 1 year in both diploid and triploid oysters. Chronobiological analyses of daily, tidal and lunar rhythms were performed according the annual change of the photoperiod. In parallel, growth and gametogenesis status were measured and spawning events were detected by valvometry. The results showed that triploids had reduced gametogenesis, without spawning events, and approximately three times more growth than diploids. These differences in physiological efforts could explain the result that photoperiod (daylength and/or direction of daylength) differentially drives and modulates seasonal behavior of diploid and triploid oysters. Most differences were observed during long days (spring and summer), where triploids showed longer valve opening duration but lower opening amplitude, stronger daily rhythm and weaker tidal rhythm. During this period, diploids did major gametogenesis and spawning whereas triploids did maximal growth. Differences were also observed in terms of moonlight rhythmicity and neap-spring tidal cycle rhythmicity. We suggest that the seasonal change of photoperiod differentially synchronizes oyster behavior and biological rhythms according to physiological needs based on ploidy.

  15. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  16. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  17. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  18. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  19. Invasive range expansion by the Humboldt squid, Dosidicus gigas, in the eastern North Pacific.

    PubMed

    Zeidberg, Louis D; Robison, Bruce H

    2007-07-31

    A unique 16-year time series of deep video surveys in Monterey Bay reveals that the Humboldt squid, Dosidicus gigas, has substantially expanded its perennial geographic range in the eastern North Pacific by invading the waters off central California. This sustained range expansion coincides with changes in climate-linked oceanographic conditions and a reduction in competing top predators. It is also coincident with a decline in the abundance of Pacific hake, the most important commercial groundfish species off western North America. Recognizing the interactive effects of multiple changes in the environment is an issue of growing concern in ocean conservation and sustainability research.

  20. Purification, crystallization and preliminary X-ray structure analysis of the banana lectin from Musa paradisiaca.

    PubMed

    Singh, D D; Saikrishnan, K; Kumar, Prashant; Dauter, Z; Sekar, K; Surolia, A; Vijayan, M

    2004-11-01

    The banana lectin from Musa paradisiaca, MW 29.4 kDa, has been isolated, purified and crystallized. The trigonal crystals contain one dimeric molecule in the asymmetric unit. The structure has been solved using molecular replacement to a resolution of 3 A. The structure of the subunit is similar to that of jacalin-like lectins.

  1. Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jens, Jason; Raghunathan, Kannan; Vago, Frank

    2010-01-12

    EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 {angstrom} and belonged to space group P2{sub 1}, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 {angstrom}.

  2. Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

    PubMed Central

    Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis

    2009-01-01

    EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 Å and belonged to space group P21, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 Å. PMID:19478449

  3. Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.

    PubMed

    Kellosalo, Juho; Kajander, Tommi; Honkanen, Riina; Goldman, Adrian

    2013-02-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.

  4. Chemical looping integration with a carbon dioxide gas purification unit

    DOEpatents

    Andrus, Jr., Herbert E.; Jukkola, Glen D.; Thibeault, Paul R.; Liljedahl, Gregory N.

    2017-01-24

    A chemical looping system that contains an oxidizer and a reducer is in fluid communication with a gas purification unit. The gas purification unit has at least one compressor, at least one dryer; and at least one distillation purification system; where the gas purification unit is operative to separate carbon dioxide from other contaminants present in the flue gas stream; and where the gas purification unit is operative to recycle the contaminants to the chemical looping system in the form of a vent gas that provides lift for reactants in the reducer.

  5. Moissanite anvil cell design for giga-pascal nuclear magnetic resonance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meier, Thomas; Herzig, Tobias; Haase, Jürgen

    2014-04-15

    A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with external-force vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. {sup 1}H, {sup 23}Na, {sup 27}Al, {sup 69}Ga, and {supmore » 71}Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.« less

  6. Purification of functionalized DNA origami nanostructures.

    PubMed

    Shaw, Alan; Benson, Erik; Högberg, Björn

    2015-05-26

    The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of water-soluble chlorophyll-binding protein from Chenopodium album

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohtsuki, Takayuki; Ohshima, Shigeru; Uchida, Akira, E-mail: auchida@biomol.sci.toho-u.ac.jp

    2007-09-01

    A water-soluble chlorophyll-binding protein with photoconvertibility from C. album was extracted, purified and crystallized in a darkroom. The crystal diffracted to around 2.0 Å resolution. A water-soluble chlorophyll-binding protein (WSCP) with photoconvertibility from Chenopodium album was extracted, purified and crystallized in a darkroom. Green crystals suitable for data collection appeared in about 10 d. A native data set was collected to 2.0 Å resolution at 100 K. The space group of the crystal was determined to be orthorhombic I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.13, b = 60.59, c = 107.21 Å. Preliminary analysis ofmore » the X-ray data indicated that there is one molecule per asymmetric unit.« less

  8. Crystallization Physics in Biomacromolecular Systems

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2003-01-01

    the defects responsible for poor resolution are not identified. Conformational changes are one of them. Biocrystallization in microgravity reportedly results in 20% cases of better crystals. The mechanism of how lack of convection can do this is still not clear. Lower supersaturation, self-purification &om preferentially trapped homologous impurities and step bunching are viable hypotheses.

  9. Causes and effects of a highly successful marine invasion: Case-study of the introduced Pacific oyster Crassostrea gigas in continental NW European estuaries

    NASA Astrophysics Data System (ADS)

    Troost, Karin

    2010-10-01

    Since the 1960's, the Pacific oyster Crassostrea gigas has been introduced for mariculture at several locations within NW Europe. The oyster established itself everywhere and expanded rapidly throughout the receiving ecosystems, forming extensive and dense reef structures. It became clear that the Pacific oyster induced major changes in NW European estuaries. This paper reviews the causes of the Pacific oyster's remarkably successful establishment and spread in The Netherlands and neighbouring countries, and includes a comprehensive review of consequences for the receiving communities. Ecosystem engineering by C. gigas and a relative lack of natural enemies in receiving ecosystems are identified as the most important characteristics facilitating the invader's successful establishment and expansion. The Pacific oyster's large filtration capacity and eco-engineering characteristics induced many changes in receiving ecosystems. Different estuaries are affected differently; in the Dutch Oosterschelde estuary expanding stocks saturate the carrying capacity whereas in the Wadden Sea no such problems exist. In general, the Pacific oyster seems to fit well within continental NW European estuarine ecosystems and there is no evidence that the invader outcompetes native bivalves. C. gigas induces changes in plankton composition, habitat heterogeneity and biodiversity, carrying capacity, food webs and parasite life cycles. The case of the Pacific oyster in NW European estuaries is only one example in an increasing series of biological invasions mediated by human activities. This case-study will contribute to further elucidating general mechanisms in marine invasions; invasions that sometimes appear a threat, but can also contribute to ecological complexity.

  10. Antimicrobial Peptide Production and Purification.

    PubMed

    Suda, Srinivas; Field, Des; Barron, Niall

    2017-01-01

    Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

  11. Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishimura, Mitsuhiro; Protein Research Group, RIKEN Yokohama Institute, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045; Kaminishi, Tatsuya

    2007-11-01

    A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to spacemore » group P3{sub 1}21 or P3{sub 2}21.« less

  12. Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lyashenko, Andrey V.; Zhukhlistova, Nadegda E.; Gabdoulkhakov, Azat G.

    2006-10-01

    The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented. Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccasemore » structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; R{sub free} = 23.835; r.m.s.d. bond lengths, 0.06 Å; r.m.s.d. bond angles, 1.07°) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002 ▶) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO{sub 2} substituents.« less

  13. Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Barranco-Medina, Sergio; López-Jaramillo, Francisco Javier, E-mail: fjljara@ugr.es; Bernier-Villamor, Laura

    2006-07-01

    The isolation, purification, crystallization and molecular-replacement solution of mitochondrial type II peroxiredoxin from P. sativum is reported. A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 Å from a single crystal flash-cooledmore » at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 Å, α = 102.90, β = 104.40, γ = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.« less

  14. The cytochemical and ultrastructural characteristics of phagocytes in the Pacific oyster Crassostrea gigas.

    PubMed

    Jiang, Shuai; Jia, Zhihao; Xin, Lusheng; Sun, Ying; Zhang, Ran; Wang, Weilin; Wang, Lingling; Song, Linsheng

    2016-08-01

    Phagocytes have been proved to play vital roles in the innate immune response. However, the cellular characteristics of phagocytes in invertebrates, especially in molluscs, remain largely unknown. In the present study, fluorescence activated cell sorting (FACS) was employed to sort the phagocytes from the non-phagocytic haemocytes of the Pacific oyster Crassostrea gigas. The cytochemical staining analysis revealed that phagocytes were positive staining for α-naphthyl acetate esterase and myeloperoxidase, while negative staining for toluidine blue and periodic acid-Schiff. The non-phagocytic haemocytes exhibited positive staining for periodic acid-Schiff, weak positive staining for toluidine blue, but negative staining for α-naphthyl acetate esterase and myeloperoxidase. In addition, phagocytes exhibited ultrastructural cellular features similar to those of macrophages, with large cell diameter, rough cell membrane and extended pseudopodia revealed by the scanning electron microscopy, while the non-phagocytic haemocytes exhibited small cell diameter, smooth cell surface and round spherical shape. Transmission electron microscopy further demonstrated that phagocytes were abundant of cytoplasmic bodies and mitochondria, while non-phagocytic haemocytes were characterized as the comparatively large cell nucleus with contorted and condensed heterochromatin adherent to the nuclear envelope. Moreover, compared with non-phagocytic haemocytes, phagocytes exhibited significantly higher levels of intracellular cytokines, including tumor necrosis factor, interferon-like protein and interleukin-17, and significantly higher abundance of lysosome and reactive oxygen species, which were of great importance to the activation of immune response and pathogen clearance. Taken together, these findings revealed the different cytochemical and ultrastructural features between phagocytes and non-phagocytic haemocytes in C. gigas, which would provide an important clue to investigate the

  15. Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

    PubMed

    Bredow, Melissa; Tomalty, Heather E; Walker, Virginia K

    2017-05-05

    Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the

  16. A new method of auxiliary purification for motor vehicle exhaust.

    PubMed

    Li, Dingqi

    2018-07-01

    As a result of the limitations of current purification technologies, purification efficiency is relatively low, particularly during startup or in the case of other abnormal automobile exhaust. Therefore, a new method of auxiliary purification is proposed in this paper. The acidic solution of potassium permanganate can oxidize carbon monoxide, nitrogen oxides and sulfur dioxide at relatively high temperatures and the alkaline solution of potassium permanganate can selectively absorb nitrogen oxide and sulfur dioxide. Therefore, we carried out the experiment using a solution of potassium permanganate and sulfuric acid as well as a solution of sodium carbonate and potassium permanganate, which served as the reagents for the auxiliary purification. The results of the test showed that after auxiliary purification by the acidic solution of potassium permanganate and the alkaline solution of potassium permanganate, the concentrations of carbon monoxide, hydrocarbons, nitrogen oxides and solid particles in the emissions were considerably lower than the concentrations prior to purification. It is possible to reduce the motor vehicle exhaust by the auxiliary purification of the solutions.

  17. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jin, Tengchuan; Chen, Yu-Wei; Howard, Andrew

    2008-07-01

    The crystallization of ginnacin, the 11S seed storage protein from G. biloba, is reported. Ginkgo biloba, a well known ‘living fossil’ native to China, is grown worldwide as an ornamental shade plant. Medicinal and nutritional uses of G. biloba in Asia have a long history. However, ginkgo seed proteins have not been well studied at the biochemical and molecular level. In this study, the G. biloba 11S seed storage protein ginnacin was purified by sequential anion-exchange and gel-filtration chromatography. A crystallization screen was performed and well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution hasmore » been obtained. There are six protomers in an asymmetric unit. Structure refinement is currently in progress.« less

  18. Solvent screening for a hard-to-dissolve molecular crystal.

    PubMed

    Maiti, A; Pagoria, P F; Gash, A E; Han, T Y; Orme, C A; Gee, R H; Fried, L E

    2008-09-01

    Materials with a high-degree of inter- and intra-molecular hydrogen bonding generally have limited solubility in conventional organic solvents. This presents a problem for the dissolution, manipulation and purification of these materials. Using a state-of-the-art density-functional-theory based quantum chemical solvation model we systematically evaluated solvents for a known hydrogen-bonded molecular crystal. This, coupled with direct solubility measurements, uncovered a class of ionic liquids involving fluoride anions that possess more than two orders of magnitude higher solvation power as compared with the best conventional solvents. The crystal structure of one such ionic liquid, determined by X-ray diffraction spectroscopy, indicates that F- ions are stabilized through H-bonded chains with water. The presence of coordinating water in such ionic liquids seems to facilitate the dissolution process by keeping the chemical activity of the F- ions in check.

  19. Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi

    2006-12-01

    UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the productmore » complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.« less

  20. High pressure treatment changes spoilage characteristics and shelf life of Pacific oysters ( Crassostrea gigas) during refrigerated storage

    NASA Astrophysics Data System (ADS)

    Cao, Rong; Zhao, Ling; Liu, Qi

    2017-04-01

    The effects of high pressure (HP) treatment on spoilage characteristic and shelf life extension of Pacific oysters ( Crassostrea gigas) during refrigerated storage were studied. Results showed that HP treatment of 275 MPa for 3 min or 300 MPa for 2 min could achieve 100% full release of oyster adductor muscle, pressures higher than 350 MPa caused excessive release as the shells of oysters were broken, thus use of higher pressures should be cautious in oyster processing industry because of its adverse impact on the appearance of shells. HP treatment (300 MPa, 2 min) was proper for the shucking of Pacific oyster ( Crassostrea gigas) in China. This treatment caused no organoleptic disadvantage. Moreover, HP treatment resulted in obvious differences in biochemical spoilage indicators (pH, TVB-N and TBARS) changes and volatile compounds profile determined by electronic nose during storage. HP treatment (300 MPa, 2 min) also led to a reduction of aerobic bacterial count (APC) by 1.27 log cycles. Furthermore, the APC values of oysters treated by HP were always lower than those of the control samples during storage. Based on the organoleptic, biochemical and microbiological indicators, shelf life of 6-8 d for control and 12 d for HP-treated oysters could be expected. HP treatment showed great potential in oyster processing and preservation.

  1. Effect of Amidated Low-Methoxyl Pectin on Physicochemical Characteristics of Jumbo Squid (Dosidicus gigas) Mantle Muscle Gels

    PubMed Central

    2017-01-01

    Summary Jumbo squid (Dosidicus gigas) muscle proteins show low functionality with limited use in gel products. This work aims to assess the influence of adding the natural and commercially available fibre, amidated low-methoxyl pectin (at 0.5, 1.0, 1.5, 2.0 and 3.0%), on the physicochemical and functional characteristics of jumbo squid (Dosidicus gigas) mantle muscle gels. The addition of 0.5% fibre showed an immediate effect on the gel texture profile analysis, improving hardness (p<0.05) from (3.4±0.7) N of the control (no added fibre) to (5.2±0.9) N, and increasing elasticity (p≥0.05). Shear force was significant only at 3.0% fibre addition. Water holding capacity also improved (p<0.05) with fibre addition (from 75% in the control to 90–95% after the treatments). Whiteness was affected (p<0.05) when 3.0% fibre was added. Differential scanning calorimetry showed two endothermic transition peaks in the gels. The second peak (actin) increased (p<0.05) by 1–2 °C with fibre addition. Therefore, the present study demonstrates that amidated low-methoxyl pectin (0.5–3.0%) is an excellent ingredient to improve jumbo squid mantle muscle protein functionality, increasing the gel texture and water retention characteristics. PMID:29089853

  2. Applications of thermal-gradients method for the optimization of α-amylase crystallization conditions based on dynamic and static light scattering data

    NASA Astrophysics Data System (ADS)

    Delboni, L. F.; Iulek, J.; Burger, R.; da Silva, A. C. R.; Moreno, A.

    2002-02-01

    The expression, purification, crystallization, and characterization by X-ray diffraction of α-amylase are described here. Dynamic and static light scattering methods with a temperature controller was used to optimize the crystallization conditions of α-amylase from Bacillus stearothermophilus an important enzyme in many fields of industrial activity. After applying thermal gradients for growing crystals, X-ray cryo-crystallographic methods were employed for the data collection. Crystals grown by these thermal-gradients diffracted up to a maximum resolution of 3.8 Å, which allowed the determination of the unit cell constants as follows: a=61.7 Å, b=86.7 Å, c=92.2 Å and space group C222 (or C222 1).

  3. Single-step affinity purification for fungal proteomics.

    PubMed

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  4. Separation and purification of γ-aminobutyric acid from fermentation broth by flocculation and chromatographic methodologies.

    PubMed

    Gao, Qiang; Duan, Qiang; Wang, Depei; Zhang, Yunze; Zheng, Chunyang

    2013-02-27

    To date, the multifunctional γ-aminobutyric acid (GABA) is mainly produced by microbial fermentation in industry. The purpose of this study was to find an effective method for separation and purification of 31.2 g/L initial GABA from the fermentation broth of Enterococcus raffinosus TCCC11660. To remove the impurities from fermentation broth, flocculation pretreatment using chitosan and sodium alginate was first implemented to facilitate subsequent filtration. Ultrafiltration followed two discontinuous diafiltration steps to effectively remove proteins and macromolecular pigments, and the resulting permeate was further decolored by DA201-CII resin at a high decoloration ratio and GABA recovery. Subsequently, ion exchange chromatography (IEC) with Amberlite 200C resin and gradient elution were applied for GABA separation from glutamate and arginine. Finally, GABA crystals of 99.1% purity were prepared via warm ethanol precipitation twice. Overall, our results reveal that the successive process including flocculation, filtration, ultrafiltration, decoloration, IEC, and crystallization is promising for scale-up GABA extraction from fermentation broth.

  5. Ice-shell purification of ice-binding proteins.

    PubMed

    Marshall, Craig J; Basu, Koli; Davies, Peter L

    2016-06-01

    Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Cloning, expression, purification and preliminary crystallographic analysis of the short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Harmer, Nicholas J., E-mail: nic@cryst.bioc.cam.ac.uk; King, Jerry D.; Department of Veterinary Medicine, Cambridge CB3 0ES

    2007-08-01

    The expression, purification, and crystallisation of the short-chain dehydrogenases WbmF, WbmG and WbmH from B. bronchiseptica are described. Native diffraction data to 1.5, 2.0, and 2.2 Å were obtained for the three proteins, together with complexes with nucleotides. The short-chain dehydrogenase enzymes WbmF, WbmG and WbmH from Bordetella bronchiseptica were cloned into Escherichia coli expression vectors, overexpressed and purified to homogeneity. Crystals of all three wild-type enzymes were obtained using vapour-diffusion crystallization with high-molecular-weight PEGs as a primary precipitant at alkaline pH. Some of the crystallization conditions permitted the soaking of crystals with cofactors and nucleotides or nucleotide sugars, whichmore » are possible substrate compounds, and further conditions provided co-complexes of two of the proteins with these compounds. The crystals diffracted to resolutions of between 1.50 and 2.40 Å at synchrotron X-ray sources. The synchrotron data obtained were sufficient to determine eight structures of the three enzymes in complex with a variety of cofactors and substrate molecules.« less

  7. Invasive range expansion by the Humboldt squid, Dosidicus gigas, in the eastern North Pacific

    PubMed Central

    Zeidberg, Louis D.; Robison, Bruce H.

    2007-01-01

    A unique 16-year time series of deep video surveys in Monterey Bay reveals that the Humboldt squid, Dosidicus gigas, has substantially expanded its perennial geographic range in the eastern North Pacific by invading the waters off central California. This sustained range expansion coincides with changes in climate-linked oceanographic conditions and a reduction in competing top predators. It is also coincident with a decline in the abundance of Pacific hake, the most important commercial groundfish species off western North America. Recognizing the interactive effects of multiple changes in the environment is an issue of growing concern in ocean conservation and sustainability research. PMID:17646649

  8. Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Delfosse, Vanessa; Hugonnet, Jean-Emmanuel; Sougakoff, Wladimir

    The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported. The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02more » Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry.« less

  9. Purification, crystallization and preliminary crystallographic analysis of Est25: a ketoprofen-specific hormone-sensitive lipase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, SeungBum; Joo, Sangbum; Yoon, Hyun C.

    2007-07-01

    Est25, a ketoprofen-specific hormone-sensitive lipase from a metagenomic library, was crystallized and diffraction data were collected to 1.49 Å resolution. Ketoprofen, a nonsteroidal anti-inflammatory drug, inhibits the synthesis of prostaglandin. A novel hydrolase (Est25) with high ketoprofen specificity has previously been identified using a metagenomic library from environmental samples. Recombinant Est25 protein with a histidine tag at the N-terminus was expressed in Escherichia coli and purified in a homogenous form. Est25 was crystallized from 2.4 M sodium malonate pH 7.0 and X-ray diffraction data were collected to 1.49 Å using synchrotron radiation. The crystals belong to the monoclinic space groupmore » C2, with unit-cell parameters a = 197.8, b = 95.2, c = 99.4 Å, β = 97.1°.« less

  10. Monitoring of antisolvent crystallization of sodium scutellarein by combined FBRM-PVM-NIR.

    PubMed

    Liu, Xuesong; Sun, Di; Wang, Feng; Wu, Yongjiang; Chen, Yong; Wang, Longhu

    2011-06-01

    Antisolvent crystallization can be used as an alternative to cooling or evaporation for the separation and purification of solid product in the pharmaceutical industry. To improve the process understanding of antisolvent crystallization, the use of in-line tools is vital. In this study, the process analytical technology (PAT) tools including focused beam reflectance measurement (FBRM), particle video microscope (PVM), and near-infrared spectroscopy (NIRS) were utilized to monitor antisolvent crystallization of sodium scutellarein. FBRM was used to monitor chord count and chord length distribution of sodium scutellarein particles in the crystallizer, and PVM, as an in-line video camera, provided pictures imaging particle shape and dimension. In addition, a quantitative model of PLS was established by in-line NIRS to detect the concentration of sodium scutellarein in the solvent and good calibration statistics were obtained (r(2) = 0.976) with the residual predictive deviation value of 11.3. The discussion over sensitivities, strengths, and weaknesses of the PAT tools may be helpful in selection of suitable PAT techniques. These in-line techniques eliminate the need for sample preparation and offer a time-saving approach to understand and monitor antisolvent crystallization process. Copyright © 2011 Wiley-Liss, Inc.

  11. Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nakamura, Tsutomu; Ishikawa, Kazuhiko; Hagihara, Yoshihisa

    The expression, purification and preliminary X-ray diffraction studies of a chitin-binding domain of the chitinase from P. furiosus are reported. The crystallization and preliminary X-ray diffraction analysis of the chitin-binding domain of chitinase from a hyperthermophilic archaeon, Pyrococcus furiosus, are reported. The recombinant protein was prepared using an Escherichia coli overexpression system and was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 1.70 Å resolution. The crystal belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2. The unit-cell parameters were determined to be a = b = 48.8, c = 85.0 Å.

  12. Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin from Homo sapiens.

    PubMed

    Aikawa, Yoshiki; Kida, Hiroshi; Nishitani, Yuichi; Miki, Kunio

    2015-09-01

    Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å.

  13. Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishitani, Yuichi; Maruyama, Daisuke; Nonaka, Tsuyoshi

    2006-04-01

    Preliminary X-ray diffraction studies on N-acetylglucosamine-phosphate mutase from C. albicans are reported. N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.

  14. Monogamy, polygamy, and other properties of entanglement of purification

    NASA Astrophysics Data System (ADS)

    Bagchi, Shrobona; Pati, Arun Kumar

    2015-04-01

    For bipartite pure and mixed quantum states, in addition to the quantum mutual information, there is another measure of total correlation, namely, the entanglement of purification. We study the monogamy, polygamy, and additivity properties of the entanglement of purification for pure and mixed states. In this paper, we show that, in contrast to the quantum mutual information which is strictly monogamous for any tripartite pure states, the entanglement of purification is polygamous for the same. This shows that there can be genuinely two types of total correlation across any bipartite cross in a pure tripartite state. Furthermore, we find the lower bound and actual values of the entanglement of purification for different classes of tripartite and higher-dimensional bipartite mixed states. Thereafter, we show that if entanglement of purification is not additive on tensor product states, it is actually subadditive. Using these results, we identify some states which are additive on tensor products for entanglement of purification. The implications of these findings on the quantum advantage of dense coding are briefly discussed, whereby we show that for tripartite pure states, it is strictly monogamous and if it is nonadditive, then it is superadditive on tensor product states.

  15. Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rathinaswamy, Priya; Pundle, Archana V.; Prabhune, Asmita A.

    An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented. Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2.more » Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å{sup 3} Da{sup −1}, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.« less

  16. Purification, crystallization and preliminary X-ray diffraction analysis of aspartate semialdehyde dehydrogenase (Rv3708c) from Mycobacterium tuberculosis

    PubMed Central

    Vyas, Rajan; Kumar, Vijay; Panjikar, Santosh; Karthikeyan, Subramanian; Kishan, K. V. Radha; Tewari, Rupinder; Weiss, Manfred S.

    2008-01-01

    Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B. PMID:18323599

  17. Cytochemical investigation of the digestive gland of two strombidae species (Strombus gigas and Strombus pugilis) in relation to the nutrition.

    PubMed

    Volland, Jean-Marie; Gros, Olivier

    2012-10-01

    Strombus gigas and Strombus pugilis are threatened species and aquaculture represents a good alternative solution to the fishing. In this study, we highlighted the intracellular digestion process in the digestive gland of two Strombidae species, S. gigas and Strombuspugilis, by the cytochemical characterization of two lysosomal enzymes: acid phosphatase and arylsulfatase. In order to check the efficiency of artificial food digestion, we conducted the characterization on freshly collected, starved and artificially fed individuals of S. pugilis. TEM observations of digestive gland sections from freshly collected individuals of both species revealed the presence of acid phosphatase and arylsulfatase activity mostly located in the apical third of digestive cells. Both enzymes were also detected in artificially fed individuals. In response to the starvation, acid phosphatase is not produced anymore by digestive cells, while arylsulfatase is still present. To our knowledge, this is the first cytochemical validation of intracellular digestion of artificial food in Strombidae. This study highlights the intracellular digestion of artificial food developed for Strombidae aquaculture. Moreover, we have shown that the lysosomal activity could be used as a feed index. Copyright © 2012 Wiley Periodicals, Inc.

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain

    PubMed Central

    Rodamilans, Bernardo; Montoya, Guillermo

    2007-01-01

    DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P21, with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 Å, α = γ = 90, β = 101.02°, and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). PMID:17401195

  19. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the DDX3 RNA helicase domain.

    PubMed

    Rodamilans, Bernardo; Montoya, Guillermo

    2007-04-01

    DDX3 is a human RNA helicase that is involved in RNA processing and important human diseases. This enzyme belongs to the DEAD-box protein family, the members of which are characterized by the presence of nine conserved motifs including the Asp-Glu-Ala-Asp motif that defines the family. DDX3 has two distinct domains: an ATP-binding domain in the central region of the protein and a helicase domain in the carboxy-terminal region. The helicase domain of DDX3 was cloned and overexpressed in Escherichia coli. Crystallization experiments yielded crystals that were suitable for X-ray diffraction analysis. The final crystallization conditions were a reservoir solution consisting of 2 M ammonium sulfate, 0.1 M imidazole pH 6.4 plus 5 mM spermine tetrahydrochloride and a protein solution containing 10 mM HEPES, 500 mM ammonium sulfate pH 8.0. The crystals of the helicase domain belong to the monoclinic space group P2(1), with unit-cell parameters a = 43.85, b = 60.72, c = 88.39 A, alpha = gamma = 90, beta = 101.02 degrees , and contained three molecules per asymmetric unit. These crystals diffracted to a resolution limit of 2.2 A using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).

  20. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brzezinski, Krzysztof; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan; Bujacz, Grzegorz

    2008-07-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified.more » Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.« less

  1. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa

    PubMed Central

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-01-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC. PMID:26625288

  2. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

  3. Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin from Homo sapiens

    PubMed Central

    Aikawa, Yoshiki; Kida, Hiroshi; Nishitani, Yuichi; Miki, Kunio

    2015-01-01

    Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å. PMID:26323306

  4. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman

    2007-02-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K{sub 2}) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetricmore » unit.« less

  5. Purification, crystallization and preliminary X-ray diffraction studies of D-tagatose 3-epimerase from Pseudomonas cichorii.

    PubMed

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-02-01

    D-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of D-psicose has not been reported with epimerases other than P. cichorii D-TE and D-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 A, beta = 102.82 degrees . Diffraction data were collected to 2.5 A resolution. The asymmetric unit is expected to contain four molecules.

  6. Necessity of purification during bacterial DNA extraction with environmental soils

    PubMed Central

    Choi, Jung-Hyun

    2017-01-01

    Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content. PMID:28793754

  7. Decursin from Angelica gigas mitigates amnesia induced by scopolamine in mice.

    PubMed

    Kang, So Young; Lee, Ki Yong; Park, Mi Jung; Kim, Young Chul; Markelonis, George J; Oh, Tae H; Kim, Young Choong

    2003-01-01

    We previously reported that a total methanolic extract of the underground part of Angelica gigas Nakai (Umbelliferae) (here-in-after abbreviated AG) significantly inhibited acetylcholinesterase (AChE) activity. We characterized 12 coumarin derivatives including both decursin and decursinol from extracts of AG. In this study, we evaluated the anti-amnestic activity of decursin, a major coumarin constituent isolated from AG, in vivo using ICR mice with amnesia induced by scopolamine (1 mg/kg body weight, s.c.). Decursin, when administered to mice at 1 and 5 mg/kg body weight i.p., significantly ameliorated scopolamine-induced amnesia as measured in both the passive avoidance test and the Morris water maze test. Moreover, decursin significantly inhibited AChE activity by 34% in the hippocampus of treated mice. These results indicate that decursin may exert anti-amnestic activity in vivo through inhibition of AChE activity in the hippocampus.

  8. Californium purification and electrodeposition

    DOE PAGES

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; ...

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO 3 and HCl, and Eichrom TEVA resin in NH 4SCN. The TEVA NH 4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

  9. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    PubMed

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

  10. Purification of Carbon Nanotubes: Alternative Methods

    NASA Technical Reports Server (NTRS)

    Files, Bradley; Scott, Carl; Gorelik, Olga; Nikolaev, Pasha; Hulse, Lou; Arepalli, Sivaram

    2000-01-01

    Traditional carbon nanotube purification process involves nitric acid refluxing and cross flow filtration using surfactant TritonX. This is believed to result in damage to nanotubes and surfactant residue on nanotube surface. Alternative purification procedures involving solvent extraction, thermal zone refining and nitric acid refiuxing are used in the current study. The effect of duration and type of solvent to dissolve impurities including fullerenes and P ACs (polyaromatic compounds) are monitored by nuclear magnetic reasonance, high performance liquid chromatography, and thermogravimetric analysis. Thermal zone refining yielded sample areas rich in nanotubes as seen by scanning electric microscopy. Refluxing in boiling nitric acid seem to improve the nanotube content. Different procedural steps are needed to purify samples produced by laser process compared to arc process. These alternative methods of nanotube purification will be presented along with results from supporting analytical techniques.

  11. Hyperentanglement purification using imperfect spatial entanglement.

    PubMed

    Wang, Tie-Jun; Mi, Si-Chen; Wang, Chuan

    2017-02-06

    As the interaction between the photons and the environment which will make the entangled photon pairs in less entangled states or even in mixed states, the security and the efficiency of quantum communication will decrease. We present an efficient hyperentanglement purification protocol that distills nonlocal high-fidelity hyper-entangled Bell states in both polarization and spatial-mode degrees of freedom from ensembles of two-photon system in mixed states using linear optics. Here, we consider the influence of the photon loss in the channel which generally is ignored in the conventional entanglement purification and hyperentanglement purification (HEP) schemes. Compared with previous HEP schemes, our HEP scheme decreases the requirement for nonlocal resources by employing high-dimensional mode-check measurement, and leads to a higher fidelity, especially in the range where the conventional HEP schemes become invalid but our scheme still can work.

  12. Controlled and in situ target strengths of the jumbo squid Dosidicus gigas and identification of potential acoustic scattering sources.

    PubMed

    Benoit-Bird, Kelly J; Gilly, William F; Au, Whitlow W L; Mate, Bruce

    2008-03-01

    This study presents the first target strength measurements of Dosidicus gigas, a large squid that is a key predator, a significant prey, and the target of an important fishery. Target strength of live, tethered squid was related to mantle length with values standardized to the length squared of -62.0, -67.4, -67.9, and -67.6 dB at 38, 70, 120, and 200 kHz, respectively. There were relatively small differences in target strength between dorsal and anterior aspects and none between live and freshly dead squid. Potential scattering mechanisms in squid have been long debated. Here, the reproductive organs had little effect on squid target strength. These data support the hypothesis that the pen may be an important source of squid acoustic scattering. The beak, eyes, and arms, probably via the sucker rings, also play a role in acoustic scattering though their effects were small and frequency specific. An unexpected source of scattering was the cranium of the squid which provided a target strength nearly as high as that of the entire squid though the mechanism remains unclear. Our in situ measurements of the target strength of free-swimming squid support the use of the values presented here in D. gigas assessment studies.

  13. Revision of the camel spider genus Eremocosta Roewer and a description of the female Eremocosta gigas Roewer (Arachnida, Solifugae).

    PubMed

    Cushing, Paula E; Channiago, Felix; Brookhart, Jack O

    2018-03-29

    A recent phylogenetic analysis demonstrated that the solifuge genus Eremocosta (Eremobatidae), although not monophyletic, formed a strongly supported group, rendered paraphyletic by the exclusion of E. acuitlapanensis, which we herein determine is misplaced in this genus. We revise the taxonomy of the genus Eremocosta. Nine species of the 13 currently placed in the genus are retained, E. bajaensis (Muma 1986), E. calexicensis (Muma 1951), E. formidabilus (Simon 1879), E. gigas Roewer 1934, E. gigasella (Muma 1970), E. spinipalpis (Kraepelin 1899), E. striata (Putnam 1883), and E. titania (Muma 1951). Eremocosta fusca (Muma 1986) and E. montezuma (Roewer 1934) are returned to the genus Eremorhax along with E. arenarum. Eremocosta hystrix and Eremocosta acuitlapanensis (Vázquez Gaviño-Rojas 2000) are transferred to Eremobates. We re-evaluated E. nigrimana (Pocock 1895) and determined that, since the type shows the ventrodistal concavity (VDC) diagnostic for the genus Eremocosta, it should be retained in that genus; however, because the type locality is identified as Afghanistan, far outside the range of any Eremobatidae, its status and placement remain uncertain. Eremocosta robusta (Roewer 1934) was designated nomen dubium by Muma and we maintain this designation. We provide a key to the species of Eremocosta and provide a description of the female of E. gigas.

  14. [Progress in isolation and purification of porcine islets].

    PubMed

    Zhu, Haitao; Yu, Liang; Wang, Bo

    2012-08-01

    To review the common methods of isolation and purification of porcine islets and research progress. Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.

  15. Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trincão, José; Sousa Silva, Marta; Barata, Lídia

    2006-08-01

    A glyoxalase II from L. infantum was cloned, purified and crystallized and its structure was solved by X-ray crystallography. In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222{sub 1} (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyondmore » 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypano-somatids.« less

  16. Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.

    PubMed

    Rouse, Sarah L; Hawthorne, Wlliam J; Lambert, Sebastian; Morgan, Marc L; Hare, Stephen A; Matthews, Stephen

    2016-12-01

    Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF 106-430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.

  17. Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raghunathan, Kannan; Vago, Frank S.; Ball, Terry

    2010-01-12

    EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 {angstrom}. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.1 {angstrom}{sup 3} Da{sup -1}, corresponding to 41.5% solvent content.

  18. Technological assumptions for biogas purification.

    PubMed

    Makareviciene, Violeta; Sendzikiene, Egle

    2015-01-01

    Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

  19. Cloning, expression, purification, crystallization and preliminary X-ray studies of argininosuccinate lyase (Rv1659) from Mycobacterium tuberculosis

    PubMed Central

    Paul, A.; Mishra, A.; Surolia, A.; Vijayan, M.

    2013-01-01

    The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P212121. Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule. PMID:24316845

  20. Genomic analysis of NF-κB signaling pathway reveals its complexity in Crassostrea gigas.

    PubMed

    Yu, Mingjia; Chen, Jianming; Bao, Yongbo; Li, Jun

    2018-01-01

    NF-κB signaling pathway is an evolutionarily conserved pathway that plays highly important roles in several developmental, cellular and immune response processes. With the recent release of the draft Pacific oyster (Crassostra gigas) genome sequence, we have sought to identify the various components of the NF-κB signaling pathway in these mollusks and investigate their gene structure. We further constructed phylogenetic trees to establish the evolutionary relationship of the oyster proteins with their homologues in vertebrates and invertebrates using BLASTX and neighbor-joining method. We report the presence of two classic NF-κB/Rel homologues in the pacific oyster namely Cgp100 and CgRel, which possess characteristic RHD domain and a consensus nuclear localization signal, similar to mammalian homologues and an additional CgRel-like protein, unique to C. gigas. Further, in addition to two classical IκB homologues, CgIκB1 and CgIκB2, we have identified three atypical IκB family members namely CgIκB3, CgIκB4 and CgBCL3 which lack the IκB degradation motif and consist of only one exon that might have arisen by retrotransposition from CgIκB1. Finally, we report the presence of three IKKs and one NEMO genes in oyster genome, named CgIKK1, CgIKK2, CgIKK3 and CgNEMO, respectively. While CgIKK1 and CgIKK3 domain structure is similar to their mammalian homologues, CgIKK2 was found to lack the HLH and NBD domains. Overall, the high conservation of the NF-κB/Rel, IκB and IKK family components in the pacific oyster and their structural similarity to the vertebrate and invertebrate homologues underline the functional importance of this pathway in regulation of critical cellular processes across species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Efficient purification of paclitaxel from yews using high-performance displacement chromatography technique.

    PubMed

    Watchueng, Jean; Kamnaing, Pierre; Gao, Jin-Ming; Kiyota, Taira; Yeboah, Faustinus; Konishi, Yasuo

    2011-05-20

    Paclitaxel was purified using high-performance displacement chromatography (HPDC) technique, but not by the mechanism of HPDC. On small scale, paclitaxel was extracted with methanol from dry needles of Taxus canadensis and was enriched by extracting with chloroform after removing water-soluble hydrophilic components and hexane-soluble hydrophobic components. Then, 93-99% purity of paclitaxel was obtained using the HPDC technique. On large scale, taxanes were enriched by solvent partitioning between acetic acid/MeOH/H(2)O and hexane and extracted with CH(2)Cl(2). Taxanes except paclitaxel were further removed by extracting with methanol-water-trifluoroacetic acid (1.0:98.9:0.1, v/v/v). Applying HPDC technique to water-insoluble substances is problematic as this method requires a highly aqueous solvent system. In order to overcome this incompatibility, a system was set up where paclitaxel, although in low concentration, was extracted by methanol-water-trifluoroacetic acid (10.0:89.9:0.1, v/v/v). Recycling the extracting solvent to ensure minimal volume, the extracted paclitaxel was adsorbed on a C(18) trap column. A C(18) column of 4.6mm internal diameter was then connected to the trap column. The HPDC technique was thus carried out using an isocratic acetonitrile-water-trifluoroacetic acid (30.0:69.9:0.1, v/v/v) mobile phase consisting of a displacer cetylpyridinium trifluoroacetate (3mg/mL). Paclitaxel was co-eluted with the displacer and spontaneously crystallized. The crystal (114mg) showed 99.4% purity and only 10% of paclitaxel in the starting crude extract was lost during the enrichment/purification processes. This large scale purification method was successfully applied to purify paclitaxel from Chinese yew in small scale, suggesting general applicability of the method. This is the first report of purifying a water-insoluble natural product using HPDC technique. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

  2. Development and validation of an ultra-performance convergence chromatography method for the quality control of Angelica gigas Nakai.

    PubMed

    Kim, Hyo Seon; Chun, Jin Mi; Kwon, Bo-In; Lee, A-Reum; Kim, Ho Kyoung; Lee, A Yeong

    2016-10-01

    Ultra-performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra-performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro-Phenyl column (2.1 mm × 150 mm, 1.7 μm) with a run time of 4 min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high-performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004 μg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012 μg/mL, respectively. The two components showed good regression (correlation coefficient (r 2 ) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75-102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Electrospun magnetically separable calcium ferrite nanofibers for photocatalytic water purification

    NASA Astrophysics Data System (ADS)

    EL-Rafei, A. M.; El-Kalliny, Amer S.; Gad-Allah, Tarek A.

    2017-04-01

    Three-dimensional random calcium ferrite, CaFe2O4, nanofibers (NFs) were successfully prepared via the electrospinning method. The effect of calcination temperature on the characteristics of the as-spun NFs was investigated. X-ray diffraction analysis showed that CaFe2O4 phase crystallized as a main phase at 700 °C and as a sole phase at 1000 °C. Field emission scanning electron microscopy emphasized that CaFe2O4 NFs were fabricated with diameters in the range of 50-150 nm and each fiber was composed of 20-50 nm grains. Magnetic hysteresis loops revealed superparamagnetic behavior for the prepared NFs. These NFs produced active hydroxyl radicals under simulated solar light irradiation making them recommendable for photocatalysis applications in water purification. In the meantime, these NFs can be easily separated from the treated water by applying an external magnetic field.

  4. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    PubMed Central

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-01-01

    d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-­psicose has not been reported with epimerases other than P. cichorii D-­TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules. PMID:17277456

  5. Large scale preparation and crystallization of neuron-specific enolase.

    PubMed

    Ishioka, N; Isobe, T; Kadoya, T; Okuyama, T; Nakajima, T

    1984-03-01

    A simple method has been developed for the large scale purification of neuron-specific enolase [EC 4.2.1.11]. The method consists of ammonium sulfate fractionation of brain extract, and two subsequent column chromatography steps on DEAE Sephadex A-50. The chromatography was performed on a short (25 cm height) and thick (8.5 cm inside diameter) column unit that was specially devised for the large scale preparation. The purified enolase was crystallized in 0.05 M imidazole-HCl buffer containing 1.6 M ammonium sulfate (pH 6.39), with a yield of 0.9 g/kg of bovine brain tissue.

  6. Impacts of ocean acidification on gene expression and biomineralisation in the Pacific oyster Crassostrea gigas Thunberg, 1793

    NASA Astrophysics Data System (ADS)

    Bagusche, F.; Pouvreau, S.; Trueman, C.; Long, S.; Hauton, C.

    2012-04-01

    The published evidence of impacts of ocean acidification and on marine calcifiers has emphasized the need to understand the molecular mechanisms of biomineralisation. Crassostrea gigas is an ideal organism to examine these processes as: 1) the hatchery rearing of larval stages is well constrained, 2) studies have established an ontogenetic switch in deposition of carbonate polymorphs from aragonite in larval shells to calcite in adults and 3) it is a globally-important commercial species. Research summarized in this presentation will identify some of the molecular mechanisms involved in calcification processes during ontogeny of Crassostrea gigas, as well as possible impacts of changes in environmental conditions such as temperature and pH. Data will be presented from a quantitative real-time PCR study of the changes in gene expression during development in different environments. Additionally scanning electron microscopy and infrared spectroscopy analyses of shell microstructures and composition will be summarised to correlate changes in gene expression with end-point differences in shell structure. Preliminary results suggest that changes in the environmental conditions lead to differences in expression patterns of genes involved in biomineralisation processes. The combined effects of ambient seawater temperature and low pH show the greatest negative effect on larval shell development, identified as malformations, eroded shell surfaces and a significant decrease in shell size. However, the effect of higher seawater temperature seems to amend the effects of ocean acidification on larval shell development.

  7. Chromogenic behaviors of the Humboldt squid (Dosidicus gigas) studied in situ with an animal-borne video package.

    PubMed

    Rosen, Hannah; Gilly, William; Bell, Lauren; Abernathy, Kyler; Marshall, Greg

    2015-01-15

    Dosidicus gigas (Humboldt or jumbo flying squid) is an economically and ecologically influential species, yet little is known about its natural behaviors because of difficulties in studying this active predator in its oceanic environment. By using an animal-borne video package, National Geographic's Crittercam, we were able to observe natural behaviors in free-swimming D. gigas in the Gulf of California with a focus on color-generating (chromogenic) behaviors. We documented two dynamic displays without artificial lighting at depths of up to 70 m. One dynamic pattern, termed 'flashing' is characterized by a global oscillation (2-4 Hz) of body color between white and red. Flashing was almost always observed when other squid were visible in the video frame, and this behavior presumably represents intraspecific signaling. Amplitude and frequency of flashing can be modulated, and the phase relationship with another squid can also be rapidly altered. Another dynamic display termed 'flickering' was observed whenever flashing was not occurring. This behavior is characterized by irregular wave-like activity in neighboring patches of chromatophores, and the resulting patterns mimic reflections of down-welled light in the water column, suggesting that this behavior may provide a dynamic type of camouflage. Rapid and global pauses in flickering, often before a flashing episode, indicate that flickering is under inhibitory neural control. Although flashing and flickering have not been described in other squid, functional similarities are evident with other species. © 2015. Published by The Company of Biologists Ltd.

  8. Structural studies on Desulfovibrio gigas cytochrome c3 by two-dimensional 1H-nuclear-magnetic-resonance spectroscopy.

    PubMed Central

    Piçarra-Pereira, M A; Turner, D L; LeGall, J; Xavier, A V

    1993-01-01

    Several aromatic amino acid residues and haem resonances in the fully reduced form of Desulfovibrio gigas cytochrome c3 are assigned, using two-dimensional 1H n.m.r., on the basis of the interactions between the protons of the aromatic amino acids and the haem protons as well as the intrahaem distances known from the X-ray structure [Kissinger (1989) Ph.D. Thesis, Washington State University]. The interhaem interactions observed in the n.m.r. spectra are in full agreement with the D. gigas X-ray structure and also with the n.m.r. data from Desulfovibrio vulgaris (Hildenborough) [Turner, Salgueiro, LeGall and Xavier (1992) Eur. J. Biochem. 210, 931-936]. The good correlation between the calculated ring-current shifts and the observed chemical shifts strongly supports the present assignments. Observation of the two-dimensional nuclear-Overhauser-enhancement spectra of the protein in the reduced, intermediate and fully oxidized stages led to the ordering of the haems in terms of their midpoint redox potentials and their identification in the X-ray structure. The first haem to oxidize is haem I, followed by haems II, III and IV, numbered according to the Cys ligand positions in the amino acid sequences [Mathews (1985) Prog. Biophys. Mol. Biol. 54, 1-56]. Although the haem core architecture is the same for the different Desulfovibrio cytochromes c3, the order of redox potentials is different. PMID:8397514

  9. Ostreid herpesvirus type 1 genomic diversity in wild populations of Pacific oyster Crassostrea gigas from Italian coasts.

    PubMed

    Burioli, E A V; Prearo, M; Riina, M V; Bona, M C; Fioravanti, M L; Arcangeli, G; Houssin, M

    2016-06-01

    Ostreid herpesvirus 1 (OsHV-1) is a significant pathogen affecting the young Pacific oyster Crassostrea gigas, worldwide. A new variant, OsHV-1 μVar, has been associated with recurrent mortality events in Europe since 2008. Epidemiological data collection is key for global risk assessment; however little is known about health status and genotypes present in European wild oyster beds. Most studies to date have involved only cultivated individuals during mortality events, and reported low genotype diversity. With this study, conducted along the Italian coasts, we investigated for the first time the presence of OsHV-1 in European natural oyster beds. Analysis of three genomic regions revealed the presence of at least nine different genotypes, including two variants close to the OsHV-1 reference, known since the early 1990s but with no European record reported since 2010, and highlights relevant genotype diversity in natural environment. Phylogenetic analysis distinguished two distinct clusters and geographical distribution of genotypes, with the exception of a variant very closely related to the μVar, which appeared the single genotype present in all the Adriatic sites. Interestingly, these wild symptom free populations could represent, in Europe, an accessible alternative to the import of OsHV-1-resistant oyster strains from the East Pacific, the native area of C. gigas, avoiding the high-risk of non-native marine species and new pathogen introductions. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Comprehensive HPTLC Fingerprinting for Quality Control of an Herbal Drug - The Case of Angelica gigas Root.

    PubMed

    Frommenwiler, Débora Arruda; Kim, Jonghwan; Yook, Chang-Soo; Tran, Thi Thu Trang; Cañigueral, Salvador; Reich, Eike

    2018-04-01

    The quality of herbal drugs is usually controlled using several tests recommended in a monograph. HPTLC is the method of choice for identification in many pharmacopoeias. If combined with a suitable reference material for comparison, HPTLC can provide information beyond identification and thus may simplify quality control. This paper describes, as a proof of concept, how HPTLC can be applied to define specifications for an herbal reference material and to control the quality of an herbal drug according to these specifications. Based on multiple batches of cultivated Angelica gigas root, a specific HPTLC method for identification was optimized. This method can distinguish 27 related species. It also can detect the presence of mixtures of A. gigas with two other Angelica species traded as "Dang gui" and is suitable as well for quantitative assessment of samples in a test for minimum content of the sum of decursin and decursinol angelate. The new concept of "comprehensive HPTLC fingerprinting" is proposed: HPTLC fingerprints (images), which are used for identification, are converted into peak profiles and the intensities of selected zones are quantitatively compared to those of the corresponding zones of the reference material. Following a collaborative trial involving three laboratories in three countries, the method was applied to check the quality of further candidates for establishing an appropriate reference material. In conclusion, this case demonstrates that a single HPTLC analysis can provide information about identity, purity, and minimum content of markers of an herbal drug. Georg Thieme Verlag KG Stuttgart · New York.

  11. Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom.

    PubMed

    Ullah, Anwar; Magalhães, Geraldo Santana; Masood, Rehana; Mariutti, Ricardo Barros; Coronado, Monika Aparecida; Murakami, Mário Tyago; Barbaro, Katia Cristina; Arni, Raghuvir Krishnaswamy

    2014-10-01

    Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Å and diffracted to a maximum resolution of 2.6 Å.

  12. Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom

    PubMed Central

    Ullah, Anwar; Magalhães, Geraldo Santana; Masood, Rehana; Mariutti, Ricardo Barros; Coronado, Monika Aparecida; Murakami, Mário Tyago; Barbaro, Katia Cristina; Arni, Raghuvir Krishnaswamy

    2014-01-01

    Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Å and diffracted to a maximum resolution of 2.6 Å. PMID:25286953

  13. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase

    PubMed Central

    dos Reis, Caio Vinicius; Bernardes, Amanda; Polikarpov, Igor

    2013-01-01

    Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7 Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63 Å. PMID:23695585

  14. GigaGauss solenoidal magnetic field inside bubbles excited in under-dense plasma

    PubMed Central

    Lécz, Zs.; Konoplev, I. V.; Seryi, A.; Andreev, A.

    2016-01-01

    This paper proposes a novel and effective method for generating GigaGauss level, solenoidal quasi-static magnetic fields in under-dense plasma using screw-shaped high intensity laser pulses. This method produces large solenoidal fields that move with the driving laser pulse and are collinear with the accelerated electrons. This is in contrast with already known techniques which rely on interactions with over-dense or solid targets and generates radial or toroidal magnetic field localized at the stationary target. The solenoidal field is quasi-stationary in the reference frame of the laser pulse and can be used for guiding electron beams. It can also provide synchrotron radiation beam emittance cooling for laser-plasma accelerated electron and positron beams, opening up novel opportunities for designs of the light sources, free electron lasers, and high energy colliders based on laser plasma acceleration. PMID:27796327

  15. GigaGauss solenoidal magnetic field inside bubbles excited in under-dense plasma

    NASA Astrophysics Data System (ADS)

    Lécz, Zs.; Konoplev, I. V.; Seryi, A.; Andreev, A.

    2016-10-01

    This paper proposes a novel and effective method for generating GigaGauss level, solenoidal quasi-static magnetic fields in under-dense plasma using screw-shaped high intensity laser pulses. This method produces large solenoidal fields that move with the driving laser pulse and are collinear with the accelerated electrons. This is in contrast with already known techniques which rely on interactions with over-dense or solid targets and generates radial or toroidal magnetic field localized at the stationary target. The solenoidal field is quasi-stationary in the reference frame of the laser pulse and can be used for guiding electron beams. It can also provide synchrotron radiation beam emittance cooling for laser-plasma accelerated electron and positron beams, opening up novel opportunities for designs of the light sources, free electron lasers, and high energy colliders based on laser plasma acceleration.

  16. Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    López-Jaramillo, F. J., E-mail: javier@lec.ugr.es; Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, E-18071; Pérez-Banderas, F.

    The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purificationmore » glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated.« less

  17. A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories.

    PubMed

    Hughes, Laura; Wilkins, Kimberly; Goldsmith, Cynthia S; Smith, Scott; Hudson, Paul; Patel, Nishi; Karem, Kevin; Damon, Inger; Li, Yu; Olson, Victoria A; Satheshkumar, P S

    2017-05-01

    Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron ® . Genetron ® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield. Published by Elsevier B.V.

  18. Rapid and inexpensive analysis of genetic variability in Arapaima gigas by PCR multiplex panel of eight microsatellites.

    PubMed

    Hamoy, I G; Santos, E J M; Santos, S E B

    2008-01-22

    The aim of the present study was the development of a multiplex genotyping panel of eight microsatellite markers of Arapaima gigas, previously described. Specific primer pairs were developed, each one of them marked with either FAM-6, HEX or NED. The amplification conditions using the new primers were standardized for a single reaction. The results obtained demonstrate high heterozygosity (average of 0.69) in a Lower Amazon population. The multiplex system described can thus be considered a fast, efficient and inexpensive method for the investigation of genetic variability in Arapaima populations.

  19. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

  20. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

  1. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

  2. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

  3. 21 CFR 884.6170 - Assisted reproduction water and water purification systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Assisted reproduction water and water purification... Devices § 884.6170 Assisted reproduction water and water purification systems. (a) Identification. Assisted reproduction water purification systems are devices specifically intended to generate high quality...

  4. Purification of boron nitride nanotubes via polymer wrapping

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Jin-Hyuk; Kim, Jaewoo; WCI Quantum Beam based Radiation Research Center, Korea Atomic Energy Research Institute, 1045 Daedukdaero, Daejeon 305-353

    2013-03-15

    Highlights: ► Surface modification of boron nitride nanotubes using polymeric materials. ► Surface-modified BNNT was purified with a simple dilution-centrifugation step. ► Surface-modified BNNT can be directly used for polymer composite fabrication ► Degree of purification was analyzed by Raman spectroscopy. - Abstract: Boron nitride nanotubes (BNNT) synthesized by a ball milling-annealing were surface-modified using three different types of polymeric materials. Those materials were chosen depending on future applications especially in polymer nanocomposite fabrications. We found that the surface-modified BNNT can be purified with a simple dilution-centrifugation step, which would be suitable for large-scale purification. Degree of purification was monitoredmore » by means of the center peak position and FWHM of E{sub 2g} mode of BNNT in Raman spectra. As the purification of BNNT develops, the peak position was up-shifted while FWHM of the peak was narrowed.« less

  5. Analysis of Giga-size Earth Observation Data in Open Source GRASS GIS 7 - from Desktop to On-line Solutions.

    NASA Astrophysics Data System (ADS)

    Stepinski, T. F.; Mitasova, H.; Jasiewicz, J.; Neteler, M.; Gebbert, S.

    2014-12-01

    GRASS GIS is a leading open source GIS for geospatial analysis and modeling. In addition to being utilized as a desktop GIS it also serves as a processing engine for high performance geospatial computing for applications in diverse disciplines. The newly released GRASS GIS 7 supports big data analysis including temporal framework, image segmentation, watershed analysis, synchronized 2D/3D animations and many others. This presentation will focus on new GRASS GIS 7-powered tools for geoprocessing giga-size earth observation (EO) data using spatial pattern analysis. Pattern-based analysis connects to human visual perception of space as well as makes geoprocessing of giga-size EO data possible in an efficient and robust manner. GeoPAT is a collection of GRASS GIS 7 modules that fully integrates procedures for pattern representation of EO data and patterns similarity calculations with standard GIS tasks of mapping, maps overlay, segmentation, classification(Fig 1a), change detections etc. GeoPAT works very well on a desktop but it also underpins several GeoWeb applications (http://sil.uc.edu/ ) which allow users to do analysis on selected EO datasets without the need to download them. The GRASS GIS 7 temporal framework and high resolution visualizations will be illustrated using time series of giga-size, lidar-based digital elevation models representing the dynamics of North Carolina barrier islands over the past 15 years. The temporal framework supports efficient raster and vector data series analysis and simplifies data input for visual analysis of dynamic landscapes (Fig. 1b) allowing users to rapidly identify vulnerable locations, changes in built environment and eroding coastlines. Numerous improvements in GRASS GIS 7 were implemented to support terabyte size data processing for reconstruction of MODIS land surface temperature (LST) at 250m resolution using multiple regressions and PCA (Fig. 1c) . The new MODIS LST series (http://gis.cri.fmach.it/eurolst/) includes

  6. Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

    PubMed Central

    Lezin, George; Kuehn, Michael R.; Brunelli, Luca

    2011-01-01

    Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

  7. 24 CFR 203.52 - Acceptance of individual residential water purification equipment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... residential water purification equipment. 203.52 Section 203.52 Housing and Urban Development Regulations... water purification equipment. If a property otherwise eligible for insurance under this part does not have access to a continuing supply of safe and potable water without the use of a water purification...

  8. 24 CFR 203.52 - Acceptance of individual residential water purification equipment.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... residential water purification equipment. 203.52 Section 203.52 Housing and Urban Development Regulations... water purification equipment. If a property otherwise eligible for insurance under this part does not have access to a continuing supply of safe and potable water without the use of a water purification...

  9. 24 CFR 203.52 - Acceptance of individual residential water purification equipment.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... residential water purification equipment. 203.52 Section 203.52 Housing and Urban Development Regulations... water purification equipment. If a property otherwise eligible for insurance under this part does not have access to a continuing supply of safe and potable water without the use of a water purification...

  10. 24 CFR 203.52 - Acceptance of individual residential water purification equipment.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... residential water purification equipment. 203.52 Section 203.52 Housing and Urban Development Regulations... water purification equipment. If a property otherwise eligible for insurance under this part does not have access to a continuing supply of safe and potable water without the use of a water purification...

  11. Genomics Study of the Exposure Effect of Gymnodinium catenatum, a Paralyzing Toxin Producer, on Crassostrea gigas' Defense System and Detoxification Genes

    PubMed Central

    García-Lagunas, Norma; Romero-Geraldo, Reyna; Hernández-Saavedra, Norma Y.

    2013-01-01

    Background Crassostrea gigas accumulates paralytic shellfish toxins (PST) associated with red tide species as Gymnodinium catenatum. Previous studies demonstrated bivalves show variable feeding responses to toxic algae at physiological level; recently, only one study has reported biochemical changes in the transcript level of the genes involved in C. gigas stress response. Principal Findings We found that 24 h feeding on toxic dinoflagellate cells (acute exposure) induced a significant decrease in clearance rate and expression level changes of the genes involved in antioxidant defense (copper/zinc superoxide dismutase, Cu/Zn-SOD), cell detoxification (glutathione S-transferase, GST and cytochrome P450, CPY450), intermediate immune response activation (lipopolysaccharide and beta glucan binding protein, LGBP), and stress responses (glutamine synthetase, GS) in Pacific oysters compared to the effects with the non-toxic microalga Isochrysis galbana. A sub-chronic exposure feeding on toxic dinoflagellate cells for seven and fourteen days (30×103 cells mL−1) showed higher gene expression levels. A significant increase was observed in Cu/Zn-SOD, GST, and LGBP at day 7 and a major increase in GS and CPY450 at day 14. We also observed that oysters fed only with G. catenatum (3×103 cells mL−1) produced a significant increase on the transcription level than in a mixed diet (3×103 cells mL−1 of G. catenatum+0.75×106 cells mL−1 I. galbana) in all the analyzed genes. Conclusions Our results provide gene expression data of PST producer dinoflagellate G. catenatum toxic effects on C. gigas, a commercially important bivalve. Over expressed genes indicate the activation of a potent protective mechanism, whose response depends on both cell concentration and exposure time against these toxic microalgae. Given the importance of dinoflagellate blooms in coastal environments, these results provide a more comprehensive overview of how oysters respond to stress generated by

  12. Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

    PubMed

    Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

    2006-09-01

    An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

  13. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  14. Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman–Birk inhibitor from Oryza sativa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, Yi-Hung; Li, Hsin-Tai; Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan

    2006-06-01

    Rice Bowman–Birk inhibitor was expressed and crystallized. Bowman–Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 Å, the unit cell belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 74.37, b = 96.69, cmore » = 100.36 Å. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.« less

  15. Crystallization and preliminary X-ray diffraction analysis of FabG from Yersinia pestis.

    PubMed

    Nanson, Jeffrey David; Forwood, Jade Kenneth

    2014-01-01

    The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported. Recombinant FabG was expressed, purified to homogeneity and crystallized via the hanging-drop vapour-diffusion technique. Diffraction data were collected at the Australian Synchrotron to 2.30 Å resolution. The crystal displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 68.22, b = 98.68, c = 169.84 Å, and four ypFabG molecules in the asymmetric unit.

  16. L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.

    PubMed

    Krintel, Christian; Frydenvang, Karla; Ceravalls de Rabassa, Anna; Kaern, Anne M; Gajhede, Michael; Pickering, Darryl S; Kastrup, Jette S

    2014-05-01

    In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp). © 2014 FEBS.

  17. Temporal and maternal effects on reproductive ecology of the giant gartersnake (Thamnophis gigas)

    USGS Publications Warehouse

    Halstead, Brian J.; Wylie, Glenn D.; Casazza, Michael L.; Coates, Peter S.

    2009-01-01

    We used mixed-effects models to examine relationships of reproductive characteristics of the giant gartersnake (Thamnophis gigas) to improve population modeling and conservation planning for this species. Neonates from larger litters had lower mass, and mass of neonates also was affected by random variation among mothers. Length of mother did not affect relative mass of litters; however, our data suggest that longer mothers expended less reproductive effort per offspring than shorter mothers. We detected random variation in length of neonates among mothers, but these lengths were not related to length of mother or size of litter. Mean size of litter varied among years, but little evidence existed for a relationship between size of litter or mass of litter and length of mother. Sex ratios of neonates did not differ from 1:1.

  18. Automated multi-dimensional purification of tagged proteins.

    PubMed

    Sigrell, Jill A; Eklund, Pär; Galin, Markus; Hedkvist, Lotta; Liljedahl, Pia; Johansson, Christine Markeland; Pless, Thomas; Torstenson, Karin

    2003-01-01

    The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

  19. Crystal-field effects in fluoride crystals for optical refrigeration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hehlen, Markus P

    2010-01-01

    The field of optical refrigeration of rare-earth-doped solids has recently seen an important breakthrough. The cooling of a YLiF{sub 4} (YLF) crystal doped with 5 mol% Yb3+ to 155 K by Seletskiy et al [NPhot] has surpassed the lowest temperatures ({approx}170 K for {approx}100 mW cooling capacity) that are practical with commercial multi-stage thermoelectric coolers (TEC) [Glaister]. This record performance has advanced laser cooling into an application relevant regime and has put first practical optical cryocoolers within reach. The result is also relevant from a material perspective since for the first time, an Yb3+-doped crystal has outperformed an Yb3+-doped glass.more » The record temperature of 208 K was held by the Yb3+-doped fluorozirconate glass ZBLAN. Advanced purification and glass fabrication methods currently under development are expected to also advance ZBLAN:Yb3+ to sub-TEC temperatures. However, recent achievements with YLF:Yb3+ illustrate that crystalline materials may have two potentially game-changing advantajes over glassy materials. First, the crystalline environment reduces the inhomogeneous broadening of the Yb3+ electronic transitions as compared to a glassy matrix. The respective sharpening of the crystal-field transitions increases the peak absorption cross section at the laser excitation wavelength and allows for more efficient pumping of the Yb3+ ions, particularly at low temperatures. Second, many detrimental impurities present in the starting materials tend to be excluded from the crystal during its slow growth process, in contrast to a glass where all impurities present in the starting materials are included in the glass when it is formed by temperature quenching a melt. The ultra high purity required for laser cooling materials [PRB] therefore may be easier to realize in crystals than in glasses. Laser cooling occurs by laser excitation of a rare-earth ion followed by anti-Stokes luminescence. Each such laser-cooling cycle

  20. Isolation, purification, crystallization and preliminary X-ray studies of two 30 kDa proteins from silkworm haemolymph.

    PubMed

    Pietrzyk, Agnieszka J; Bujacz, Anna; Łochyńska, Małgorzata; Jaskólski, Mariusz; Bujacz, Grzegorz

    2011-03-01

    Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.

  1. Zooming to the centre of the Milky Way - GigaGalaxy Zoom phase 2

    NASA Astrophysics Data System (ADS)

    2009-09-01

    The second of three images of ESO's GigaGalaxy Zoom project has just been released online. It is a new and wonderful 340-million-pixel vista of the central parts of our home galaxy as seen from ESO's Paranal Observatory with an amateur telescope. This 34 by 20-degree wide image provides us with a view as experienced by amateur astronomers around the world. However, its incredible beauty and appeal owe much to the quality of the observing site and the skills of Stéphane Guisard, the world-renowned astrophotographer, who is also an ESO engineer. This second image directly benefits from the quality of Paranal's sky, one of the best on the planet, where ESO's Very Large Telescope is located. In addition, Guisard has drawn on his professional expertise as an optical engineer specialising in telescopes, a rare combination in the world of astrophotographers. Guisard, as head of the optical engineering team at Paranal, is responsible for ensuring that the Very Large Telescope has the best optical performance possible. To create this stunning, true-colour mosaic of the Galactic Centre region, Guisard assembled about 1200 individual images, totalling more than 200 hours of exposure time, collected over 29 nights, during Guisard's free time, while working during the day at Paranal [1]. The image shows the region spanning the sky from the constellation of Sagittarius (the Archer) to Scorpius (the Scorpion). The very colourful Rho Ophiuchi and Antares region is a prominent feature to the right, although much darker areas, such as the Pipe and Snake nebulae also stand out. The dusty lane of our Milky Way runs obliquely through the image, dotted with remarkable bright, reddish nebulae, such as the Lagoon and the Trifid Nebulae, as well as NGC 6357 and NGC 6334. This dark lane also hosts the very centre of our Galaxy, where a supermassive black hole is lurking. "The area I have depicted in this image is an incredibly rich region of the sky, and the one I find most beautiful

  2. Rapid expansion of the invasive oyster Crassostrea gigas at its northern distribution limit in Europe: Naturally dispersed or introduced?

    PubMed Central

    Anglès d’Auriac, Marc B.; Norling, Pia; Lapègue, Sylvie; Staalstrøm, André; Hjermann, Dag Ø.; Thaulow, Jens

    2017-01-01

    The Pacific oyster, Crassostrea gigas, was introduced to Europe for aquaculture purposes, and has had a rapid and unforeseen northward expansion in northern Europe. The recent dramatic increase in number of C. gigas populations along the species’ northern distribution limit has questioned the efficiency of Skagerrak as a dispersal barrier for transport and survival of larvae. We investigated the genetic connectivity and possible spreading patterns between Pacific oyster populations on the southern Norwegian coast (4 localities) and Swedish and Danish populations by means of DNA microsatellite analysis of adult oysters, and by simulating larvae drift. In the simulations we used a 3D oceanographic model to explore the influence of recent climate change (1990–2010) on development, survival, and successful spreading of Danish and Swedish Pacific oyster larvae to Norwegian coastal waters. The simulations indicated adequate temperature conditions for development, survival, and settlement of larvae across the Skagerrak in warm years since 2000. However, microsatellite genotyping revealed genetic differences between the Norwegian populations, and between the Norwegian populations and the Swedish and Danish populations, the latter two populations being more similar. This patchwork pattern of genetic dissimilarity among the Norwegian populations points towards multiple local introduction routes rather than the commonly assumed unidirectional entry of larvae drifted from Denmark and Sweden. Alternative origins of introduction and implications for management, such as forecasting and possible mitigation actions, are discussed. PMID:28486520

  3. Improving the large scale purification of the HIV microbicide, griffithsin.

    PubMed

    Fuqua, Joshua L; Wanga, Valentine; Palmer, Kenneth E

    2015-02-22

    Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of

  4. Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin.

    PubMed

    Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

    2012-04-01

    β(2)-Microglobulin (β(2)m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide-MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β(2)m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β(2)m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3(2)21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β(2)m and IgSF in the AIS. © 2012 International Union of Crystallography. All rights reserved.

  5. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

    PubMed

    Ma, Zheng; Fung, Victor; D'Orso, Iván

    2017-01-26

    The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

  6. Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

    2006-08-01

    Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

  7. One-step purification of a functional, constitutively activated form of visual arrestin.

    PubMed

    Huang, Li; Mao, Xiang; Abdulaev, Najmoutin G; Ngo, Tony; Liu, Wei; Ridge, Kevin D

    2012-03-01

    Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗). The results show that both prodomain/Profinity eXact™ fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale™ Mini Profinity eXact™ cartridge on the Profinia™ purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho∗. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. An effective purification method using large bottles for human pancreatic islet isolation

    PubMed Central

    Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A.; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F.; Grayburn, Paul A.; Matsumoto, Shinichi

    2012-01-01

    The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification. PMID:23221740

  9. [Determination of proximal chemical composition of squid (dosidicus gigas) and development of gel products].

    PubMed

    Abugoch, L; Guarda, A; Pérez, L M; Paredes, M P

    1999-06-01

    The good nutritional properties of meat from big squid (Dosidicus gigas) living on the Chilean coast, was determined through its proximal composition 70 cal/100 g fresh meat; 82.23 +/- 0.98% moisture; 15.32 +/- 0.93% protein; 1.31 +/- 0.12% ashes; 0.87 +/- 0.18% fat and 0.27% NNE (non-nitrogen extract). The big squid meat was used to develop a gel product which contained NaCl and TPP. It was necessary to use additives for gel preparation, such as carragenin or alginate or egg albumin, due to the lack of gelation properties of squid meat. Formulations containing egg albumin showed the highest gel force measured by penetration as compared to those that contained carragenin or alginate.

  10. D1 dopamine receptor is involved in shell formation in larvae of Pacific oyster Crassostrea gigas.

    PubMed

    Liu, Zhaoqun; Wang, Lingling; Yan, Yunchen; Zheng, Yan; Ge, Wenjing; Li, Meijia; Wang, Weilin; Song, Xiaorui; Song, Linsheng

    2018-07-01

    Dopamine (DA), a significant member of catecholamines, is reported to induce biomineralization of calcium carbonate vaterite microspheres via dopamine receptor (DR) in bivalves, implying the modulation of dopaminergic system on shell formation during larval development. In this research, a homologue of D1 type DR (CgD1DR-1) was identified from oyster Crassostrea gigas, whose full length cDNA was 1197 bp. It was widely expressed in various tissues of C. gigas, with the significantly higher levels in hepatopancreas, mantle, muscle and gill. During developmental stages, the mRNA transcripts of CgD1DR-1 in D-shape larvae were obviously higher (p < 0.05) than those in trochophore and umbo larvae, and CO 2 exposure could inhibit the synthesis of DA and mRNA expression of CgD1DR-1. After cell transfection and DA treatment, intracellular cAMP in cells with the expression of CgD1DR-1 increased significantly (p < 0.05). Furthermore, the incubation with SCH 23390 for the blockage of CgD1DR-1 significantly restrained the expressions of six shell formation-related genes including CgTyrosinase-1, CgTyrosinase-3, CgChitinaseLP, CgAMC, CgBMP and CgBMPR in trochophore and D-shape larvae. These results jointly suggested that DA together with its receptor CgD1DR-1 might be involved in shell formation during oyster larval development from trochophore to D-shape larvae, and CO 2 -induced ocean acidification (OA) might influence marine bivalves by inhibiting the DA-D1DR pathway to prohibit their shell formation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1995-01-01

    During the fourth semi-annual period under this grant we have pursued the following activities: (1) crystal growth morphology and kinetics studies with tetragonal lysozyme. These clearly revealed the influence of higher molecular weight protein impurities on interface shape; (2) characterization of the purity and further purification of lysozyme solutions. These efforts have, for the first time, resulted in lysozyme free of higher molecular weight components; (3) continuation of the salt repartitioning studies with Seikagaku lysozyme, which has a lower protein impurity content that Sigma stock. These efforts confirmed our earlier findings of higher salt contents in smaller crystals. However, less salt is in corporated into the crystals grown from Seikagaku stock. This strongly suggests a dependence of salt repartitioning on the concentration of protein impurities in lysozyme. To test this hypothesis, repartitioning studies with the high purity lysozyme prepared in-house will be begun shortly; (4) numerical modelling of the interaction between bulk transport and interface kinetics. These simulations have produced interface shapes which are in good agreement with out experimental observations; and (5) light scattering studies on under- and supersaturated lysozyme solutions. A consistent interpretation of the static and dynamic data leaves little doubt that pre-nucleation clusters, claimed to exist even in undersaturated solutions, are not present. The article: 'Growth morphology response to nutrient and impurity nonuniformities' is attached.

  12. Morphometric partitioning of the respiratory surface area and diffusion capacity of the gills and swim bladder in juvenile Amazonian air-breathing fish, Arapaima gigas.

    PubMed

    Fernandes, Marisa Narciso; da Cruz, André Luis; da Costa, Oscar Tadeu Ferreira; Perry, Steven Franklin

    2012-09-01

    The gills and the respiratory swim bladders of juvenile specimens (mean body mass 100g) of the basal teleost Arapaima gigas (Cuvier 1829) were evaluated using stereological methods in vertical sections. The surface areas, harmonic mean barrier thicknesses and morphometric diffusing capacities for oxygen and carbon dioxide were estimated. The average respiratory surface area of the swim bladder (2173 cm² kg⁻¹) exceeded that of the gills (780 cm² kg⁻¹) by a factor of 2.79. Due to the extremely thin air-blood barrier in the swim bladder (harmonic mean 0.22 μm) and the much thicker water-blood barrier of the gills (9.61 μm), the morphometric diffusing capacity for oxygen and carbon dioxide was 88 times greater in the swim bladder than in the gills. These data clearly indicate the importance of the swim bladder, even in juvenile A. gigas that still engage in aquatic respiration. Because of the much greater diffusion constant of CO₂ than O₂ in water, the gills also remain important for CO₂ release. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Cesium hafnium chloride: A high light yield, non-hygroscopic cubic crystal scintillator for gamma spectroscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burger, Arnold, E-mail: aburger@fisk.edu; Department of Physics and Astronomy, Vanderbilt University, Nashville, Tennessee 37235; Rowe, Emmanuel

    We report on the scintillation properties of Cs{sub 2}HfCl{sub 6} (cesium hafnium chloride or CHC) as an example of a little-known class of non-hygroscopic compounds having the generic cubic crystal structure of K{sub 2}PtCl{sub 6}. The crystals are easily growable from the melt using the Bridgman method with minimal precursor treatments or purification. CHC scintillation is centered at 400 nm, with a principal decay time of 4.37 μs and a light yield of up to 54 000 photons/MeV when measured using a silicon CCD photodetector. The light yield is the highest ever reported for an undoped crystal, and CHC also exhibits excellent lightmore » yield nonproportionality. These desirable properties allowed us to build and test CHC gamma-ray spectrometers providing energy resolution of 3.3% at 662 keV.« less

  14. Purification of Logic-Qubit Entanglement

    PubMed Central

    Zhou, Lan; Sheng, Yu-Bo

    2016-01-01

    Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network. PMID:27377165

  15. Purification of Logic-Qubit Entanglement.

    PubMed

    Zhou, Lan; Sheng, Yu-Bo

    2016-07-05

    Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network.

  16. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  17. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of MCAT from Staphylococcus aureus.

    PubMed

    Hong, Seung Kon; Kim, Kook Han; Kim, Eunice EunKyeong

    2010-01-01

    Malonyl-CoA:acyl-carrier protein transacylase (MCAT), encoded by the fabd gene, is a key enzyme in type II fatty-acid biosynthesis. It is responsible for transferring the malonyl group from malonyl-CoA to the holo acyl-carrier protein (ACP). Since the type II system differs from the type I system that mammals use, it has received enormous attention as a possible antibiotic target. In particular, only a single isoform of MCAT has been reported and a continuous coupled enzyme assay has been developed. MCAT from Staphylococcus aureus was overexpressed in Escherichia coli and the protein was purified and crystallized. Diffraction data were collected to 1.2 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 41.608, b = 86.717, c = 43.163 A, alpha = gamma = 90, beta = 106.330 degrees . The asymmetric unit contains one SaMCAT molecule.

  18. Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

    PubMed

    Liu, Keyang; Wen, Zhanbo; Li, Na; Yang, Wenhui; Hu, Lingfei; Wang, Jie; Yin, Zhe; Dong, Xiaokai; Li, Jinsong

    2012-12-01

    Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Virus purification by CsCl density gradient using general centrifugation.

    PubMed

    Nasukawa, Tadahiro; Uchiyama, Jumpei; Taharaguchi, Satoshi; Ota, Sumire; Ujihara, Takako; Matsuzaki, Shigenobu; Murakami, Hironobu; Mizukami, Keijirou; Sakaguchi, Masahiro

    2017-11-01

    Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

  20. Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes.

    PubMed

    Linke, Christian; Siemens, Nikolai; Middleditch, Martin J; Kreikemeyer, Bernd; Baker, Edward N

    2012-07-01

    The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205 kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P2(1) and P2(1)2(1)2(1) in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52-357 of Epf. Complete data sets were collected to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron.

  1. Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes

    PubMed Central

    Linke, Christian; Siemens, Nikolai; Middleditch, Martin J.; Kreikemeyer, Bernd; Baker, Edward N.

    2012-01-01

    The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205 kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P21 and P212121 in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52–357 of Epf. Complete data sets were collected to 2.0 and 1.6 Å resolution, respectively, at the Australian Synchrotron. PMID:22750867

  2. Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas

    PubMed Central

    Boullot, Floriane; Castrec, Justine; Bidault, Adeline; Dantas, Natanael; Payton, Laura; Perrigault, Mickael; Tran, Damien; Amzil, Zouher; Boudry, Pierre; Soudant, Philippe; Hégaret, Hélène; Fabioux, Caroline

    2017-01-01

    Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters. PMID:28106838

  3. A preliminary analysis of trace-elemental signatures in statoliths of different spawning cohorts for Dosidicus gigas off EEZ waters of Chile

    NASA Astrophysics Data System (ADS)

    Liu, Bilin; Chen, Xinjun; Fang, Zhou; Hu, Song; Song, Qian

    2015-12-01

    We applied solution-based ICP-MS method to quantify the trace-elemental signatures in statoliths of jumbo flying squid, Dosidius gigas, which were collected from the waters off northern and central Chile during the scientific surveys carried out by Chinese squid jigging vessels in 2007 and 2008. The age and spawning date of the squid were back-calculated based on daily increments in statoliths. Eight elemental ratios (Sr/Ca, Ba/Ca, Mg/Ca, Mn/Ca, Na/Ca, Fe/Ca, Cu/Ca and Zn/Ca) were analyzed. It was found that Sr is the second most abundant element next to Ca, followed by Na, Fe, Mg, Zn, Cu, Ba and Mn. There was no significant relationship between element/Ca and sea surface temperature (SST) and sea surface salinity (SSS), although weak negative or positive tendency was found. MANOVA analysis showed that multivariate elemental signatures did not differ among the cohorts spawned in spring, autumn and winter, and no significant difference was found between the northern and central sampling locations. Classification results showed that all individuals of each spawned cohorts were correctly classified. This study demonstrates that the elemental signatures in D. gigas statoliths are potentially a useful tool to improve our understanding of its population structure and habitat environment.

  4. Influence of indigenous minor components on fat crystal network of fully hydrogenated palm kernel oil and fully hydrogenated coconut oil.

    PubMed

    Chai, Xiu-Hang; Meng, Zong; Cao, Pei-Rang; Liang, Xin-Yu; Piatko, Michael; Campbell, Shawn; Koon Lo, Seong; Liu, Yuan-Fa

    2018-07-30

    Purification of triglycerides from fully hydrogenated palm kernel oil (FHPKO) and fully hydrogenated coconut oil (FHCNO) was performed by a chromatographic method. Lipid composition, thermal properties, polymorphism, isothermal crystallization behaviour, nanostructure and microstructure of FHPKO, FHPKO-triacylglycerol (TAG), FHCNO and FHCNO-TAG were evaluated. Removal of minor components had no effect on triglycerides composition. However, the presence of the minor components did increase the slip melting point and promote onset of crystallization. Furthermore, the thickness of the nanoscale crystals increased, and polymorphic transformation from β' to β occurred in FHPKO after the removal of minor components, and from α to β' in FHCNO. Sharp changes in the values of the Avrami constant K and exponent n suggested that the presence of minor components changed the crystal growth mechanism. The PLM results indicated that a coarser crystal structure with lower fractal dimension appeared after the removal of minor components from both FHPKO and FHCNO. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia).

    PubMed

    Sathya Moorthy, Pon; Neelagandan, K; Balasubramanian, M; Ponnuswamy, M N

    2009-02-01

    Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 A resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 A, alpha = 78.742, beta = 89.819, gamma = 65.320 degrees .

  6. Expression, purification, crystallization and initial crystallographic characterization of the p-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kwon, Soo-Young; Kang, Beom Sik; Kim, Ghyung-Hwa

    2007-11-01

    PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction and plays an important role in the biodegradation of aromatic compounds. PHBH from Corynebacterium glutamicum was crystallized using the hanging-drop vapour-diffusion method in the presence of NaH{sub 2}PO{sub 4} and K{sub 2}HPO{sub 4} as precipitants. X-ray diffraction data were collectedmore » to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 94.72, c = 359.68 Å, γ = 120°. The asymmetric unit contains two molecules, corresponding to a packing density of 2.65 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.« less

  7. Waste water biological purification plants of dairy products industry and energy management

    NASA Astrophysics Data System (ADS)

    Stepanov, Sergey; Solkina, Olga; Stepanov, Alexander; Zhukova, Maria

    2017-10-01

    The paper presents results of engineering and economical comparison of waste water biological purification plants of dairy products industry. Three methods of purification are compared: traditional biological purification with the use of secondary clarifiers and afterpurification through granular-bed filters, biomembrane technology and physical-and-chemical treatment together with biomembrane technology for new construction conditions. The improvement of the biological purification technology using nitro-denitrification and membrane un-mixing of sludge mixture is a promising trend in this area. In these calculations, an energy management which is widely applied abroad was used. The descriptions of the three methods are illustrated with structural schemes. Costs of equipment and production areas are taken from manufacturers’ data. The research is aimed at an engineering and economical comparison of new constructions of waste water purification of dairy products industry. The experiment demonstrates advantages of biomembrane technology in waste water purification. This technology offers prospects of 122 million rubles cost saving during 25 years of operation when compared with of the technology of preparatory reagent flotation and of 13.7 million rubles cost saving compared to the option of traditional biological purification.

  8. Inert gas enhanced laser-assisted purification of platinum electron-beam-induced deposits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon

    Electron-beam-induced deposition patterns, with composition of PtC 5, were purified using a pulsed laser-induced purification reaction to erode the amorphous carbon matrix and form pure platinum deposits. Enhanced mobility of residual H 2O molecules via a localized injection of inert Ar–H 2 (4%) is attributed to be the reactive gas species for purification of the deposits. Surface purification of deposits was realized at laser exposure times as low as 0.1 s. The ex situ purification reaction in the deposit interior was shown to be rate-limited by reactive gas diffusion into the deposit, and deposit contraction associated with the purification processmore » caused some loss of shape retention. To circumvent the intrinsic flaws of the ex situ anneal process, in situ deposition and purification techniques were explored that resemble a direct write atomic layer deposition (ALD) process. First, we explored a laser-assisted electron-beam-induced deposition (LAEBID) process augmented with reactive gas that resulted in a 75% carbon reduction compared to standard EBID. Lastly, a sequential deposition plus purification process was also developed and resulted in deposition of pure platinum deposits with high fidelity and shape retention.« less

  9. Inert gas enhanced laser-assisted purification of platinum electron-beam-induced deposits

    DOE PAGES

    Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon; ...

    2015-06-30

    Electron-beam-induced deposition patterns, with composition of PtC 5, were purified using a pulsed laser-induced purification reaction to erode the amorphous carbon matrix and form pure platinum deposits. Enhanced mobility of residual H 2O molecules via a localized injection of inert Ar–H 2 (4%) is attributed to be the reactive gas species for purification of the deposits. Surface purification of deposits was realized at laser exposure times as low as 0.1 s. The ex situ purification reaction in the deposit interior was shown to be rate-limited by reactive gas diffusion into the deposit, and deposit contraction associated with the purification processmore » caused some loss of shape retention. To circumvent the intrinsic flaws of the ex situ anneal process, in situ deposition and purification techniques were explored that resemble a direct write atomic layer deposition (ALD) process. First, we explored a laser-assisted electron-beam-induced deposition (LAEBID) process augmented with reactive gas that resulted in a 75% carbon reduction compared to standard EBID. Lastly, a sequential deposition plus purification process was also developed and resulted in deposition of pure platinum deposits with high fidelity and shape retention.« less

  10. Purification of organic acids by chromatography with strong anionic resins: Investigation of uptake mechanisms.

    PubMed

    Lemaire, Julien; Blanc, Claire-Line; Lutin, Florence; Théoleyre, Marc-André; Stambouli, Moncef; Pareau, Dominique

    2016-08-05

    Bio-based organic acids are promising renewable carbon sources for the chemical industry. However energy-consuming purification processes are used, like distillation or crystallization, to reach high purities required in some applications. That is why preparative chromatography was studied as an alternative separation technique. In a previous work dealing with the purification of lactic, succinic and citric acids, the Langmuir model was insufficient to explain the elution profiles obtained with a strong anionic resin. Consequently the Langmuir model was coupled with a usual ion-exchange model to take into account the retention of their conjugate bases (<2%), which are commonly neglected at low pH (<1.5). Elution simulations with both uptake mechanisms fitted very well with experimental pulse tests. Only two parameters were optimized (equilibrium constant of acid uptake and ion-exchange selectivity coefficient of conjugate base) and their value were coherent with experimental and resin suppliers' data. These results confirmed that the singular tailing and apparent delay observed with succinic and citric acids can be explained by the high affinity of succinate and citrate for resin cationic sites. The model was implemented in a preparative chromatography simulation program in order to optimize operating parameters of our pilot-scale ISMB unit (Improved Simulated Moving Bed). The comparison with experimental ISMB profiles was conclusive. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

    PubMed

    Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

    2014-05-01

    Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.

  12. Aptamer facilitated purification of functional proteins.

    PubMed

    Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

    2018-01-15

    DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Purification of swine haptoglobin by affinity chromatography.

    PubMed Central

    Eurell, T E; Hall, W F; Bane, D P

    1990-01-01

    A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin. Images Fig. 2. Fig. 3. PMID:2123414

  14. Cell purification: a new challenge for biobanks.

    PubMed

    Almeida, Maria; García-Montero, Andres C; Orfao, Alberto

    2014-01-01

    Performing '-omics' analyses on heterogeneous biological tissue samples, such as blood or bone marrow, can lead to biased or even erroneous results, particularly when the targeted cells and/or molecules are present at relatively low percentages/amounts. In such cases, whole sample analysis will most probably dilute and mask the features of the cell and/or molecules of interest, and this will negatively impact the results and their interpretation. Therefore, frequently it is critically important to have well-characterized and high-quality purified cell populations for the reliable detection of subtle variations in their specific features, such as gene expression profile, protein expression pattern and metabolic status. Biobanks are technological platforms which aim to provide researchers access to a large number of high-quality biological samples and their associated data, particularly to support high-quality scientific and clinical research projects, and such projects will benefit enormously by having access to high-quality purified cell populations or their biological components (e.g. DNA, RNA, proteins). Therefore, a clear opportunity exists for preparative cell sorting techniques in biobanks. Although multiple different cell purification approaches exist or are under development (e.g. cell purification techniques based on cell adherence, density and/or cell size properties, methods based on antibody binding as well as new lab-on-a-chip purification techniques), the choice for a specific technology depends on multiple variables, including cell recovery, purity and yield, among others. In addition, most cell purification approaches are not well suited for high-throughput (HT) purification of multiple cell populations coexisting in a sample. Here we review the most (currently) used cell sorting methods that may be applied for sample preparation in biobanks. For the different approaches, technical considerations about their advantages and limitations are highlighted

  15. Intensification of oily waste waters purification by means of liquid atomization

    NASA Astrophysics Data System (ADS)

    Eskin, A. A.; Tkach, N. S.; Kim, M. I.; Zakharov, G. A.

    2017-10-01

    In this research, a possibility of using liquid atomization for improving the efficiency of purification of wastewater by different methods has been studied. By the introduced method and an experimental setup for wastewater purification, saturation rate increases with its purification by means of dissolved air flotation. Liquid atomization under excess pressure allows to gain a large interfacial area between the saturated liquid and air, which may increase the rate of purified liquid saturation almost twice, compared to the existing methods of saturation. Current disadvantages of liquid atomization used for intensification of wastewater purification include high energy cost and secondary emulsion of polluting agents. It is also known that by means of liquid atomization a process of ozonizing can be intensified. Large contact surface between the purified liquid and ozone-air mixture increases the oxidizing efficiency, which allows to diminish ozone discharge. Liquid atomization may be used for purification of wastewaters by ultraviolet radiation. Small drops of liquid will be proportionally treated by ultraviolet, which makes it possible to do purification even of turbid wastewaters. High-speed liquid motion will prevent the pollution of quartz tubes of ultraviolet lamps.

  16. Recent Advances in Nanoporous Membranes for Water Purification

    PubMed Central

    Wang, Zhuqing; Colombi Ciacchi, Lucio

    2018-01-01

    Nanoporous materials exhibit wide applications in the fields of electrocatalysis, nanodevice fabrication, energy, and environmental science, as well as analytical science. In this review, we present a summary of recent studies on nanoporous membranes for water purification application. The types and fabrication strategies of various nanoporous membranes are first introduced, and then the fabricated nanoporous membranes for removing various water pollutants, such as salt, metallic ions, anions, nanoparticles, organic chemicals, and biological substrates, are demonstrated and discussed. This work will be valuable for readers to understand the design and fabrication of various nanoporous membranes, and their potential purification mechanisms towards different water pollutants. In addition, it will be helpful for developing new nanoporous materials for quick, economic, and high-performance water purification. PMID:29370128

  17. Crystal Growth by Physical Vapor Transport: Experiments and Simulation Dynamics

    NASA Technical Reports Server (NTRS)

    Ramachandran, N.; Worlikar, A.; Su, Ching-Hua; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Crystal growth from the vapor phase has various advantages over melt growth. The main advantage is from a lower processing temperature, which makes the process more amenable in instances where the melting temperature of the crystal is high. Other benefits stem from the inherent purification mechanism in the process due to differences in the vapor pressures of the native elements and impurities, and the enhanced interfacial morphological stability during the growth process. Further, the implementation of PVT growth in closed ampoules affords experimental simplicity with minimal needs for complex process control, which makes it an ideal candidate for space investigations in systems where gravity tends to have undesirable effects on the growth process. Bulk growth of wide band gap II-VI semiconductors by PVT has been developed and refined over the past several years at NASA MSFC. A new modeling approach for PVT has also been recently formulated and its validation and testing is the main objective of this work.

  18. Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yoshida, Ayako; Tomita, Takeo; Kuzuyama, Tomohisa

    2007-02-01

    To elucidate the mechanism of regulation of aspartate kinase, the regulatory subunit (the β subunit of T. thermophilus aspartate kinase) was crystallized in the presence of the inhibitor threonine. Aspartate kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the β subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 Å at a synchrotron source. The crystal belongs to the cubic spacemore » group P4{sub 3}32 or P4{sub 1}32, with unit-cell parameters a = b = c = 141.8 Å.« less

  19. Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine† †Electronic supplementary information (ESI) available: NMR spectra of selected product, mass spectra of selected products, crystallization information, and experimental procedures are supplied. See DOI: 10.1039/c7sc00905d Click here for additional data file.

    PubMed Central

    Loren, Bradley P.; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang

    2017-01-01

    A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis. PMID:28979759

  20. Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

    PubMed Central

    Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

    2008-01-01

    Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056