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Sample records for antifreeze protein prepared

  1. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030 ...

  2. Antifreeze Proteins of Bacteria

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 12. Antifreeze Proteins of Bacteria. M K Chattopadhyay. General Article Volume 12 Issue 12 December 2007 pp 25-30. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/012/12/0025-0030. Keywords.

  3. Natural and Artificial Antifreeze Proteins

    Science.gov (United States)

    Arai, Soichi; Hirao, Noriko

    In the blood of winter polar fish an antifreeze glycoprotein (AFGP) occurs which acts to protect the fish from freezing to death. The AFGP has a unique hydrophilic hydrophobic conformation, involved in non-colligative depression of the freezing temperature of water without altering the melting point of ice. This phenomenon is reportedly a reflection of the ice crystal growth inhibition by the adsorption of the AFGP onto a-axial surfaces of the ice crystal. The authors, on the other hand, have developed an enzymatically modified protein (EMG-12) by covalent attachment of leucine dodecyl ester to the C-terminal position of gelation with the aid of a reverse reaction catalyzed by a protease. EMG-12, having a hydrophilic-hydrophobic structure, is highly surface-active and acts to stabilize a supercooling state of water by antinucleation. Discussions are made on similarities and dissimilarities of structure-function relationships of these natural and artificial antifreeze proteins. The significance of using them as antifreeze agents is also discussed.

  4. Antifreeze proteins of teleost fishes.

    Science.gov (United States)

    Fletcher, G L; Hew, C L; Davies, P L

    2001-01-01

    Marine teleosts at high latitudes can encounter ice-laden seawater that is approximately 1 degrees C colder than the colligative freezing point of their body fluids. They avoid freezing by producing small antifreeze proteins (AFPs) that adsorb to ice and halt its growth, thereby producing an additional non-colligative lowering of the freezing point. AFPs are typically secreted by the liver into the blood. Recently, however, it has become clear that AFP isoforms are produced in the epidermis (skin, scales, fin, and gills) and may serve as a first line of defense against ice propagation into the fish. The basis for the adsorption of AFPs to ice is something of a mystery and is complicated by the extreme structural diversity of the five antifreeze types. Despite the recent acquisition of several AFP three-dimensional structures and the definition of their ice-binding sites by mutagenesis, no common ice-binding motif or even theme is apparent except that surface-surface complementarity is important for binding. The remarkable diversity of antifreeze types and their seemingly haphazard phylogenetic distribution suggest that these proteins might have evolved recently in response to sea level glaciation occurring just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Not surprisingly, the expression of AFP genes from different origins can also be quite dissimilar. The most intensively studied system is that of the winter flounder, which has a built-in annual cycle of antifreeze expression controlled by growth hormone (GH) release from the pituitary in tune with seasonal cues. The signal transduction pathway, transcription factors, and promoter elements involved in this process are just beginning to be characterized.

  5. Antivirulence Properties of an Antifreeze Protein

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    Martin Heisig

    2014-10-01

    Full Text Available As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

  6. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Overwintering plants secrete antifreeze proteins (AFPs) to provide freezing tolerance. These proteins bind to and inhibit the growth of ice crystals that are formed in the apoplast during subzero temperatures. Antifreeze activity has been detected in more than 60 plants and AFPs have been purified from 15 of these, including ...

  7. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas

    2014-01-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face...... of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active...

  8. Antifreeze Proteins from Diverse Organisms and their Applications: An Overview.

    Science.gov (United States)

    Cheung, Randy Chi Fai; Ng, Tzi Bun; Wong, Jack Ho

    2017-01-01

    Antifreeze proteins are ice-binding or ice-structuring proteins that prevent water from freezing by adsorbing to the ice surface and stopping the growth of minute ice crystals to large crystals in a non-colligative manner. The antifreeze proteins are found in species like fish, arthropods, plants, algae, fungi, yeasts and bacteria. The diversity, distribution and classification of antifreeze proteins were highlighted in this review. Antifreeze proteins help the organisms adapt to and survive in subzero temperature environments. The distribution of antifreeze proteins in different species appears to be the outcome of a combination of independent evolutionary events, probably the convergent evolution or horizontal gene transfer. Benefits can be derived from the frost resistance of these organisms. Their potential applications have been recognized in food processing, cryopreservation, cryosurgery, fishery and agricultural industries and anti-icing materials development. This review includes information on the current understanding of antifreeze proteins. A discussion on interactions and mechanisms involving ice recognition and adsorption was also included. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

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    Chen Wang

    Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

  10. Antifreeze Protein Mimetic Metallohelices with Potent Ice Recrystallization Inhibition Activity.

    Science.gov (United States)

    Mitchell, Daniel E; Clarkson, Guy; Fox, David J; Vipond, Rebecca A; Scott, Peter; Gibson, Matthew I

    2017-07-26

    Antifreeze proteins are produced by extremophile species to control ice formation and growth, and they have potential applications in many fields. There are few examples of synthetic materials which can reproduce their potent ice recrystallization inhibition property. We report that self-assembled enantiomerically pure, amphipathic metallohelicies inhibited ice growth at just 20 μM. Structure-property relationships and calculations support the hypothesis that amphipathicity is the key motif for activity. This opens up a new field of metallo-organic antifreeze protein mimetics and provides insight into the origins of ice-growth inhibition.

  11. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... 0.52°C. TH. Zhang et al. 2011. 4. Daucus carrota. Tap. Root extract. 36. Apoplastic. Polygalacturonase inhibitor protein. N-glycosylated,. TH-0.36 at. 150 μ ...... na R 2011 A framework for classification of antifreeze proteins in over wintering plants based on their sequence and structural features. J. Bioinform.

  12. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Antifreeze proteins (AFPs) are found in a wide range of species including fishes, plants, etc. They have very characteristic feature that inhibit the growth and recrystallization of ice that forms in intercellular spaces. Two expressed sequence tags (ESTs) were previously identified from salt stress cDNA library in Fragilariopsis ...

  13. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    In this paper, seventeen different fish Antifreeze Proteins (AFPs) retrieved from Swiss-Prot database are analysed and characterized using In silico tools. ... Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Maha Vidyalaya (Deemed University), Enathur, Kanchipuram 631 561; EXCEL and Polymer ...

  14. Structure and application of antifreeze proteins from Antarctic bacteria.

    Science.gov (United States)

    Muñoz, Patricio A; Márquez, Sebastián L; González-Nilo, Fernando D; Márquez-Miranda, Valeria; Blamey, Jenny M

    2017-08-07

    Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface. Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to β-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice. AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a β-helix structure, and

  15. MINING THE PROTEIN REPERTOIRE OF A HIMALAYAN SHRUB, HIPPOPHAE RHAMNOIDES FOR ANTIFREEZE PROTEINS

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    Bhavana Sharma

    2016-09-01

    Full Text Available Seabuckthorn (Hippophae rhamnoides, a cold desert shrub, can survive freezing temperature conditions and is considered as frost and drought tolerant. Earlier, antifreeze activity was reported in seabuckthorn seedlings grown under laboratory conditions. No reports are available on the cold hardiness of this huge bioresource available naturally in the Himalayan region. Detection of antifreeze activity in leaves and berries (splat assay and nanoliter osmometer confirmed the presence of putative antifreeze proteins (AFPs which may help in the survival of this plant under freezing conditions. Flower shaped ice crystals were observed in both leaves and berries while hexagonal ice crystals in seedlings indicated comparatively higher antifreeze activity in the wild parts. Splat assay results confirmed highest IRI activity in leaf (a 2.75 fold decrease in mean grain size of ice crystal after annealing followed by berry (with 1.75 fold decrease and least in the seedlings (with 1.5 fold decrease. Gel filtration chromatography resolved leaf fractions exhibited antifreeze activity in 34, 36 and 41 kDa while in berry fractions a 41 kDa polypeptide showed antifreeze activity. This is the first report showing presence of AFPs in (H. rhamnoides leaf and berry. Shotgun proteomic analysis using Q-Exactive Orbitrap High Resolution Mass Spectrometer and functional annotation of leaf and berry proteins revealed their association with primary, secondary metabolism, defence/stress, redox regulation, signalling and structural remodelling majority of which are affected during cold stress. Further purification of these AFPs could open gates for commercial utilization of this plant growing abundantly in Himalayan regions of India.

  16. Two domains of RD3 antifreeze protein diffuse independently.

    Science.gov (United States)

    Holland, Nolan B; Nishimiya, Yoshiyuki; Tsuda, Sakae; Sönnichsen, Frank D

    2008-06-03

    Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.

  17. Solvation structure of ice-binding antifreeze proteins

    Science.gov (United States)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  18. Antifreeze proteins: Adsorption to ice, silica and gas hydrates

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Huang; Brown, Alan; Wathen, Brent; Ripmeester, John A.; Walker, VIrginia K.

    2005-07-01

    Certain organisms survive under freezing conditions that could otherwise prove fatal by the synthesis of antifreeze proteins (AFPs). AFPs adsorb to the surface of microscopic ice crystals and prevent further ice growth, resulting in a noncolligative freezing point depression. Type I AFP from the winter flounder (wfAFP) is an alfa-helical, alanine-rich serum protein that helps protect against innoculative freezing from ice-laden seas. The AFP of a moth from the boreal forest, Choristoneura fumiferana (Cf), is a beta-helical threonine-rich protein that helps prevent freezing at the overwintering, caterpillar stage. In contrast, the beta-roll AFP from the grass, Lolium perenne (Lp), confers little freezing point depression and the plants readily freeze. Remarkably, AFPs also adsorb to tetrahyrofuran (THF) hydrate, changing the hydrate's octahedral morphology and, as well, inhibiting the growth of THF and gas hydrates. The hyperactive CfAFP, with 30-100 times the activity of wfAFP toward ice, showed far greater nucleation inhibition for THF hydrate than did a commercial hydrate inhibitor, poly(N-vinylpyrrolidone) (PVP). Active AFPs were also judged to be superior to PVP in that they inhibited the memory effect, a phenomenon whereby hydrate reforms at a faster rate soon after melting. An inactive mutant wfAFP, with an amino acid substitution at the ice-binding site, also reduced the growth of THF hydrate but was ineffective at suppressing hydrate reformation. These results suggest that the molecular properties important for ice adsorption and inhibition of hydrate reformation may be similar, and are distinct from those required for hydrate growth inhibition. The different AFPs also show markedly different aggregations on a third hydrophilic substrate, silica. Together these studies suggest that AFP adsorption to ice, hydrates and silica depends on the overall structure, specific residues and protein-protein interactions. (Author)

  19. Using Antifreeze Proteins to understand ice microstructure evolution

    Science.gov (United States)

    Bayer-Giraldi, Maddalena; Azuma, Nobuhiko; Takata, Morimasa; Weikusat, Christian; Kondo, Hidemasa; Kipfstuhl, Sepp

    2017-04-01

    Polar ice sheets are considered a unique climate archive. The chemical analysis of its impurities and the development of its microstructure with depth give insight in past climate conditions as well as in the development of the ice sheet with time and deformation. Microstructural patterns like small grain size observed in specific depths are thought to be linked to the retarding effect of impurities on ice grain growth. Clear evidence of size or chemical composition of the impurities causing this effect is missing, but in this context a major role of nanoparticles has been suggested. In order to shed light on different mechanisms by which nanoparticles can control microstructure development we used antifreeze proteins (AFPs) as proxies for particles in ice. These proteins are small nanoparticles, approx. 5 nm in size, with the special characteristics of firmly binding to ice through several hydrogen bonds. We used AFPs from the sea-ice microalgae Fragilariopsis cylindrus (fcAFPs) in bubble-free, small-grained polycrystalline ice obtained by the phase-transition size refinement method. We explain how fcAFP bind to ice by presenting the 3-D-protein structure model inferred by X-ray structure analysis, and show the importance of the chemical interaction between particles and ice in controlling normal grain growth, comparing fcAFPs to other protein nanoparticles. We used modifications of fcAFPs for particle localization through fluorescence spectroscopy. Furthermore, the effect of fcAFPs on the driving factors for ice deformation during creep, i.e. on internal dislocations due to incorporation within the lattice and on the mobility of grain boundaries due to pinning, makes these proteins particularly interesting in studying the process of ice deformation.

  20. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    Energy Technology Data Exchange (ETDEWEB)

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  1. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.

    Science.gov (United States)

    Olijve, Luuk L C; Meister, Konrad; DeVries, Arthur L; Duman, John G; Guo, Shuaiqi; Bakker, Huib J; Voets, Ilja K

    2016-04-05

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

  2. Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.

    Science.gov (United States)

    Scotter, Andrew J; Kuntz, Douglas A; Saul, Michelle; Graham, Laurie A; Davies, Peter L; Rose, David R

    2006-06-01

    We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.

  3. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

    DEFF Research Database (Denmark)

    Kristiansen, E; Ramløv, Hans; Højrup, Peter

    2011-01-01

    beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs......Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn...

  4. Cloning, expression, and activity of type IV antifreeze protein from cultured subtropical olive flounder (Paralichthys olivaceus

    Directory of Open Access Journals (Sweden)

    Jong Kyu Lee

    2016-10-01

    Full Text Available Abstract Antifreeze proteins (AFPs lower the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals via an adsorption-inhibition mechanism. However, the function of type IV AFP (AFP IV is questionable, as its antifreeze activity is on the verge of detectable limits, its physiological concentration in adult fish blood is too low to function as a biological antifreeze, and its homologues are present even in fish from tropic oceans as well as freshwater. Therefore, we speculated that AFP IV may have gained antifreeze activity not by selective pressure but by chance. To test this hypothesis, we cloned, expressed, and assayed AFP IV from cultured subtropical olive flounder (Paralichthys olivaceus, which do not require antifreeze protein for survival. Among the identified expressed sequence tags of the flounder liver sample, a 5′-deleted complementary DNA (cDNA sequence similar to the afp4 gene of the longhorn sculpin was identified, and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP IV shared 55, 53, 52, and 49 % identity with those of Pleuragramma antarcticum, Myoxocephalus octodecemspinosus, Myoxocephalus scorpius, and Notothenia coriiceps, respectively. Furthermore, the genomic structure of this gene was conserved with those of other known AFP IVs. Notably, the recombinant AFP IV showed a weak but distinct thermal hysteresis of 0.07 ± 0.01 °C at the concentration of 0.5 mg/mL, and ice crystals in an AFP IV solution grew star-shaped, which are very similar to those obtained from other polar AFP IVs. Taken together, our results do not support the hypothesis of evolution of AFP IV by selective pressure, suggesting that the antifreeze activity of AFP IV may have been gained by chance.

  5. Cloning and expression of a novel antifreeze protein AFP72 from the beetle Tenebrio molitor.

    Science.gov (United States)

    Yan, Qing-Hua; Yang, Li; Wang, Qing; Zhang, Hui-Rong; Shao, Qiang

    2012-01-01

    A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coil TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein displayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.

  6. Applications of type I antifreeze proteins: studies with model membranes & cryoprotectant properties.

    Science.gov (United States)

    Inglis, Steven R; Turner, Jennifer J; Harding, Margaret M

    2006-12-01

    Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), found in the body fluids of many species of polar fish allow them to survive in waters colder than the equilibrium freezing point of their blood and other internal fluids. Despite their structural diversity, all AF(G)Ps kinetically depress the temperature at which ice grows in a non-colligative manner and hence exhibit thermal hysteresis. AF(G)Ps also share the ability to interact with and protect mammalian cells and tissues from hypothermic damage (e.g., improved storage of human blood platelets at low temperatures), and are able to stabilize or disrupt membrane composition during low temperature and freezing stress (e.g., cryoprotectant properties in stabilization of sperm and oocytes). This review will summarize studies of AFPs with phospholipids and plant lipids, proposed mechanisms for inhibition of leakage from membranes, and cryoprotectant studies with biological samples. The major focus will be on the alpha-helical type I antifreeze proteins, and synthetic mutants, that have been most widely studied. For completeness, data on glycoproteins will also be presented. While a number of models to explain stabilization and destabilization of different lipid systems have been proposed, it is currently not possible to predict whether a particular AFP will stabilize or destabilize a given lipid system. Furthermore the relationship between the antifreeze property of thermal hysteresis and membrane stabilization is unknown. This lack of detailed knowledge about how AFPs function in the presence of different types of materials has hampered progress toward the development of antifreezes for cold storage of cells, tissues, and organs.

  7. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    Science.gov (United States)

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. HEAT INDUCIBLE EXPRESSION OF ANTIFREEZE PROTEIN GENES FROM THE BEETLES Tenebrio molitor AND Microdera punctipennis.

    Science.gov (United States)

    Li, Jieqiong; Ma, Wenjing; Ma, Ji

    2016-01-01

    Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis. To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real-time quantitative PCR. Mild low temperatures (5~15 degree C), high temperature (38~47 degree C for M. punctipennis, or 37~42 degree C for T. molitor) and temperature difference (10~30 degree C) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25 degree C. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5 degree C and 15 degree C for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25 degree C. High temperature (37~42 degree C) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10 degree C was effective in stimulating Tmafps expression. The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat

  9. Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

    Directory of Open Access Journals (Sweden)

    Hak Jun Kim

    2017-01-01

    Full Text Available Antifreeze proteins (AFPs are biological antifreezes with unique properties, including thermal hysteresis(TH,ice recrystallization inhibition(IRI,and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification, storage temperature, and types of biological sample type.

  10. Electro-Optical Properties Characterization of Fish Type III Antifreeze Protein

    OpenAIRE

    Salvay, Andrés G.; Santos, Javier; Howard, Eduardo I.

    2007-01-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa—or 14-kDa tandem—globular proteins. In the present work, we study ...

  11. Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

    Science.gov (United States)

    Duman, John G

    2015-06-01

    Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants. © 2015. Published by The Company of Biologists Ltd.

  12. An insight into the molecular basis for convergent evolution in fish antifreeze Proteins.

    Science.gov (United States)

    Nath, Abhigyan; Chaube, Radha; Subbiah, Karthikeyan

    2013-08-01

    Antifreeze proteins (AFPs) prevent the growth of ice-crystals in order to enable certain organisms to survive under sub-zero temperature surroundings. These AFPs have evolved from different types of proteins without having any significant structural and sequence similarities among them. However, all the AFPs perform the same function of anti-freeze activity and are a classical example of convergent evolution. We have analyzed fish AFPs at the sequence level, the residue level and the physicochemical property group composition to discover molecular basis for this convergent evolution. Our study on amino acid distribution does not reveal any distinctive feature among AFPs, but comparative study of the AFPs with their close non-AFP homologs based on the physicochemical property group residues revealed some useful information. In particular (a) there is a similar pattern of avoidance and preference of amino acids in Fish AFP subtypes II, III and IV-Aromatic residues are avoided whereas small residues are preferred, (b) like other psychrophilic proteins, AFPs have a similar pattern of preference/avoidance for most of the residues except for Ile, Leu and Arg, and (c) most of the computed amino acids in preferred list are the key functional residues as obtained in previous predicted model of Doxey et al. For the first time this study revealed common patterns of avoidance/preference in fish AFP subtypes II, III and IV. These avoidance/preference lists can further facilitate the identification of key functional residues and can shed more light into the mechanism of antifreeze function. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

    International Nuclear Information System (INIS)

    Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

    2009-01-01

    Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

  14. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-08

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  15. Operation of Kelvin Effect in the Activities of an Antifreeze Protein: A Molecular Dynamics Simulation Study.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2018-03-29

    Ice growth and melting inhibition activities of antifreeze proteins (AFPs) are better explained by the adsorption-inhibition mechanism. Inhibition occurs as a result of the Kelvin effect induced by adsorbed protein molecules onto the surface of seed ice crystal. However, the Kelvin effect has not been explored by the state-of-the-art experimental techniques. In this work, atomistic molecular dynamics simulations have been carried out with Tenebrio molitor antifreeze protein ( TmAFP) placed at ice-water interface to probe the Kelvin effect in the mechanism of AFPs. Simulations show that, below equilibrium melting temperature, ice growth is inhibited through the convex ice-water interface formation toward the water phase and, above equilibrium melting temperature, ice melting is inhibited through the concave ice-water interface formation inward to ice phase. Simulations further reveal that the radius of curvature of the interface formed to stop the ice growth increases with decrease in the degree of supercooling. Our results are in qualitative agreement with the theoretical prediction of the Kelvin effect and thus reveal its operation in the activities of AFPs.

  16. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    GRAVY, Grand Average Hydropathy. structure of protein (3D coordinates data). The 3D structure of AFPs Q01758 and P05140 were gener- ated by homology modelling using Esypred34 server. The similar 3D structures (for the AFPs Q01758 and. P05140 sequences) in the Protein Data bank. (www.rscb.org) were identified ...

  17. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  18. Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

    Directory of Open Access Journals (Sweden)

    Srisulak Dheeranupattana

    2005-07-01

    Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

  19. Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Johnsen, Johannes Lørup; Kristiansen, Erlend

    2014-01-01

    , the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show......The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect...... that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths...

  20. An Effective Antifreeze Protein Predictor with Ensemble Classifiers and Comprehensive Sequence Descriptors

    Directory of Open Access Journals (Sweden)

    Runtao Yang

    2015-09-01

    Full Text Available Antifreeze proteins (AFPs play a pivotal role in the antifreeze effect of overwintering organisms. They have a wide range of applications in numerous fields, such as improving the production of crops and the quality of frozen foods. Accurate identification of AFPs may provide important clues to decipher the underlying mechanisms of AFPs in ice-binding and to facilitate the selection of the most appropriate AFPs for several applications. Based on an ensemble learning technique, this study proposes an AFP identification system called AFP-Ensemble. In this system, random forest classifiers are trained by different training subsets and then aggregated into a consensus classifier by majority voting. The resulting predictor yields a sensitivity of 0.892, a specificity of 0.940, an accuracy of 0.938 and a balanced accuracy of 0.916 on an independent dataset, which are far better than the results obtained by previous methods. These results reveal that AFP-Ensemble is an effective and promising predictor for large-scale determination of AFPs. The detailed feature analysis in this study may give useful insights into the molecular mechanisms of AFP-ice interactions and provide guidance for the related experimental validation. A web server has been designed to implement the proposed method.

  1. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    Directory of Open Access Journals (Sweden)

    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  2. The mysteries of memory effect and its elimination with antifreeze proteins

    Energy Technology Data Exchange (ETDEWEB)

    Walker, V.; Gordienko, R.; Kuiper, M.; Huva, E.; Wu, Z. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology; Zeng, H.; Ripmeester, J. [Queen' s Univ., Kingston, ON (Canada). Dept. of Biology]|[National Research Council of Canada, Ottawa, ON (Canada). Steacie Inst. for Molecular Sciences

    2008-07-01

    With the decline in easily accessible and conventional hydrocarbon supplies, exploration will focus on hydrocarbons in deep offshore waters, in permafrost or in crystalline water as gas hydrates. Crystallization of water or water-encaged gas molecules takes place when nuclei reach a critical size, but the crystal growth may be inhibited by certain antifreeze proteins (AFPs). In this study, the authors hypothesized that the crystal lattice of gas hydrates may act as an alternative for substrate antifreeze proteins (AFPs). AFP-mediated inhibition of ice and clathrate hydrate crystallization was examined. Since the AFPs had a notable ability to eliminate the memory effect (ME) or the faster reformation of clathrate hydrates after melting, the authors were prompted to examine heterogeneous nucleation. Silica, served as a model nucleator hydrophilic surface. Quartz crystal microbalance-dissipation (QCM-D) experiments showed that an active AFP was tightly adsorbed to the silica surface. However, polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), 2 commercial hydrate kinetic inhibitors that do not eliminate ME, were not as tightly adsorbed. A mutant AFP inhibited tetrahydrofuran clathrate hydrate growth, but not ME. QCM-D analysis showed that adsorption of the mutant AFP was more similar to PVCap than the active AFP. It was concluded that although there is no evidence for memory in ice reformation, the crystallization of ice and hydrates, and the elimination of the more rapid recrystallization of hydrates, can be mediated by the same proteins. The properties of adsorbed layers can be effectively monitored by QCM-D. These study results provided useful information about the inhibition mechanism of heterogeneous nucleation of clathrate hydrate. The technique facilitates the screening of potential low dose hydrate inhibitors and residues in AFPs that are involved in silica adsorption. 24 refs., 1 tab., 4 figs.

  3. Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

    DEFF Research Database (Denmark)

    Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans

    2014-01-01

    Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform...... this is the first report of dimerization of AFP type III proteins....

  4. The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III).

    Science.gov (United States)

    Deller, R C; Carter, B M; Zampetakis, I; Scarpa, F; Perriman, A W

    2018-01-01

    The aim of this study was to examine the effect of chemical cationization on the structure and function of antifreeze protein III (AFP III) over an extreme temperature range (-40°C to +90°C) using far-UV synchrotron radiation circular dichroism (SRCD) and ice recrystallization inhibition (IRI) assays. Chemical cationization was able to produce a modified AFP III with a net cationic charge at physiological pH that had enhanced resistance to denaturation at elevated temperatures, with no immediate negative impact on protein structure at subzero temperatures. Furthermore, cationized AFP III retained an IRI activity similar to that of native AFP III. Consequently, chemical cationization may provide a pathway to the development of more robust antifreeze proteins as supplementary cryoprotectants in the cryopreservation of clinically relevant cells. Copyright © 2017. Published by Elsevier Inc.

  5. Growth-melt asymmetry in ice crystals under the influence of spruce budworm antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Pertaya, Natalya [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); Celik, Yeliz [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); DiPrinzio, Carlos L [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); Wettlaufer, J S [Department of Geology and Geophysics, Yale University, New Haven, CT 06520-8109 (United States); Davies, Peter L [Department of Biochemistry, Queen' s University, Kingston, ON K7L 3N6 (Canada); Braslavsky, Ido [Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States)

    2007-10-17

    Here we describe studies of the crystallization behavior of ice in an aqueous solution of spruce budworm antifreeze protein (sbwAFP) at atmospheric pressure. SbwAFP is an ice binding protein with high thermal hysteresis activity, which helps protect Choristoneura fumiferana (spruce budworm) larvae from freezing as they overwinter in the spruce and fir forests of the north eastern United States and Canada. Different types of ice binding proteins have been found in many other species. They have a wide range of applications in cryomedicine and cryopreservation, as well as the potential to protect plants and vegetables from frost damage through genetic engineering. However, there is much to learn regarding the mechanism of action of ice binding proteins. In our experiments, a solution containing sbwAFP was rapidly frozen and then melted back, thereby allowing us to produce small single crystals. These maintained their hexagonal shapes during cooling within the thermal hysteresis gap. Melt-growth-melt sequences in low concentrations of sbwAFP reveal the same shape transitions as are found in pure ice crystals at low temperature (-22 deg. C) and high pressure (2000 bar) (Cahoon et al 2006 Phys. Rev. Lett. 96 255502); while both growth and melt shapes display faceted hexagonal morphology, they are rotated 30 deg. relative to one another. Moreover, the initial melt shape and orientation is recovered in the sequence. To visualize the binding of sbwAFP to ice, we labeled the antifreeze protein with enhanced green fluorescent protein (eGFP) and observed the sbwAFP-GFP molecules directly on ice crystals using confocal microscopy. When cooling the ice crystals, facets form on the six primary prism planes (slowest growing planes) that are evenly decorated with sbwAFP-GFP. During melting, apparent facets form on secondary prism planes (fastest melting planes), leaving residual sbwAFP at the six corners of the hexagon. Thus, the same general growth-melt behavior of an apparently

  6. Balance between hydration enthalpy and entropy is important for ice binding surfaces in Antifreeze Proteins.

    Science.gov (United States)

    Schauperl, Michael; Podewitz, Maren; Ortner, Teresa S; Waibl, Franz; Thoeny, Alexander; Loerting, Thomas; Liedl, Klaus R

    2017-09-19

    Antifreeze Proteins (AFPs) inhibit the growth of an ice crystal by binding to it. The detailed binding mechanism is, however, still not fully understood. We investigated three AFPs using Molecular Dynamics simulations in combination with Grid Inhomogeneous Solvation Theory, exploring their hydration thermodynamics. The observed enthalpic and entropic differences between the ice-binding sites and the inactive surface reveal key properties essential for proteins in order to bind ice: While entropic contributions are similar for all sites, the enthalpic gain for all ice-binding sites is lower than for the rest of the protein surface. In contrast to most of the recently published studies, our analyses show that enthalpic interactions are as important as an ice-like pre-ordering. Based on these observations, we propose a new, thermodynamically more refined mechanism of the ice recognition process showing that the appropriate balance between entropy and enthalpy facilitates ice-binding of proteins. Especially, high enthalpic interactions between the protein surface and water can hinder the ice-binding activity.

  7. Influence of Block Copolymerization on the Antifreeze Protein Mimetic Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol).

    Science.gov (United States)

    Congdon, Thomas R; Notman, Rebecca; Gibson, Matthew I

    2016-09-12

    Antifreeze (glyco) proteins are produced by many cold-acclimatized species to enable them to survive subzero temperatures. These proteins have multiple macroscopic effects on ice crystal growth which makes them appealing for low-temperature applications-from cellular cryopreservation to food storage. Poly(vinyl alcohol) has remarkable ice recrystallization inhibition activity, but its mode of action is uncertain as is the extent at which it can be incorporated into other high-order structures. Here the synthesis and characterization of well-defined block copolymers containing poly(vinyl alcohol) and poly(vinylpyrrolidone) by RAFT/MADIX polymerization is reported, as new antifreeze protein mimetics. The effect of adding a large second hydrophilic block is studied across a range of compositions, and it is found to be a passive component in ice recrystallization inhibition assays, enabling retention of all activity. In the extreme case, a block copolymer with only 10% poly(vinyl alcohol) was found to retain all activity, where statistical copolymers of PVA lose all activity with very minor changes to composition. These findings present a new method to increase the complexity of antifreeze protein mimetic materials, while retaining activity, and also to help understand the underlying mechanisms of action.

  8. Electro-optical properties characterization of fish type III antifreeze protein.

    Science.gov (United States)

    Salvay, Andrés G; Santos, Javier; Howard, Eduardo I

    2007-12-01

    Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa-or 14-kDa tandem-globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.

  9. Antifreeze poisoning

    Science.gov (United States)

    The poisonous ingredients in antifreeze are: Ethylene glycol Methanol Propylene glycol ... For ethylene glycol: Death may occur within the first 24 hours. If ... little as 2 tablespoons (1 ounce or 30 milliliters) can kill a ...

  10. Fluorescence and confocal microscopy studies of the ice surface - antifreeze protein interactions.

    Science.gov (United States)

    Pertaya, N.; Thomson, E.; Davies, P. L.; Braslavsky, I.

    2005-03-01

    Biomineralization is a phenomenon in which biological material influences mineral growth on the molecular level. A compelling example involves antifreeze proteins (AFPs) known to prevent fish and insects from freezing. AFPs have many potential applications in agriculture, biomedical science, and can be used as a model platform to understand biomineralization processes for future nanotechnology applications. Here we describe a new approach to study the interaction between AFPs and ice using fluorescence and confocal microscopy combined with a unique ice growth cell. After conjugating green fluorescent protein (GFP) to Type III AFP, we imaged the fluorescence signal around and inside of the ice crystals that emerged from the cooled AFP-GFP solution, and have observed an enhanced fluorescence signal at the edge of the ice crystal. In a second cell we observed a dramatic change in the ice growth morphology when AFPs were introduced into an initially pure system. Further developments of these methods will permit the direct imaging of the location and concentration of the AFPs on ice surfaces and enable a better understanding of their operation. Supported by CIHR, the Bosack and Kruger Foundation, Ohio and Yale Universities.

  11. ANTIFREEZE PROTEINS IN PLANTS: AN OVERVIEW WITH AN INSIGHT INTO THE DETECTION TECHNIQUES INCLUDING NANOBIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    Bhavana Sharma

    2014-08-01

    Full Text Available Antifreeze proteins (AFPs are a class of polypeptides which enables various organisms to survive subzero temperatures and have been found in vertebrates, invertebrates, plants, fungi and lichens. AFPs possess the characteristic thermal hysteresis (TH and ice recrystallization inhibition (IRI properties which allow them to adsorb the surface of ice crystals and inhibit their growth and recrystallization. AFPs are also known as ice restructuring proteins due to their ability to modify ice crystal morphology which leads to formation of hexagonal shape ice crystals in the presence of AFPs and disc shape AFPs in its absence. AFPs have various applications in medical, agricultural, industrial and biotechnological field. This review provides an overview of the AFPs, their TH and IRI properties and potential biotechnological applications of AFPs. Various conventional detection methods like Capillary assay and Differential Scanning Calorimetry (DSC with their advantages and disadvantages are discussed in detail along with the commonly used Splat assay and Nanoliter osmometer. Moreover, a novel, high-throughput and efficient nanobiotechnological method for AFP detection is also discussed. The method is based on colorimetric detection of freeze-labile gold nanoparticles and can provide an alternative to overcome the limitations of conventional methods by providing quick and easy way to screen AFPs in multiple systems simultaneously

  12. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  13. Calorimetric determination of inhibition of ice crystal growth by antifreeze protein in hydroxyethyl starch solutions.

    Science.gov (United States)

    Hansen, T N; Carpenter, J F

    1993-01-01

    Differential scanning calorimetry and cryomicroscopy were used to investigate the effects of type I antifreeze protein (AFP) from winter flounder on 58% solutions of hydroxyethyl starch. The glass, devitrification, and melt transitions noted during rewarming were unaffected by 100 micrograms/ml AFP. Isothermal annealing experiments were undertaken to detect the effects of AFP-induced inhibition of ice crystal growth using calorimetry. A premelt endothermic peak was detected during warming after the annealing procedure. Increasing the duration or the temperature of the annealing for the temperature range from -28 and -18 degrees C resulted in a gradual increase in the enthalpy of the premelt endotherm. This transition was unaffected by 100 micrograms/ml AFP. Annealing between -18 and -10 degrees C resulted in a gradual decrease in the premelt peak enthalpy. This process was inhibited by 100 micrograms/ml AFP. Cryomicroscopic examination of the samples revealed that AFP inhibited ice recrystallization during isothermal annealing at -10 degrees C. Annealing at lower temperatures resulted in minimal ice recrystallization and no visible effect of AFP. Thus, the 100 micrograms/ml AFP to have a detectable influence on thermal events in the calorimeter, conditions must be used that result in significant ice growth without AFP and visible inhibition of this process by AFP. Images FIGURE 8 PMID:7690257

  14. Lateral transfer of a lectin-like antifreeze protein gene in fishes.

    Directory of Open Access Journals (Sweden)

    Laurie A Graham

    Full Text Available Fishes living in icy seawater are usually protected from freezing by endogenous antifreeze proteins (AFPs that bind to ice crystals and stop them from growing. The scattered distribution of five highly diverse AFP types across phylogenetically disparate fish species is puzzling. The appearance of radically different AFPs in closely related species has been attributed to the rapid, independent evolution of these proteins in response to natural selection caused by sea level glaciations within the last 20 million years. In at least one instance the same type of simple repetitive AFP has independently originated in two distant species by convergent evolution. But, the isolated occurrence of three very similar type II AFPs in three distantly related species (herring, smelt and sea raven cannot be explained by this mechanism. These globular, lectin-like AFPs have a unique disulfide-bonding pattern, and share up to 85% identity in their amino acid sequences, with regions of even higher identity in their genes. A thorough search of current databases failed to find a homolog in any other species with greater than 40% amino acid sequence identity. Consistent with this result, genomic Southern blots showed the lectin-like AFP gene was absent from all other fish species tested. The remarkable conservation of both intron and exon sequences, the lack of correlation between evolutionary distance and mutation rate, and the pattern of silent vs non-silent codon changes make it unlikely that the gene for this AFP pre-existed but was lost from most branches of the teleost radiation. We propose instead that lateral gene transfer has resulted in the occurrence of the type II AFPs in herring, smelt and sea raven and allowed these species to survive in an otherwise lethal niche.

  15. Synthetic antifreeze peptide

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  16. De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

    Directory of Open Access Journals (Sweden)

    Ricardo Vera Bravo

    2011-12-01

    Full Text Available A new strategy is presented for the designand synthesis of peptides that exhibitice-binding and antifreeze activity. Apennant-type dendrimer polypeptidescaffold combining an α-helical backbonewith four short β-strand branches wassynthesized in solid phase using Fmocchemistry in a divergent approach. The51-residue dendrimer was characterizedby reverse phase high performance liquidchromatography, mass spectrometry andcircular dichroism. Each β-strand branchcontained three overlapping TXT aminoacid repeats, an ice-binding motif foundin the ice-binding face of the sprucebudworm (Choristoneura fumiferanaand beetle (Tenebrio molitor antifreezeproteins. Ice crystals in the presence ofthe polypeptide monomer displayed flat,hexagonal plate morphology, similar tothat produced by weakly active antifreezeproteins. An oxidized dimeric form of thedendrimer polypeptide also produced flathexagonal ice crystals and was capableof inhibiting ice crystal growth upontemperature reduction, a phenomenontermed thermal hysteresis, a definingproperty of antifreeze proteins. Linkageof the pennant-type dendrimer to a trifunctionalcascade-type polypeptideproduced a trimeric macromolecule thatgave flat hexagonal ice crystals withhigher thermal hysteresis activity thanthe dimer or monomer and an ice crystal burst pattern similar to that producedby samples containing insect antifreezeproteins. This macromolecule was alsocapable of inhibiting ice recrystallization.

  17. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules.

    Science.gov (United States)

    Mitchell, Daniel E; Congdon, Thomas; Rodger, Alison; Gibson, Matthew I

    2015-10-26

    Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used 'splat' methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics.

  18. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance {sup 13}C NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daley, Margaret E.; Sykes, Brian D. [University of Alberta, Department of Biochemistry, CIHR Group in Protein Structure and Function and Protein Engineering Network of Centres of Excellence (Canada)

    2004-06-15

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance {sup 13}C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the {sup 1}H-{sup 13}C NOE were determined in this study. The C{alpha}H relaxation measurements were compared to the previously measured {sup 15}N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the {chi}{sub 1} dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than {+-}25 deg.

  19. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

    Directory of Open Access Journals (Sweden)

    Jaewang Lee

    Full Text Available Ovarian tissue (OT cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs on mouse ovarian tissue cryopreservation and transplantation.Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III and concentration (0.1, 1, 10 mg/mL used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs and repair (DDR, respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control. Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL

  20. Reversible binding of the HPLC6 isoform of type I antifreeze proteins to ice surfaces and the antifreeze mechanism studied by multiple quantum filtering-spin exchange NMR experiment.

    Science.gov (United States)

    Ba, Yong; Wongskhaluang, Jeff; Li, Jiabo

    2003-01-15

    Antifreeze proteins (AFPs) protect organisms from freezing damage by inhibiting the growth of seed-ice crystals. It has long been hypothesized that irreversible binding of AFPs to ice surfaces is responsible for inhibiting the growth of seed-ice crystals as such a mechanism supports the popularly accepted Kelvin effect for the explanation of local freezing-point depression. However, whether the binding is reversible or irreversible is still under debate due to the lack of direct experimental evidence. Here, we report the first direct experimental result, by using the newly developed multiple quantum (MQ) filtering-spin exchange NMR experiment, that shows that the binding of HPLC6 peptides to ice surfaces is reversible. It was found that the reversible process can be explained by the model of monolayer adsorption. These results suggest that the Kelvin effect is not suitable for explaining the antifreeze mechanism, and direct interactions between the peptides and the ice-surface binding sites are the driving forces for the binding of AFPs to ice surfaces. We propose that there exists a concentration gradient of AFP from an ice-binding surface to the solution due to the affinity of ice surfaces to AFPs. This concentration gradient creates a dense layer of AFP in contact with the ice-binding surface, which depresses the local freezing point because of the colligative property, but not the Kelvin effect.

  1. The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis.

    Science.gov (United States)

    Duman, John G.; Serianni, Anthony S.

    2002-01-01

    Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. (13)NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be approximately 8.5 degrees C.

  2. Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production.

    Science.gov (United States)

    Lewis, Johanne M; Ewart, K Vanya; Driedzic, William R

    2004-01-01

    Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.

  3. A kinetic description of antifreeze glycoprotein activity.

    Science.gov (United States)

    Burcham, T S; Osuga, D T; Yeh, Y; Feeney, R E

    1986-05-15

    The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.

  4. Saccharide antifreeze compositions

    Energy Technology Data Exchange (ETDEWEB)

    Walters, Kent; Duman, John G; Serianni, Anthony S

    2013-12-10

    The invention provides an antifreeze glycolipid compounds and composition comprising a polysaccharide moiety of Formula I; ##STR00001## wherein D-Manp represents a D-mannopyranose moiety, D-Xylp represents a D-xylopyranose moiety, and n is about 5 to about 70; and one or more lipid moieties covalently linked to the polysaccharide moiety of Formula I or electrostatically associated with the polysaccaride moiety for Formula I. The antifreeze glycolipid compounds and compositions can be used for a variety of industrial, agricultural, medical, and cosmetic applications where recrystallization-inhibition, cyroprotection, or cryopreservation is desired. The antifreeze glycolipid compounds or compositions can be used as, for example, as cryoprotectants for tissue preservation and transplantation, improving the texture of processed frozen food and frozen meats, frostbit protection, crop protection, and green alternatives for land vehicle antifreeze and aircraft de-icing.

  5. Crystallization and preliminary X-ray crystallographic analysis of Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein

    Energy Technology Data Exchange (ETDEWEB)

    Nishimiya, Yoshiyuki; Kondo, Hidemasa [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Yasui, Masanori [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Division of Biological Sciences, Graduate School of Science, Hokkaido University, N8W5, Kita, Sapporo 060-0808 (Japan); Sugimoto, Hiroshi [Biometal Science Laboratory, Riken SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Noro, Natsuko; Sato, Ryoko [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Suzuki, Mamoru [Insititute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Miura, Ai [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Tsuda, Sakae, E-mail: s.tsuda@aist.go.jp [Functional Protein Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517 (Japan); Division of Biological Sciences, Graduate School of Science, Hokkaido University, N8W5, Kita, Sapporo 060-0808 (Japan)

    2006-06-01

    Crystallization and X-ray data analyses were successful for both Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein. The resolution of the crystal was 1.35 Å for the Ca{sup 2+}-independent species, and was 1.25 and 1.06 Å for the Ca{sup 2+}-dependent species in the Ca{sup 2+}-free and -bound states, respectively. Ca{sup 2+}-independent and Ca{sup 2+}-dependent species of the type II antifreeze protein (AFP) were both crystallized using the hanging-drop vapour-diffusion method. It appeared that the crystal of the Ca{sup 2+}-independent species from Brachyosis rostratus belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 43.3, b = 48.4, c = 59.7 Å, and diffraction data were collected to 1.34 Å resolution. For the Ca{sup 2+}-dependent type II AFP species from Hypomesus nipponensis, crystallization was carried out for its Ca{sup 2+}-free and Ca{sup 2+}-bound states. 1.25 Å resolution data were collected from the crystal in the Ca{sup 2+}-free state, which exhibited P3{sub 1}21 (or P3{sub 2}21) symmetry, with unit-cell parameters a = b = 66.0, c = 50.3 Å. Data collection could be extended to 1.06 Å resolution for the crystal in the Ca{sup 2+} -bound state, which appeared to be isomorphous to the crystal in the Ca{sup 2+}-free state (unit-cell parameters a = b = 66.0, c = 49.8 Å). These data will allow us to determine the high-resolution structures of the two species of type II AFP.

  6. Antifreeze glycoprotein agents: structural requirements for activity.

    Science.gov (United States)

    Carvajal-Rondanelli, Patricio A; Marshall, Sergio H; Guzman, Fanny

    2011-11-01

    Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs. Copyright © 2011 Society of Chemical Industry.

  7. An open source cryostage and software analysis method for detection of antifreeze activity

    DEFF Research Database (Denmark)

    Lørup Buch, Johannes; Ramløv, H

    2016-01-01

    The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method....... The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing...

  8. Stable transmission and transcription of newfoundland ocean pout type III fish antifreeze protein (AFP) gene in transgenic mice and hypothermic storage of transgenic ovary and testis.

    Science.gov (United States)

    Bagis, Haydar; Aktoprakligil, Digdem; Mercan, Hande Odaman; Yurdusev, Nevzat; Turgut, Gazi; Sekmen, Sakir; Arat, Sezen; Cetin, Seyfettin

    2006-11-01

    Here we describe the generation of transgenic mice carrying type III fish antifreeze protein (AFP) gene and evaluate whether AFP type III protects transgenic mouse ovaries and testes from hypothermic storage. AFPs exist in many different organisms. In fish, AFPs protect the host from freezing at temperatures below the colligative freezing point by adsorbing to the surface of nucleating ice crystals and inhibiting their growth. The transgenic expression of AFP holds great promise for conferring freeze-resistant plant and animal species. AFP also exhibits a potential for the cryopreservation of tissues and cells. In this study, we have generated 42 founder mice harboring the Newfoundland ocean pout (OP5A) type III AFP transgene and established one transgenic line (the line #6). This study demonstrated that AFP gene construct has been stably transmitted to the mouse progeny in the F3 generations in the line #6. Furthermore, the presence of AFP transcripts was confirmed by RT-PCR analysis on cDNAs from liver, kidney, ovarian, and testis tissues of the mouse from F3 generation in this line. These results indicate that ocean pout type III AFP gene could be integrated and transmitted to the next generation and stably transcribed in transgenic mice. In histological analysis of testis and ovarian tissues of nontransgenic control and AFP transgenic mice it has been shown that both tissues of AFP transgenic mice were protected from hypothermic storage (+4 degrees C). The AFP III transgenic mice obtained for the first time in this study would be useful for investigating the biological functions of AFP in mammalian systems and also its potential role in cryopreservation.

  9. Testing antifreeze protein from the longhorn beetle Rhagium mordax as a kinetic gas hydrate inhibitor using a high-pressure micro differential scanning calorimeter

    DEFF Research Database (Denmark)

    Daraboina, Nagu; Perfeldt, Christine Malmos; von Solms, Nicolas

    2015-01-01

    pressure micro differential scanning calorimeter HP-mu DSC VII (Setaram Inc.) containing two 50 cc high pressure cells (maximum operating pressure 40 MPa; temperature range -40 to 120 degrees C) was employed to observe methane hydrate formation and decomposition in the presence of hyperactive antifreeze...

  10. Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins.

    Science.gov (United States)

    Amir, Gabriel; Rubinsky, Boris; Basheer, Sheick Yousif; Horowitz, Liana; Jonathan, Leor; Feinberg, Micha S; Smolinsky, Aram K; Lavee, Jacob

    2005-11-01

    Freeze-tolerant fish survive sub-zero temperatures by non-colligatively lowering the freezing temperature of their body fluids using anti-freeze proteins (AFPs). We sought to evaluate and compare the effects of prolonged sub-zero cryopreservation of transplanted rat hearts using AFP I or AFP III. Two heterotopic rat heart transplantation protocols were used. In Protocol 1 (n = 104), hearts (n = 8/group) were preserved for 12, 18 and 24 hours in University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C, UW/AFP I at -1.3 degrees C and UW/AFP III at -1.3 degrees C, with and without nucleation. Post-operative evaluation consisted of visual viability scoring of the hearts after 60 minutes. Protocol 2 (n = 58) involved evaluation of 24-hour post-transplant viability, echocardiography (fractional shortening [FS], left ventricular end-systolic and -diastolic diameter [ESD, EDD] and anterior and posterior wall systolic and diastolic thickness [AWT-S, AWT-D, PWT-S, PWT-D]), TUNEL staining and electron microscopy (EM) findings for hearts preserved for 18, 21 and 24 hours in UW at 4 degrees C or UW/AFP III at -1.3 degrees C. Hearts preserved in UW at -1.3 degrees C with nucleation froze and died. Three of 8 hearts preserved in UW at 4 degrees C for 24 hours died, whereas all hearts preserved at -1.3 degrees C survived. Hearts preserved in UW/AFP for 18 and 24 hours at -1.3 degrees C had superior viability scores compared with those in UW at 4 degrees C. Hearts in AFP III at -1.3 degrees C displayed greater AWT-S and AWT-D (3.5 +/- 0.2 vs 2.4 +/- 0.2, p hour preservation. In the 21-hour preservation group, AFP-treated hearts displayed improved echocardiographic systolic contraction indices, including: improved FS (27 +/- 3.7 vs 15 +/- 4, p = 0.04); diminished ESD (0.28 +/- 0.57 vs 0.47 +/- 0.6, p zero cryopreservation, AFPs protect the heart from freezing, improve survival and hemodynamics, and reduce apoptotic cell death.

  11. Hydration layer dynamics and association mechanisms of food and antifreeze proteins : A Molecular Dynamics and Transition Path Sampling study

    NARCIS (Netherlands)

    Brotzakis, Z.F.

    2017-01-01

    By the time the reader reads this line, billions of protein association events just occurred in our body, such as the ones regulating cell communication, signaling pathways, or in initiating a self-assembly processes, such as tissue fabrication, etc. The timescale of such transitions is slow,

  12. Synthetic antifreeze peptide and synthetic gene coding for its production

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  13. Antifreeze life cycle assessment (LCA

    Directory of Open Access Journals (Sweden)

    Kesić Jelena

    2005-01-01

    Full Text Available Antifreeze based on ethylene glycol is a commonly used commercial product The classification of ethylene glycol as a toxic material increased the disposal costs for used antifreeze and life cycle assessment became a necessity. Life Cycle Assessment (LCA considers the identification and quantification of raw materials and energy inputs and waste outputs during the whole life cycle of the analyzed product. The objectives of LCA are the evaluation of impacts on the environment and improvements of processes in order to reduce and/or eliminate waste. LCA is conducted through a mathematical model derived from mass and energy balances of all the processes included in the life cycle. In all energy processes the part of energy that can be transformed into some other kind of energy is called exergy. The concept of exergy considers the quality of different types of energy and the quality of different materials. It is also a connection between energy and mass transformations. The whole life cycle can be described by the value of the total loss of exergy. The physical meaning of this value is the loss of material and energy that can be used. The results of LCA are very useful for the analyzed products and processes and for the determined conditions under which the analysis was conducted. The results of this study indicate that recycling is the most satisfactory solution for the treatment of used antifreeze regarding material and energy consumption but the re-use of antifreeze should not be neglected as a solution.

  14. A nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant Alaskan beetle Upis ceramboides

    OpenAIRE

    Walters, Kent R.; Serianni, Anthony S.; Sformo, Todd; Barnes, Brian M.; Duman, John G.

    2009-01-01

    Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly a...

  15. Scientist prepare Lysozyme Protein Crystal

    Science.gov (United States)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  16. [Antifreeze glycoproteins in fishes: structure, mode of action and possible applications].

    Science.gov (United States)

    Wöhrmann, A

    1996-02-01

    Two types of antifreezes have been isolated from polar and northern temperate fishes so far. They are either glycopeptides or peptides. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 degree below the melting point before ice crystals grow. This phenomenon is called thermal hysteresis, in contrast to the normal colligative effect of solutes. All Antarctic notothenioids (perches) investigated so far have the typical antifreeze glycoproteins (AFGP) with the tripeptide Ala-Ala-Thr and the disaccharide Gal-GalNAc. In the Antarctic silverfish Pleuragramma antarcticum there could be found a novel GlcNAc containing antifreeze glycoprotein, the PAGP. The antifreezes not only lower the freezing temperature, but they also retard recrystallization on frozen storage. Antifreeze proteins thus could be useful for biotechnology and cryomedicine in the future. Since some are now synthesized chemically or by genetic engineering, they no longer have to be isolated from fish blood.

  17. Preparing and evaluating delivery systems for proteins

    DEFF Research Database (Denmark)

    Jorgensen, L; Moeller, E H; van de Weert, M

    2006-01-01

    From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...... and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge...... for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions....

  18. Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides.

    Science.gov (United States)

    Ebbinghaus, Simon; Meister, Konrad; Prigozhin, Maxim B; Devries, Arthur L; Havenith, Martina; Dzubiella, Joachim; Gruebele, Martin

    2012-07-18

    Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Fractionation of liver proteins by preparative electrophoresis.

    Science.gov (United States)

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

    2004-02-01

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

  20. Antifreeze Glycoproteins Alter the Molecular Scale Surface Morphology of Ice

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine A.; Qiu, Roger; Yeh, Yin

    2003-03-01

    Trematomas borchgrevinki live in the harsh super-cooled waters of the Antarctic. Critical to their survival are antifreeze glycoproteins (AFGPs) that further suppress the freezing temperature of their blood serum in addition to the colligative action of salts found in the ocean. These proteins also modify ice crystal growth habits as well as inhibit recrystallization in polycrystalline ice. To date many other types of antifreeze proteins have been identified in cold weather insects, plants, and other fish, but the exact mechanism is not entirely understood. The mechanism is non-colligative since only a few mg/ml are required for ice crystal growth inhibition and a non-equilibrium melting/freezing point hysteresis is observed. Atomic force microscopy (AFM) can yield a wealth of surface information that can reveal molecular scale information of biomineralization processes. We use AFM to directly probe the surface of ice crystals grown from the vapor in the pure phase and in the presence of growth inhibitors/modifiers, AFGPs. Results show that the AFGPs heavily pin the surface of ice.

  1. Ice growth in supercooled solutions of antifreeze glycoprotein.

    Science.gov (United States)

    Harrison, K; Hallett, J; Burcham, T S; Feeney, R E; Kerr, W L; Yeh, Y

    Inhibition of ice growth in supercooled solution by certain proteins is vital to the survival of many living organisms. Some fish, native to both subzero northern and southern waters, have special proteins or glycoproteins in their blood serum that inhibit ice formation. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 K below the melting point before ice crystals grow. This phenomenon is called freezing hysteresis, in contrast to the normal colligative effect of solutes that depresses the equilibrium temperature, around which small changes lead to crystal growth or melting depending on sign. Some insects also exhibit a serum freezing hysteresis. We report the effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of these antifreeze glycoproteins (AFGPs). We find that the crystallization rate is up to five times greater than that in pure water.

  2. Efficiency of Composite Binders with Antifreezing Agents

    Science.gov (United States)

    Ogurtsova, Y. N.; Zhernovsky, I. V.; Botsman, L. N.

    2017-11-01

    One of the non-heating methods of cold-weather concreting is using concretes hardening at negative temperatures. This method consists in using chemical additives which reduce the freezing temperature of the liquid phase and provide for concrete hardening at negative temperatures. The non-heating cold-weather concreting, due to antifreezing agents, allows saving heat and electric energy at the more flexible work performance technology. At selecting the antifreezing components, the possibility of concreting at temperatures up to minus 20 °C and combination with a plasticizer contained in the composite binder were taken into account. The optimal proportions of antifreezing and complex agents produced by MC-Bauchemie Russia for fine-grained concretes were determined. So, the introduction of antifreezing and complex agents allows obtaining a structure of composite characteristic for cement stone in the conditions of below zero temperatures at using different binders; the hydration of such composite proceeded naturally. Low-water-demand binders (LWDB) based composites are characterized by a higher density and homogeneity due to a high dispersity of a binder and its complicated surface providing for a lot of crystallization centers. LWDB contains small pores keeping water in a liquid form and promoting a more complete hydration process.

  3. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis

    OpenAIRE

    Wilkins, S. P.; Blum, A. J.; Burkepile, D. E.; Rutland, T. J.; Wierzbicki, A.; Kelly, M.; Hamann, M.T.

    2002-01-01

    Polar plants and animals survive in subzero waters (−2°C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. T...

  4. Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers.

    Science.gov (United States)

    Nagel, Lilly; Plattner, Carolin; Budke, Carsten; Majer, Zsuzsanna; DeVries, Arthur L; Berkemeier, Thomas; Koop, Thomas; Sewald, Norbert

    2011-08-01

    In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4-55) of [AAT]( n )-repeating units. The threonine side chain of each unit is glycosidically linked to β-D: -galactosyl-(1 → 3)-α-N-acetyl-D: -galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.

  5. Synthesis of Structures Related to Antifreeze Glycoproteins

    OpenAIRE

    Fyrner, Timmy

    2005-01-01

    In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

  6. De novo gene evolution of antifreeze glycoproteins in codfishes revealed by whole genome sequence data.

    Science.gov (United States)

    Baalsrud, Helle Tessand; Tørresen, Ole Kristian; Hongrø Solbakken, Monica; Salzburger, Walter; Hanel, Reinhold; Jakobsen, Kjetill S; Jentoft, Sissel

    2017-12-05

    New genes can arise through duplication of a pre-existing gene or de novo from non-coding DNA, providing raw material for evolution of new functions in response to a changing environment. A prime example is the independent evolution of antifreeze glycoprotein genes (afgps) in the Arctic codfishes and Antarctic notothenioids to prevent freezing. However, the highly repetitive nature of these genes complicates studies of their organization. In notothenioids, afgps evolved from an extant gene, yet the evolutionary origin of afgps in codfishes is unknown. Here, we demonstrate that afgps in codfishes have evolved de novo from non-coding DNA 13-18 Ma, coinciding with the cooling of the Northern Hemisphere. Using whole-genome sequence data from several codfishes and notothenioids, we find higher copy number of afgp in species exposed to more severe freezing suggesting a gene dosage effect. Notably, antifreeze function is lost in one lineage of codfishes analogous to the afgp losses in non-Antarctic notothenioids. This indicates that selection can eliminate the antifreeze function when freezing is no longer imminent. Additionally, we show that evolution of afgp-assisting antifreeze potentiating protein genes (afpps) in notothenioids coincides with origin and lineage-specific losses of afgp. The origin of afgps in codfishes is one of the first examples of an essential gene born from non-coding DNA in a non-model species. Our study underlines the power of comparative genomics to uncover past molecular signatures of genome evolution, and further highlights the impact of de novo gene origin in response to a changing selection regime. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Enrichment of low-abundance brain proteins by preparative electrophoresis.

    Science.gov (United States)

    Fountoulakis, Michael; Juranville, Jean François

    2003-02-15

    Detection of low-copy-number gene products is essential for the development of novel drugs, however, it represents a major drawback of proteomics and simultaneously a scientific challenge. We studied the enrichment of rat brain cytosolic proteins by preparative electrophoresis using the PrepCell apparatus. The electrophoresis was performed in the presence of 0.1% lithium dodecyl sulfate. The proteins eluted from the gel were analyzed by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption ionization mass specrometry. Lithium dodecyl sulfate was easily exchanged against agents compatible with isoelectric focusing. Low-abundance proteins, which had not been found before, including neuronal-specific and calcium-binding proteins, were detected. In particular, low-molecular-mass proteins, such as hippocalcin, visinin-like proteins, and 14-3-3 proteins were strongly enriched by preparative electrophoresis.

  8. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  9. An automatic refolding apparatus for preparative-scale protein production.

    Directory of Open Access Journals (Sweden)

    Yanye Feng

    flexible strategy may provide a powerful tool for preparative scale protein production.

  10. Effects of preheating on ice growth in antifreeze polypeptides solutions in a narrow space

    Science.gov (United States)

    Miyamoto, T.; Nishi, N.; Waku, T.; Tanaka, N.; Hagiwara, Y.

    2016-09-01

    We conducted measurements on the unidirectional freezing of aqueous solutions of polypeptide or of winter flounder antifreeze protein. The polypeptide was based on a part of the antifreeze protein. We measured temperatures in the solutions and ice with a small thermocouple, and defined the interface temperature as the temperature at the tip of the serrated or pectinate interface. It was found that the interface temperature of these solutions was lower than that of pure water. To vary the activity of these solutes, we preheated the solutions and cooled them before conducting the measurements. We found that preheating for several hours caused further decreases in the interface temperature and a decrease in the interface velocity. In addition, the inclined interfaces became wider as a result of the preheating. Thus, the supercooled states in the solutions were enhanced by the preheating. To investigate the reasons for these changes, we measured the aggregates of the solutes in the solutions. These aggregates became larger as a result of preheating. It can therefore be concluded that these large aggregates attenuated the ice growth by their interaction with the ice surfaces.

  11. Lipid oxidation in omega-3 emulsions prepared with milk proteins

    DEFF Research Database (Denmark)

    Horn, Anna Frisenfeldt; Nielsen, Nina Skall; Andersen, Ulf

    An increasing body of evidence supports the health beneficial effects of omega-3 polyunsaturated fatty acids. Therefore, incorporation of marine oils into foods has also gained an increasing interest. However, the highly unsaturated lipids present in marine oils are prone to lipid oxidation....... The properties of the emulsifier used and the structure at the interface are therefore expected to be of great importance for oxidation in emulsions. This presentation will include results from mainly three different studies of lipid oxidation in omega-3 emulsions prepared with milk proteins and protein...

  12. Renal conservation of antifreeze peptide in Antarctic eelpout, Rhigophila dearborni.

    Science.gov (United States)

    Eastman, J T; De Vries, A L; Coalson, R E; Nordquist, R E; Boyd, R B

    1979-11-08

    In Antarctic notothenioid fishes large amounts (3% w/v) of small molecular weights of 2,600-23,500 and would be expected to be filtered into the urine, they remain in the blood because the kidneys of these fishes contain only aglomerular nephrons. Unlike the situation in most fishes, urine formation is the result of secretion rather than filtration and reabsorption. On the other hand, the peptide antifreezes in Northern Hemisphere fishes such as the winter flounder. Pseudopleuronectes americanus, are retained by the glomerular kidney even though inulin, of comparable weight, is rapidly filtered from the blood into the urine. The Antarctic eelpout (zoarcid), Rhigophila dearborni, which is unrelated to either the Antarctic notothenioids or P. americanus, also uses a peptide antifreeze (molecular weight 6,000) which is maintained at a concentration of 3% (w/v) in the blood plasma. We report here that the lack of antifreeze in the urine of R. dearborni probably reflects the fact that the glomeruli are not functional and cannot filter. We support this conclusion with morphological and physiological evidence and relate our findings to the conservation of biological antifreeze necessary for life in ice-laden polar waters.

  13. Preparation of encapsulated proteins dissolved in low viscosity fluids

    International Nuclear Information System (INIS)

    Ehrhardt, Mark R.; Flynn, Peter F.; Wand, A. Joshua

    1999-01-01

    The majority of proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. One potential approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques is to encapsulate them in a reverse micelle which is dissolved in a low viscosity fluid. Unfortunately, promising low viscosity fluids such as the short chain alkanes, supercritical carbon dioxide, and various halocarbon refrigerants all require the application of significant pressure to be kept liquefied at room temperature. Here we describe the design and use of a simple cost effective NMR tube suitable for the preparation of solutions of proteins encapsulated in reverse micelles dissolved in such fluids

  14. RAPID TEST METHOD FOR EVALUATION OF ANTIFREEZE ADDITIVE EFFICIENCY

    Directory of Open Access Journals (Sweden)

    S. V. Gushchin

    2015-01-01

    Full Text Available Usage of chemical additives while executing concrete works at negative temperatures is considered as a convenient and economical method. Range of the used antifreeze additives is rather wide. A great number of new additives are advertised but their characteristics have not been practically studied. Evaluation of the antifreeze additive efficiency is unfortunately rather long process and it does not provide comprehensive data on concrete structure formation processes. Due to this development of rapid and comprehensive methodology for construction companies is urgently required.Freezing processes of antifreeze additive aqueous solutions and hardening of cement paste with them have been investigated in the paper. The paper proposes a methodology for determination of freezing point for aqueous solutions of chemical additives of various applications. Identity of  freezing point for a chemical additive aqueous solution and cement paste with an equal concentration of the additive in the paste pore fluid has been determined while taking  calcium nitrate and sodium formate additives as an example. The paper demonstrates the possibility to evaluate efficiency of antifreeze additive action on the basis of kinetics in temperature changes of the cement paste with additives by its consecutive freezing and defrosting.  A methodology for operational evaluation in the field of chemical additive application for concreting items at negative temperatures has been offered in the paper.  The methodology does not require  deficient and expensive test-equipment. It can be applied at ordinary construction companies and it is comprehensible for personnel of low-qualification.  The paper shows the possibility to develop an original methodology for designing concrete structure which is based on operating efficiency determinations  for single and integrated antifreeze additives.

  15. Fragrance release profile from sonochemically prepared protein microsphere containers.

    Science.gov (United States)

    Tzhayik, Oshrat; Cavaco-Paulo, Artur; Gedanken, Aharon

    2012-07-01

    Protein microspheres have been prepared by sonicating a mixture of pure fragrant oil (amyl acetate (AA)) with an aqueous protein (bovine serum albumin) solution. The prepared protein spheres are nano- to micrometer sized with an encapsulation efficiency of approx. 97% for the AA present on the surface and inside the BSA capsule. Containers were found stable for more than 6 months when stored sealed at 4°C and 20°C. For the release profile measurements, we used a simple, automated and direct method. We continuously weighed the encapsulated microspheres and measured the evaporation rates. The release profiles at 15°C and 25°C display two different evaporation rates. The higher rate is the sum of a few evaporation rates, including water molecules, while the slower rate is due to the evaporation of pure AA. The changes in the evaporation rates occur upon the collapse of the container. This event coincides with the full evaporation of water. For morphological characterization we dyed the AA with Nile red, and used SEM, ESEM, Cryo-SEM, light microscopy, and confocal laser scanning microscopy measurements. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Isolation of an antifreeze peptide from the Antarctic sponge Homaxinella balfourensis.

    Science.gov (United States)

    Wilkins, S P; Blum, A J; Burkepile, D E; Rutland, T J; Wierzbicki, A; Kelly, M; Hamann, M T

    2002-12-01

    Polar plants and animals survive in subzero waters (-2 degrees C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry.

  17. Potential Antifreeze Compounds in Present-Day Martian Seepage Groundwater

    Directory of Open Access Journals (Sweden)

    Jiin-Shuh Jean

    2008-01-01

    Full Text Available Is the recently found seepage groundwater on Mars pure H2O, or mixed with salts and other antifreeze compounds? Given the surface conditions of Mars, it is unlikely that pure water could either exist in its liquid state or have shaped Mars¡¦ fluid erosional landforms (gullies, channels, and valley networks. More likely is that Mars¡¦ seepage groundwater contains antifreeze and salt compounds that resist freezing and suppress evaporation. This model better accounts for Mars¡¦ enigmatic surface erosion. This paper suggests 17 antifreeze compounds potentially present in Martian seepage groundwater. Given their liquid state and physical properties, triethylene glycol, diethylene glycol, ethylene glycol, and 1,3-propylene glycol are advanced as the most likely candidate compounds. This paper also explores how a mixing of glycol or glycerol with salts in the Martian seepage groundwater may have lowered water¡¦s freezing point and raised its boiling point, with consequences that created fluid gully and channel erosion. Ethylene glycol and related hydrocarbon compounds have been identified in Martian and other interstellar meteorites. We suggest that these compounds and their proportions to water be included for detection in future explorations.

  18. Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. Influence of preparation techniques on particle characteristics and protein delivery.

    Science.gov (United States)

    Bezemer, J M; Radersma, R; Grijpma, D W; Dijkstra, P J; van Blitterswijk, C A; Feijen, J

    2000-07-03

    The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent extraction from a water-in-oil (w/o) emulsion in ethanol or methanol did not result in the formation of microspheres, due to massive polymer precipitation caused by rapid solvent extraction in these non-solvents. In a second process, microspheres were first prepared by a water-in-oil-in-water (w/o/w) emulsion system with 4% poly(vinyl alcohol) (PVA) as stabilizer in the external phase, followed by extraction of the remaining solvent. As non-solvents ethanol, methanol and mixtures of methanol and water were employed. However, the use of alcohols in the extraction medium resulted in microspheres which gave an incomplete lysozyme release at a non-constant rate. Complete lysozyme release was obtained from microspheres prepared by an emulsification-solvent evaporation method in PBS containing poly(vinyl pyrrolidone) (PVP) or PVA as stabilizer. PVA was most effective in stabilizing the w/o/w emulsion. Perfectly spherical microspheres were produced, with high protein entrapment efficiencies. These microspheres released lysozyme at an almost constant rate for approximately 28 days. The reproducibility of the w/o/w emulsion process was demonstrated by comparing particle characteristics and release profiles of three batches, prepared under similar conditions.

  19. Production of protein hydrolysates from fish byproduct prepared by enzymatic hydrolysis

    OpenAIRE

    Murna Muzaifa; Novi Safriani; Fahrizal Zakaria

    2012-01-01

    The objective of this research was to study the production of fish protein hydrolysate (FPH) from fish by-product prepared by enzymatichydrolysis. Fish by-product were prepared using Alcalase and Flavourzyme enzyme and properties of FPH were analyzed. The resultsshowed that FPH prepared using Alcalase enzyme had greater amount of protein (82.66%) than FPH prepared using Flavourzyme enzyme(73.51%). Solubility, emulsifying and foaming properties of FPH prepared using Alcalase were also better t...

  20. SynProt: A Database for Proteins of Detergent-Resistant Synaptic Protein Preparations

    Science.gov (United States)

    Pielot, Rainer; Smalla, Karl-Heinz; Müller, Anke; Landgraf, Peter; Lehmann, Anne-Christin; Eisenschmidt, Elke; Haus, Utz-Uwe; Weismantel, Robert; Gundelfinger, Eckart D.; Dieterich, Daniela C.

    2012-01-01

    Chemical synapses are highly specialized cell–cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design. PMID:22737123

  1. Protein preparations in the development of media fragrances in technology of food products

    Directory of Open Access Journals (Sweden)

    I. N. Tolpigina

    2013-01-01

    Full Text Available Investigated the sorption properties of plant and animal proteins, common on the Russian market. Installed the recommended dosage of the preparations of animal protein and vegetable origin, as well as the biological value.

  2. Preparations and mechanical properties of low proteins radiation vulcanised natural rubber latex (RVNRL)

    International Nuclear Information System (INIS)

    Wan Manshol Wan Zin; Chai Chee Keong

    2000-01-01

    Low proteins latex can be used in RVNRL preparation. Either Formulation Rll, uses single sensitiser or Formulation Rlll, uses a combination of sensitisers are useful in low proteins RVNRL preparation. However, these RVNRL formulations produce film vulcanisates of different mechanical properties and accelerated ageing resistance

  3. Anti-freeze proteins: characteristics, mechanism of action and applications

    NARCIS (Netherlands)

    Stolle-Smits, T.; Kreuwels, M.J.J.; Wichers, H.J.

    1998-01-01

    Antivrieseiwitten hebben de eigenschap om het vriespunt te verlagen en rekristallisatie van ijs tegen te gaan. Deze eiwitten komen voor in poolvissen, insekten, planten en micro-organismen. Bovendien geven ze de ijskristallen een andere morfologie

  4. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    1. Acaenamagellanica. Prickly burr. Doucet et al. 2000. 2. Acer saccharoides. Maple. Doucet et al. 2000. 3. Agrostistenuis. Creeping bentgrass. Doucet et al. 2000. 4. Alliarapetiolata. Garlic mustard. Urrutia et al. 1992. 5. Ammopiptanthusmongolicus. Evergreen legume. Wang et al. 2003. 6. Aster cordifolius. Wood aster.

  5. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    organs.12 Chao et al13 have reported the relative effi- cacy of AFP types I, II and III in protecting the red blood cells. Numerous structure and function studies ..... Table 7. PDB templates (first 2 hits with maximum % identity) obtained using BLASTP search against the Pro- tein Data Bank. Accession number. PDB code.

  6. Studies on new antifreeze protein from the psychrophilic diatom ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... forward primer and XhoI to reverse primer for cloning into pET vector. Bacterial and plant expressions. PCR products were analyzed and resolved on 1% agarose gel. (w/v). Bands with expected lengths were excised from the gel with razor blade and purified using the QIAquick gel extraction kit. (Qiagen, CA ...

  7. Antifreeze proteins enable plants to survive in freezing conditions

    Indian Academy of Sciences (India)

    Common name. References. 28. Hordeumvulgare. Barley. Antikainen and Griffith 1997. 29. Hydrophyllumvirginianum. Virginia waterleaf. Urrutia et al. 1992. 30. Juncussquarrosus. Heath rush. Doucet et al. 2000. 31. Loliumperenne. Perennial ryegrass. Sidebottom et al. 2000; Pudney et al. 2003. 32. Northofagusantarctica.

  8. Structure/Function Studies of Insect Antifreeze Proteins

    National Research Council Canada - National Science Library

    Duman, John

    1997-01-01

    ...-x5-x6-Cys-X8-X9-Ala-X11-Th-X13 where X3 and X1 tend toward charged residues, X5 toward threonine or serine, X9 toward asparagine or aspartate, X6 toward asparagine or lysine, and X13 toward alanine...

  9. Hierarchical polymer brush nanoarrays: a versatile way to prepare multiscale patterns of proteins.

    Science.gov (United States)

    Li, Yunfeng; Zhang, Junhu; Liu, Wendong; Li, Daowei; Fang, Liping; Sun, Hongchen; Yang, Bai

    2013-03-01

    This paper presents a versatile way to prepare multiscale and gradient patterns of proteins. The protein patterns are fabricated by conjugating proteins covalently on patterns of polymer brush that are prepared by techniques combining colloidal lithography with photolithography, and two-step colloidal lithography. Taking advantages of this technique, the parameters of protein patterns, such as height, diameters, periods, and distances between two dots, can be arbitrarily tuned. In addition, the protein patterns with varies of architectures, such as microdiscs, microstripes, microrings, microtriangles, microgrids, etc., consisting of protein nanodots, are prepared and the sample size is up to 4 cm(2). The as-prepared patterns of fibronectin can promote the cell adhesion and cell location.

  10. Preparation of monoclonal antibodies against radiation-induced protein

    International Nuclear Information System (INIS)

    Nozawa, R.; Tanaka, A.; Watanabe, H.; Kitayama, S.

    1992-01-01

    We obtained the 6 monoclonal antibodies against gamma-induced proteins of Deinococcus radiodurans, and these antibodies were designated as Mab-3F, 4B, 4D, 4F, 4G and 12G. Using these antibodies, we investigated the relations between gamma-induced proteins and other stress protein in strain R1, and the induction of proteins were compared among strain R1, resistant mutant (rec1) and radiosensitive mutant (rec30). We found new 6 proteins recognized by these monoclonal antibodies which were induced after gamma-irradiation especially in strain R1 and rec 1, but not induced in strain rec30. We suppose that these proteins participate in repair of DNA damages including double strand breaks caused by gamma-irradiation. One of them was around 46kDa protein band recognized by Mab-12G, and this protein was so induced in a large quantity after irradiation that the protein could detect by gold staining. In addition to this observation, we found some proteins which were induced in R1 and rec 1 by gamma-irradiation and other stress, but not in strain rec30, such as 31kDa protein band recognized by Mab-3F, 4B and 4G, and other 11 proteins which were especially induced in irradiated strain R1. The latter proteins might be reinforcement factor to radioresistance such as GroE and DnaK, or participant in repair of damage by gamma-irradiation in strain R1. (author)

  11. Preparation of functional lupine protein fractions by dry separation

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Berghout, J.A.M.; Goot, van der A.J.; Boom, R.M.; Schutyser, M.A.I.

    2014-01-01

    Lupine protein concentrate is a promising ingredient that can be obtained by a combination of milling and air classification, generally called dry fractionation. This is a more sustainable route than conventional wet extraction and delivers a protein concentrate with native functional properties.

  12. Protein concentrates from defatted rice bran: preparation and characterization

    Directory of Open Access Journals (Sweden)

    Inajara Beatriz Brose PIOTROWICZ

    2017-10-01

    Full Text Available Abstract The goal of this work is to determine the optimal conditions for the obtaining of protein concentrates from rice bran. The effects of defatting processes applied to this product in laboratory and in industrial scales were investigated. Through two experimental designs were performed, and production conditions to obtain the protein concentrates were chosen with values of protein contents average 59.9% and 57.1%, with protein yields of 61.6% and 30.7%. The concentrates produced with industrially defatted rice bran showed higher digestibility and increased thermal stability compared with the product obtained with laboratory defatted rice bran. Both concentrates show molecular weight proteins below 50 kDa. The morphology of the precipitated proteins, analyzed by scanning electron microscopy, there was a great difference in the size of particles, which form the wet precipitates. The differences presented by the concentrates can be due to defatting processes which raw materials were submitted. Thus, studying the protein extraction conditions and knowing its characteristics is very important for the industry, because food processing requires knowing the behavior of each compound during and after processes they will be submitted.

  13. Preparation, characterization and functional properties of flax seed protein isolate.

    Science.gov (United States)

    Kaushik, Pratibha; Dowling, Kim; McKnight, Stafford; Barrow, Colin J; Wang, Bo; Adhikari, Benu

    2016-04-15

    Flaxseed protein isolate (FPI) was extracted from flaxseeds, and its amino acid composition and functional properties (solubility, thermal stability, emulsifying properties and electrostatic charge density, water holding and fat absorption capacities) were determined. The highest purity of FPI (90.6%) was achieved by extraction at 60°C. FPI had a low lysine to arginine ratio of 0.25, which is desired in heart-healthy foods and infant formulas. The denaturation temperature of FPI was 105°C. FPI had the highest emulsion activity index (375.51 m(2)/g), highest emulsion stability index (179.5 h) and zeta potential (-67.4 mV) when compared to those of other commonly used proteins, such as sodium caseinate (SC), whey protein isolate (WPI), gelatin (Gel) and soy protein isolate (SPI). The average emulsion droplet size of emulsions stabilized by these proteins was in the order SCproteins. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  14. Inhibition of Condensation Frosting by Arrays of Hygroscopic Antifreeze Drops.

    Science.gov (United States)

    Sun, Xiaoda; Damle, Viraj G; Uppal, Aastha; Linder, Rubin; Chandrashekar, Sriram; Mohan, Ajay R; Rykaczewski, Konrad

    2015-12-29

    The formation of frost and ice can have negative impacts on travel and a variety of industrial processes and is typically addressed by dispensing antifreeze substances such as salts and glycols. Despite the popularity of this anti-icing approach, some of the intricate underlying physical mechanisms are just being unraveled. For example, recent studies have shown that in addition to suppressing ice formation within its own volume, an individual salt saturated water microdroplet forms a region of inhibited condensation and condensation frosting (RIC) in its surrounding area. This occurs because salt saturated water, like most antifreeze substances, is hygroscopic and has water vapor pressure at its surface lower than water saturation pressure at the substrate. Here, we demonstrate that for macroscopic drops of propylene glycol and salt saturated water, the absolute RIC size can remain essentially unchanged for several hours. Utilizing this observation, we demonstrate that frost formation can be completely inhibited in-between microscopic and macroscopic arrays of propylene glycol and salt saturated water drops with spacing (S) smaller than twice the radius of the RIC (δ). Furthermore, by characterizing condensation frosting dynamics around various hygroscopic drop arrays, we demonstrate that they can delay complete frosting over of the samples 1.6 to 10 times longer than films of the liquids with equivalent volume. The significant delay in onset of ice nucleation achieved by dispensing propylene glycol in drops rather than in films is likely due to uniform dilution of the drops driven by thermocapillary flow. This transport mode is absent in the films, leading to faster dilution, and with that facilitated homogeneous nucleation, near the liquid-air interface.

  15. DSCG binding protein and process for preparing same

    Energy Technology Data Exchange (ETDEWEB)

    Pecht, I.; Mazurek, N.

    1987-07-28

    An essentially pure protein is described consisting essentially of the protein, (CBP), present in nature in membranes of basophile cells and in mast cells, having a molecular weight of about 60,000 +- 2,000 determined by SDS polyacrylamide electrophoresis an isoelectric point of about 3.9 and an amino acid composition of about 4 units of asparagine, 3 units of threonine and serine, 3 units glycine, 2 units alanine, 2 units proline, 1 unit cysteine, 2 units valine, 1 unit methionine, 1 unit isoleucine, 2 units leucine, 1 unit tyrosine, 1 unit phenylalanine, 2 units histamine, 2 units lysine and 1 unit arginine. The protein is able to build calcium and having a calcium dependent affinity to the disodium salt of 1,2 bis(-2 carboxychromon-5-yloxy)-2-hydroxy propane (DSCG).

  16. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption.

    Directory of Open Access Journals (Sweden)

    Kei Fujiwara

    Full Text Available Cell-free protein synthesis (CFPS is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20-30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL.

  17. Efficient preparation and analysis of membrane and membrane protein systems

    Czech Academy of Sciences Publication Activity Database

    Javanainen, M.; Martinez-Seara, Hector

    2016-01-01

    Roč. 1858, č. 10 (2016), s. 2468-2482 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : tools and software * membrane building * protein insertion * molecular dynamics * lipid bilayer Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2016

  18. FT-IR Spectra of Antifreeze Glycoproteins in Heavy Water and D2O Ice.

    Science.gov (United States)

    Tsvetkova, N. M.; Crowe, J. H.; Feeney, R. H.; Fink, W. H.; Yeh, Yin

    2000-03-01

    This work presents FT-IR studies on the antifreeze glycoprotein (AFGP)/heavy water (D2O) mixtures during freezing and melting. AFGP in the blood serum of polar fish are known to prevent ice crystal growth by a non-colligative mechanism. There are 8 known fractions of AFGP (1 8) that range in molecular mass from 33.7 to 2.6 kD respectively, each composed of alanine-alanine-threonine repeats, with a disaccharide attached to the threonine residue. The smallest peptide (AFGP-8) is structurally different from fractions 1-5 in that it contains proline substituting for alanine in certain positions. Substantial linewidth change of the D20 bending mode (ca. 1210 cm-1) was measured with solutions containing fractions 2-5 during both freezing and thawing cycles, suggesting significant coupling between protein and water molecules. At the same time, the Amide I band between 1620 and 1675 cm-1 shows that 310 helix and random coils are the main conformations of fractions 2-5 and fraction 8 in the presence of ice. In liquid state, b-sheet dominates the secondary structure of AFGP 8, whereas b-sheet and random coil are the main conformations of AFGP 2-5. These results are discussed in terms of the ability of AFGP 2-5 to affect the surface states of ice.

  19. Processing of precursor proteins by preparations of oviduct microsomes.

    Science.gov (United States)

    Thibodeau, S N; Walsh, K A

    1980-01-01

    The signal peptidases from both chicken oviduct and dog pancreas microsomes have been detected by a post-translational assay and, in the case of dog microsomes, by a cotranslational assay. This proteolytic activity is not sensitive to a variety of conventional protease inhibitors under the conditions used in these assays. Using a synthetic ester substrate, two additional activities have been observed in oviduct microsomes, but neither appears to correspond to the signal peptidase activity. Using cDNA as a probe for specific mRNAs, it was shown that polysomes, in the process of synthesizing secretory proteins, bind to microsomal membrane vesicles. This binding appears to be dependent on the nature of the translation product (presumably the specific signal peptide) rather than on the mRNA itself. In addition, pretreatment of the membrane vesicles with N-ethyl maleimide prevents the vectorial discharge and processing of several secretory proteins but does not inhibit the binding of their polysomes to membranes.

  20. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

  1. Preparation of denatured protein bone sterilized with gamma radiation

    International Nuclear Information System (INIS)

    Luna Z, D.

    2005-01-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  2. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    Science.gov (United States)

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from "Escherichia coli" inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This 7-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies,…

  3. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Science.gov (United States)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  4. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

    NARCIS (Netherlands)

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-01-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI

  5. Preparation and characterization of protein isolate from glandless and glanded cottonseed

    Science.gov (United States)

    Protein isolates were prepared from cottonseed meals recovered from both glandless and glanded cottonseed. Isolate yield was 2.5-fold greater from the glandless meal than from the glanded meal, indicating that the gossypol in the glanded meal was likely promoting the formation of protein aggregates...

  6. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    multiplexing readouts, but this has a natural limitation. High-content screening via image acquisition and analysis allows multiplexing of few parameters, but is connected to substantial time consumption and complex logistics. We report on integration of Reverse Phase Protein Arrays (RPPA)-based readouts...... into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high...

  7. Preparation and characterization of a thermoresponsive gigaporous medium for high-speed protein chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Jian-Bo, E-mail: jbqu@upc.edu.cn [State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580 (China); Chen, Yan-Li; Huan, Guan-Sheng [State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580 (China); Zhou, Wei-Qing [National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Liu, Jian-Guo; Zhu, Hu [State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580 (China); Zhang, Xiao-Yun, E-mail: zhangxy@upc.edu.cn [State Key Laboratory of Heavy Oil Processing, Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580 (China)

    2015-01-01

    Highlights: • A high-speed thermoresponsive bioseparation medium was prepared in two steps. • Non-specific adsorption of proteins on thermoresponsive medium was greatly reduced. • Separation of proteins was achieved by only adjusting column temperature. • It was able to separate proteins at the mobile phase velocity up to 2167 cm h{sup −1}. - Abstract: A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS–P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS–P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h{sup −1}. In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in ‘green’ high-speed preparative protein chromatography.

  8. Proteomic analysis of the mouse brain following protein enrichment by preparative electrophoresis.

    Science.gov (United States)

    Xixi, Elena; Dimitraki, Ploumisti; Vougas, Kostantinos; Kossida, Sofia; Lubec, Gert; Fountoulakis, Michael

    2006-04-01

    Proteomics is a powerful technology to study the identity and levels of brain proteins. Changes of protein levels as well as modifications that occur in neurological disorders may be informative for the pathogenesis of these disorders and could result in the identification of potential drug targets and disease markers. To increase the capability of characterizing complex protein profiles, protein mixtures should be separated into simpler fractions, thus increasing the likelihood of detecting low-abundance proteins. Considering that low-abundance proteins are thought to be involved in important biological processes, identification of those low-copy-number gene products appears to be a scientific challenge. In the present study, proteomic analysis of adult mouse brain tissue was performed following enrichment by preparative electrophoresis. This was performed using the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Samples were electrophoresed in a cylindrical polyacrylamide gel and the proteins of the fractions collected were first analyzed by 1-D and then by 2-DE. Protein identification was performed by MALDI-TOF-MS. The present analysis resulted in the identification of 360 different gene products. Among those were transport proteins, transcription activators, signal transduction molecules as well as proteins with a number of other functions. Preparative electrophoresis is an efficient method for the enrichment of proteins of low molecular mass and may be useful in the investigation of disorders of the central nervous system.

  9. A novel preparation technique of red (sparkling wine for protein analysis

    Directory of Open Access Journals (Sweden)

    Elisabeth I. Vogt

    2016-06-01

    Full Text Available Despite their low concentration, proteins can influence several key enological parameters such as foam stability or haze formation in (sparkling wine. Most studies focus on white (sparkling wine since the higher content of phenolic compounds in red wines impairs proteomic research. The aim of the study was the development of a method for the preparation of red (sparkling wine proteins for proteomic analysis. Three methods of sample preparation were assessed on silver stained SDS-PAGE gels and with MALDI-TOF MS. Our new method was highly suitable for the preparation of proteins for the aforementioned applications. The results showed a substantial increase in signal intensity with a simultaneous decrease in background noise. The preparation protocol consists of (i dialysis and freeze drying of the sample, (ii removal of phenolic compounds by water-saturated phenol and (iii protein precipitation by addition of ammonium acetate. Employment of this method followed by SDS-PAGE analysis allowed for silver stained gels with diminished background or streaking and clearly resolved protein bands. Analysis of spectra obtained from samples prepared according to the proposed protocol showed increased intensity and signal-to-noise ratio in MALDI-TOF MS. Furthermore it was demonstrated that this method can be applied to various kinds of grape products.

  10. Preparation, characterization, and NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

    International Nuclear Information System (INIS)

    Babu, Charles R.; Flynn, Peter F.; Wand, A. Joshua

    2003-01-01

    Encapsulating a protein in a reverse micelle and dissolving it in a low-viscosity solvent can lower the rotational correlation time of a protein and thereby provides a novel strategy for studying proteins in a variety of contexts. The preparation of the sample is a key element in this approach and is guided by a number of competing parameters. Here we examine the applicability of several strategies for the preparation and characterization of encapsulated proteins dissolved in low viscosity fluids that are suitable for high performance NMR spectroscopy. Ubiquitin is used as a model system to explore various issues such as the homogeneity of the encapsulation, characterization of the hydrodynamic performance of reverse micelles containing protein molecules, and the effective pH of the water environment of the reverse micelle

  11. Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

    Science.gov (United States)

    Shilpashree, B G; Arora, Sumit; Sharma, Vivek; Bajaj, Rajesh Kumar; Tomar, S K

    2016-04-01

    Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (Piron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (Piron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Protein distribution in lupin protein isolates from Lupinus angustifolius L. prepared by various isolation techniques.

    Science.gov (United States)

    Muranyi, Isabel S; Volke, Daniela; Hoffmann, Ralf; Eisner, Peter; Herfellner, Thomas; Brunnbauer, Markus; Koehler, Peter; Schweiggert-Weisz, Ute

    2016-09-15

    Differences in the protein distribution of various protein isolates from Lupinus angustifolius L. Vitabor were identified as affected by the isolation procedure (alkaline and/or salt-induced extraction followed by isoelectric and/or dilutive precipitation). Protein isolates extracted in alkaline solution showed higher protein yields (26.4-31.7%) compared to salt-induced extraction (19.8-30.0%) or combined alkaline and salt-induced extraction (23.3-25.6%). Chemical variations among the protein isolates especially occurred within the albumins. Protein isolates precipitated isoelectrically showed the highest contents, whereas protein isolates precipitated by dilutive showed the lowest contents of conglutin δ. Furthermore, the alkaline subunits of conglutin α and conglutin γ decreased during alkaline extraction compared to salt-induced extraction. A decrease in protein-bound polar and basic amino acids was shown after protein isolation. In contrast, the amounts of nonpolar, aliphatic, aromatic, hydroxylated and sulfur-rich amino acids were higher in the lupin protein isolates compared to the lupin flakes. However, the functional side chains could not be related to the specific molecular arrangements of the protein isolates, as a similar amino acid composition was found among the protein isolates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Antifreeze polymeric additives for fuels; Aditivos polimericos anticongelantes para combustiveis

    Energy Technology Data Exchange (ETDEWEB)

    Muniz, Aline S.; Carvalho, Agne Roani de; Sakae, George Hideki; Oliveira, Angelo R.S.; Cesar-Oliveira, Maria Aparecida F. [Universidade Federal do Parana - UFPR - Departamento de Quimica - LABPOL-Laboratorio de Polimeros Sinteticos, Centro Politecnico, Curitiba, PR (Brazil)], e-mails: mafco@ufpr.br, alinemuniz@ufpr.br

    2011-07-01

    Owing to the current interest in the reduction of environmental pollution, several researchers are seeking renewable sources of energy which can at least partially replace combustibles derived from petroleum. Diesel oil is the combustible that most seriously pollutes the environment and is thus the biodiesel that is being considered as a fuel which can be replaced by a renewable combustible; this can possibly be used in diesel engines without any modifications. However, certain problems have to be overcome with regard to the temperature at which the biodiesel should be stored and used, since there is a tendency for biodiesel to solidify at low temperatures. This suggests that there is a need for the use of anti-freeze additives. This work behind the main focus additives with only 25 ppm, were able to reduce the pour point of fuel, achieving significant results, for example, the additive M14A18 lowered the pour point (PP) of B20 to -20 degree C, showing that the use of increasing amounts of biodiesel to diesel can aggregate. The main focus of work behind the development of additives that with only 25 ppm, were able to reduce the pour point of fuel, producing significant results such as those obtained with the use of additive M14A18 which lowered the pour point of the B20 to -20 degree C, showing the possibility of using increasing amounts of biodiesel added to diesel. (author)

  14. FISH GLYCOPEPTIDE AND PEPTIDE ANTIFREEZES : THEIR INTERACTION WITH ICE AND WATER

    OpenAIRE

    Wilson, P.; Devries, A.

    1987-01-01

    Glycopeptide and peptide antifreeze agents are present in the body fluids of polar fishes and allow them to avoid freezing in ice-laden seawater. These antifreezes lower the freezing point 200 times more than predicted by colligative relations, but have little effect on the melting point of ice. They bind to ice and appear to inhibit growth by increasing the curvature of growth steps on the ice crystal surface. Such a growth would result in a substantial increase in the roughness of the surfa...

  15. Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.

    Science.gov (United States)

    To, Brian C S; Lenhoff, Abraham M

    2011-01-21

    The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Spatially selective binding of green fluorescent protein on designed organosilane nanopatterns prepared with particle lithography.

    Science.gov (United States)

    Highland, Zachary L; Garno, Jayne C

    2017-04-19

    A practical approach for preparing protein nanopatterns has been to design surface templates of nanopatterns of alkanethiols or organosilanes that will selectively bind and localize the placement of biomolecules. Particle lithography provides a way to prepare millions of protein nanopatterns with a few basic steps. For our nanopatterning strategy, organosilanes with methoxy and sulfhydryl groups were chosen as a surface template. Green fluorescent protein (GFP) was selected as a model for patterning. Areas of 2-[methoxy (polyethyleneoxy)6-9propyl]trichlorosilane (MPT-silane) are effective as a matrix for resisting the attachment of proteins, whereas nanopatterns with sulfur groups provide reactive sites for binding linker groups to connect proteins. A protocol with particle lithography was designed to make a surface template of nanopatterns of (3-mercaptopropyl)trimethoxysilane (MPTMS) surrounded by a methoxy terminated matrix. The sulfhydryl groups of the MPTMS nanopatterns were activated with a sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker. The activated regions of MPTMS furnished sites for binding GFP. Samples were characterized with atomic force microscopy after successive steps of the patterning protocol to evaluate the selectivity of protein binding. Direct views of the protein bound selectively to designated sites of MPTMS are presented, as evidence of robust and reproducible patterning. Nanoscale patterns of proteins can be used for surfaces of biochips and biosensors, and also for immunochemistry test platforms.

  17. [Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

    Science.gov (United States)

    Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

    2015-12-01

    In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

  18. the effect of partial substitution of plant protein by fishmeal prepared

    African Journals Online (AJOL)

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    THE EFFECT OF PARTIAL SUBSTITUTION OF PLANT PROTEIN BY FISHMEAL. PREPARED OUT OF COOKED AND SUN DRIED FISH OFFAL ON FEED INTAKE. AND CARCASS TRAITS OF RHODE ISLAND RED CHICKS. Asrat Tera, Tegene Neggese ∗ and Aberra Melesse. Awassa College of Agriculture, PO Box 05, ...

  19. Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling...

  20. Legume Protein Isolates for Stable Acidic Emulsions Prepared by Premix Membrane Emulsification

    NARCIS (Netherlands)

    Ladjal Ettoumi, Yakoub; Berton-Carabin, Claire; Chibane, Mohamed; Schroën, Karin

    2017-01-01

    Proteins originating from dry legumes are not that much used in food formulations, yet, they are interesting components from a sustainability point of view, and could have interesting functional properties, e.g. for emulsion preparation. Therefore, this work focuses on the potential of the water

  1. Preparation and Characterization of Water-Soluble Chitosan Nanoparticles as Protein Delivery System

    Directory of Open Access Journals (Sweden)

    Hong-liang Zhang

    2010-01-01

    Full Text Available The objective of this study was to investigate the potential of water soluble chitosan as a carrier in the preparation of protein-loaded nanoparticles. Nanoparticles were prepared by ionotropic gelation of water-soluble chitosan (WSC with sodium tripolyphosphate (TPP. Bovine serum albumin (BSA was applied as a model drug. The size and morphology of the nanoparticles were investigated as a function of the preparation conditions. The particles were spherical in shape and had a smooth surface. The size range of the nanoparticles was between 100 and 400 nm. Result of the in vitro studies showed that the WSC nanoparticles enhance and prolong the intestinal absorption of BSA. These results also indicated that WSC nanoparticles were a potential protein delivery system.

  2. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......High-throughput screening of genome wide siRNA- or compound libraries is currently applied for drug target and drug discovery. Commonly, these approaches deal with sample numbers ranging from 100,000 to several millions. Efforts to decrease costs and to increase information gained include......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

  3. Preparation

    Directory of Open Access Journals (Sweden)

    M.M. Dardir

    2014-03-01

    Full Text Available Some hexanamide-mono and di-linoleniate esters were prepared by the reaction of linolenic acid and hexanamide (derived from the reaction of hexanoic acid and diethanolamine. The chemical structure for the newly prepared hexanamide-mono and di-linoleniate esters were elucidated using elemental analysis, (FTIR, H 1NMR and chemical ionization mass spectra (CI/Ms spectroscopic techniques. The results of the spectroscopic analysis indicated that they were prepared through the right method and they have high purity. The new prepared esters have high biodegradability and lower toxicity (environmentally friendly so they were evaluated as a synthetic-based mud (ester-based mud for oil-well drilling fluids. The evaluation included study of the rheological properties, filtration and thermal properties of the ester based-muds formulated with the newly prepared esters compared to the reference commercial synthetic-based mud.

  4. Oscillations and accelerations of ice crystal growth rates in microgravity in presence of antifreeze glycoprotein impurity in supercooled water

    Science.gov (United States)

    Furukawa, Yoshinori; Nagashima, Ken; Nakatsubo, Shun-Ichi; Yoshizaki, Izumi; Tamaru, Haruka; Shimaoka, Taro; Sone, Takehiko; Yokoyama, Etsuro; Zepeda, Salvador; Terasawa, Takanori; Asakawa, Harutoshi; Murata, Ken-Ichiro; Sazaki, Gen

    2017-03-01

    The free growth of ice crystals in supercooled bulk water containing an impurity of glycoprotein, a bio-macromolecule that functions as ‘antifreeze’ in living organisms in a subzero environment, was observed under microgravity conditions on the International Space Station. We observed the acceleration and oscillation of the normal growth rates as a result of the interfacial adsorption of these protein molecules, which is a newly discovered impurity effect for crystal growth. As the convection caused by gravity may mitigate or modify this effect, secure observations of this effect were first made possible by continuous measurements of normal growth rates under long-term microgravity condition realized only in the spacecraft. Our findings will lead to a better understanding of a novel kinetic process for growth oscillation in relation to growth promotion due to the adsorption of protein molecules and will shed light on the role that crystal growth kinetics has in the onset of the mysterious antifreeze effect in living organisms, namely, how this protein may prevent fish freezing.

  5. Effects of short-time preheating on ice growth in antifreeze polypeptides solutions in a narrow space

    Science.gov (United States)

    Miyamoto, T.; Nishi, N.; Waku, T.; Tanaka, N.; Hagiwara, Y.

    2018-03-01

    We conducted experiments on the unidirectional freezing of solutions of winter flounder antifreeze protein or of a polypeptide which was based on twelve amino-acid residues of this protein. The temperature in the solutions and ice was measured with a small thermocouple. The interface temperature was defined as the temperature at the tip of the serrated or pectinate interface. The interface temperature of these solutions was lower than that of pure water. To vary this supercooling activity of these solutes, we preheated the solutions and cooled them before conducting identical experiments. It was found that short-time preheating caused further decreases in the interface temperature and interface velocities. Furthermore, the inclined interfaces and the narrow liquid regions inside the ice area became wider. To investigate the reasons for these changes, we measured aggregates of the solutes in the solutions. These aggregates were found to become larger as a result of preheating. Thus, it can be concluded that these large aggregates attenuated the ice growth by their interaction with ice. Finally, we carried out similar measurements by using pH-adjusted solutions of the protein to produce aggregates without preheating, and obtained similar supercooling enhancement by the aggregates. Thus, the effects of thermal denaturation on the supercooling were not significant in the preheating.

  6. Microencapsulation of protein drugs for drug delivery: strategy, preparation, and applications.

    Science.gov (United States)

    Ma, Guanghui

    2014-11-10

    Bio-degradable poly(lactide) (PLA)/poly(lactide-glycolide) (PLGA) and chitosan microspheres (or microcapsules) have important applications in Drug Delivery Systems (DDS) of protein/peptide drugs. By encapsulating protein/peptide drugs in the microspheres, the serum drug concentration can be maintained at a higher constant value for a prolonged time, or injection formulation can be changed to orally or mucosally administered formulation. PLA/PLGA and chitosan are most often used in injection formulation and oral formulation. However, in the preparation and applications of PLA/PLGA and chitosan microspheres containing protein/peptide drugs, the problems of broad size distribution and poor reproducibility of microspheres, and deactivation of protein during the preparation, storage and release, are still big challenges. In this article, the techniques for control of the diameter of microspheres and microcapsules will be introduced at first, then the strategies about how to maintain the bioactivity of protein drugs during preparation and drug release will be reviewed and developed in our research group. The membrane emulsification techniques including direct membrane emulsification and rapid membrane emulsification processes were developed to prepare uniform-sized microspheres, the diameter of microspheres can be controlled from submicron to 100μm by these two processes, and the reproducibility of products can be guaranteed. Furthermore, compared with conventional stirring method, the big advantages of membrane emulsification process were that the uniform microspheres with much higher encapsulation efficiency can be obtained, and the release behavior can be adjusted by selecting microsphere size. Mild membrane emulsification condition also can prevent the deactivation of proteins, which frequently occurred under high shear force in mechanical stirring, sonification, and homogenization methods. The strategies for maintaining the bioactivity of protein drug were

  7. Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.

    Directory of Open Access Journals (Sweden)

    Xabier Osteikoetxea

    Full Text Available In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody and ganglioside GM1 (cholera toxin subunit B. We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition, may prove useful for quality control of extracellular vesicle related basic and clinical studies.

  8. Nano spray drying: a novel method for preparing protein nanoparticles for protein therapy.

    Science.gov (United States)

    Lee, Sie Huey; Heng, Desmond; Ng, Wai Kiong; Chan, Hak-Kim; Tan, Reginald B H

    2011-01-17

    There has been an increasing interest in the development of protein nanotherapeutics for diseases such as cancer, diabetes and asthma. Spray drying with prior micro mixing is commonly used to obtain these powders. However, the separation and collection of protein nanoparticles with conventional spray dryer setups has been known to be extremely challenging due to its typical low collection efficiency for fine particles less than 2μm. To date, there has been no feasible approach to produce these protein nanoparticles in a single step and with high yield (>70%). In this study, we explored the feasibility of the novel Nano Spray Dryer B-90 (equipped with a vibrating mesh spray technology and an electrostatic particle collector) for the production of bovine serum albumin (BSA) nanoparticles. A statistical experimental design method (Taguchi method based on three levels, five variables L(18) orthogonal array robust design) was implemented to study the effect of and optimize the experimental conditions of: (1) spray mesh size, (2) BSA solution concentration, (3) surfactant concentration, (4) drying air flow rate and (5) inlet temperature on: (1) size and (2) morphology (axial ratio). Particle size and morphology were predominantly influenced by the spray mesh size and surfactant concentration, respectively. The drying air flow rate and inlet temperature had minimal impact. Optimized production of smooth spherical nanoparticles (median size: 460±10nm, axial ratio: 1.03±0.00, span 1.03±0.03, yield: 72±4%) was achieved using the 4μm spray mesh at BSA concentration of 0.1% (w/v), surfactant concentration of 0.05% (w/v), drying flow rate of 150L/min and inlet temperature of 120°C. The Nano Spray Dryer B-90 thus offers a new, simple and alternative approach for the production of protein nanoparticles suited for a variety of drug delivery applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Fluorescence imaging preparation methods for tissue scaffolds implanted into a green fluorescent protein porcine model.

    Science.gov (United States)

    Smith, Sarah E; White, Richard A; Grant, David A; Grant, Sheila A

    2015-10-01

    Green fluorescent protein (GFP) animal models have become increasingly popular due to their potential to enhance in vivo imaging and their application to many fields of study. We have developed a technique to observe host tissue integration into scaffolds using GFP expressing swine and fluorescence imaging. Current fluorescence imaging preparation methods cannot be translated to a full GFP animal model due to several challenges and limitations that are investigated here. We have implanted tissue scaffolds into GFP expressing swine and have prepared explanted scaffolds for fluorescence imaging using four different methods including formalin fixation and paraffin embedding, vapor fixation, freshly prepared paraformaldehyde fixation, and fresh frozen tissue. Explanted scaffolds and tissue were imaged using confocal microscopy with spectral separation to evaluate the GFP animal model for visualization of host tissue integration into explanted scaffolds. All methods except fresh frozen tissue induced autofluorescence of the scaffold, preventing visualization of detail between host tissue and scaffold fibers. Fresh frozen tissue preparation allowed for the most reliable visualization of fluorescent host tissue integration into non-fluorescent scaffolds. It was concluded that fresh frozen tissue preparation is the best method for fluorescence imaging preparation when using scaffolds implanted into GFP whole animal models.

  10. [PREPARATION OF HUMAN TISSUE PROTEIN EXTRACTS ENRICHED WITH THE SPHINGOMYELIN SYNTHASE 1].

    Science.gov (United States)

    Sudarkina, O Yu; Dergunova, L V

    2015-01-01

    Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.

  11. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

    2016-01-15

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  12. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    International Nuclear Information System (INIS)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O.; Tumpey, Terrence M.; Pushko, Peter

    2016-01-01

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  13. Single-input divergent flow IEF for preparative analysis of proteins

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Šlais, Karel

    2008-01-01

    Roč. 29, č. 22 (2008), s. 4503-4507 ISSN 0173-0835 R&D Projects: GA AV ČR IAAX00310701; GA ČR GA203/06/1179 Institutional research plan: CEZ:AV0Z40310501 Keywords : continuous divergent flow * IEF * preparative protein analysis Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.509, year: 2008

  14. Preparation and Characterization of Protein-Loaded Electrospun Fiber Mat and Its Release Kinetics.

    Science.gov (United States)

    Wen, Peng; Wen, Yan; Huang, Xiao; Zong, Min-Hua; Wu, Hong

    2017-06-14

    For the enhancement of protein's bioavailability, a specific delivery system was developed by coaxial electrospinning. Bovine serum albumin (BSA) was used as protein model, and the core-sheath fiber mat was fabricated using sodium alginate as shell layer and the BSA-loaded chitosan nanoparticle that was prepared previously as core layer. By optimizing electrospinning parameters, uniform fibers with diameters ranging from 200-600 nm were obtained, and transmission electron microscopy and confocal laser scanning microscopy revealed their core-sheath structures. Fourier transform infrared spectroscopy (FTIR) analysis demonstrated that there existed molecular interaction between components, which enhanced the mat's thermal stability and mechanic property. It was found that the predominant release mechanism of BSA from fiber mat was erosion, and little change occurred in the secondary structure of encapsulated BSA indicated by FTIR and circular dichroism analysis. The study shows that the obtained fiber mat is a potential delivery system for protein.

  15. Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiping Cai

    2008-01-01

    Full Text Available Vascular endothelial cell growth inhibitor (VEGI is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3, and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

  16. Antioxidative properties of milk protein preparations fermented by Polish strains of Lactobacillus helveticus.

    Science.gov (United States)

    Skrzypczak, Katarzyna W; Gustaw, Waldemar Z; Jabłońska-Ryś, Ewa D; Michalak-Majewska, Monika; Sławińska, Aneta; Radzki, Wojciech P; Gustaw, Klaudia M; Waśko, Adam D

    2017-01-01

    The increasing significance of food products containing substances with antioxidative activi- ties is currently being observed. This is mainly due to the fact that pathogenic changes underlying some diseases are related to the carcinogenic effects of free radicals. Antioxidative compounds play an important role in supporting and enhancing the body’s defense mechanisms, which is useful in preventing some civili- zation diseases. Unfortunately, it has been already proved that some synthetic antioxidants pose a potential risk in vivo. Therefore, antioxidant compounds derived from a natural source are extremely valuable. Milk is a source of biologically active precursors, which when enclosed in structural protein sequences are inactive. The hydrolysis process, involving bacterial proteolytic enzymes, might release biopeptides that act in various ways, including having antioxidant properties. The objective of this study was to determine the antioxidant properties of milk protein preparations fermented by Polish strains of L. helveticus. The research also focused on evaluating the dynamics of milk acidification by these strains and analyzing the textural properties of the skim milk fermented products obtained. The research studied Polish strains of L. helveticus: B734, 141, T80 and T105, which have not yet been used industrially. The antioxidant properties of 1% (w/v) solutions of milk protein preparations (skim milk powder, caseinoglycomacropeptide and α-lactoalbumin) fermented by these strains were determined by neutralizing the free radicals with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙). Moreover, solutions of skim milk powder (SMP) fermented by the microorganisms being tested were analyzed on gel electrophoresis (SDS-PAGE). The dynamics of milk acidification by these microorganisms was also analyzed L. helveticus strains were used to prepare fermented regenerated skim milk products that were subjected to texture profile analysis (TPA) performed using a TA-XT2i

  17. Preparation, aroma characteristics and volatile compounds of flavorings from enzymatic hydrolyzed rice bran protein concentrate.

    Science.gov (United States)

    Arsa, Supeeraya; Theerakulkait, Chockchai

    2018-02-19

    Rice bran is a by-product obtained from the rice milling industry. The aims of this research were to add value to rice bran by preparation of enzymatic hydrolyzed rice bran protein concentrate (HRPC) as a flavoring agent and the flavoring which was produced by HRPC has not been investigated. Different drying methods (freeze-drying and spray-drying) and fructose additions were studied for improvement of rice bran protein sensorial aroma characteristics. The most abundant amino acids in liquid HRPC (LH) were glutamic acid, arginine, aspartic acid and leucine. The intensity of desirable aromas, such as cereal-like, nut-like, milk-powder-like, sweet, and cocoa-like aroma, were higher in spray-dried HRPC powder (SHP) than in LH and freeze-dried HRPC. Volatile compounds, such as aldehydes, pyrazines and ketones, were significantly increased in HRPC powders in which fructose was added before spray-drying (SHP-F). Higher amounts of 2-methylbutanal, 3-methylbutanal, phenylacetaldehyde, 2,5-dimethylpyrazine, vanillin, 2-acetylpyrrole and maltol were detected in SHP-F. Moreover, these compounds had high odor active values, which accounted for the cocoa-like, sweet, nut-like, and milk-powder-like characteristics of SHP-F. These findings could lead to the creation of desirable aroma characteristics of rice bran protein concentrate by different preparation methods. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  18. Sulfonate-terminated carbosilane dendron-coated nanotubes: a greener point of view in protein sample preparation.

    Science.gov (United States)

    González-García, Estefanía; Gutiérrez Ulloa, Carlos E; de la Mata, Francisco Javier; Marina, María Luisa; García, María Concepción

    2017-09-01

    Reduction or removal of solvents and reagents in protein sample preparation is a requirement. Dendrimers can strongly interact with proteins and have great potential as a greener alternative to conventional methods used in protein sample preparation. This work proposes the use of single-walled carbon nanotubes (SWCNTs) functionalized with carbosilane dendrons with sulfonate groups for protein sample preparation and shows the successful application of the proposed methodology to extract proteins from a complex matrix. SEM images of nanotubes and mixtures of nanotubes and proteins were taken. Moreover, intrinsic fluorescence intensity of proteins was monitored to observe the most significant interactions at increasing dendron generations under neutral and basic pHs. Different conditions for the disruption of interactions between proteins and nanotubes after protein extraction and different concentrations of the disrupting reagent and the nanotube were also tried. Compatibility of extraction and disrupting conditions with the enzymatic digestion of proteins for obtaining bioactive peptides was also studied. Finally, sulfonate-terminated carbosilane dendron-coated SWCNTs enabled the extraction of proteins from a complex sample without using non-environmentally friendly solvents that were required so far. Graphical Abstract Green protein extraction from a complex sample employing carbosilane dendron coated nanotubes.

  19. Preparation of mesoporous silica thin films by photocalcination method and their adsorption abilities for various proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Katsuya, E-mail: katsuya-kato@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560 (Japan); Nakamura, Hitomi [National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560 (Japan); Yamauchi, Yoshihiro; Nakanishi, Kazuma; Tomita, Masahiro [Department of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570 (Japan)

    2014-07-01

    Mesoporous silica (MPS) thin film biosensor platforms were established. MPS thin films were prepared from tetraethoxysilane (TEOS) via using sol–gel and spin-coating methods using a poly-(ethylene oxide)-block-poly-(propylene oxide)-block-poly-(ethylene oxide) triblock polymer, such as P123 ((EO){sub 20}(PO){sub 70}(EO){sub 20}) or F127 ((EO){sub 106}(PO){sub 70}(EO){sub 106}), as the structure-directing agent. The MPS thin film prepared using P123 as the mesoporous template and treated via vacuum ultraviolet (VUV) irradiation to remove the triblock copolymer had a more uniform pore array than that of the corresponding film prepared via thermal treatment. Protein adsorption and enzyme-linked immunosorbent assay (ELISA) on the synthesized MPS thin films were also investigated. VUV-irradiated MPS thin films adsorbed a smaller quantity of protein A than the thermally treated films; however, the human immunoglobulin G (IgG) binding efficiency was higher on the former. In addition, protein A–IgG specific binding on MPS thin films was achieved without using a blocking reagent; i.e., nonspecific adsorption was inhibited by the uniform pore arrays of the films. Furthermore, VUV-irradiated MPS thin films exhibited high sensitivity for ELISA testing, and cytochrome c adsorbed on the MPS thin films exhibited high catalytic activity and recyclability. These results suggest that MPS thin films are attractive platforms for the development of novel biosensors. - Highlights: • VUV-treated MPS thin films with removed polymer had uniform pore. • VUV-treated MPS thin films exhibited high sensitivity by ELISA. • Cytochrome c showed the catalytic activity and recyclability on synthesized films.

  20. Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese

    OpenAIRE

    Wagh, Y. R.; Pushpadass, Heartwin A.; Emerald, F. Magdaline Eljeeva; Nath, B. Surendra

    2013-01-01

    Casein and whey protein concentrate (WPC) films, plasticized with glycerol and sorbitol independently, were prepared by casting. The film thickness, water vapour and oxygen permeation and tensile and moisture sorption properties of the films were determined. The tensile strength (TS), tensile strain (TE) and elastic modulus (EM) of the films ranged from 0.71 to 4.58 MPa, 19.22 to 66.63 % and 2.05 to 6.93 MPa, respectively. The film properties were influenced by the type of biopolymer (casein ...

  1. Preparation of Barley Storage Protein, Hordein, for Analytical Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    DEFF Research Database (Denmark)

    Doll, Hans; Andersen, Bente

    1981-01-01

    The extraction, reduction, and alkylation of barley hordein for routine electrophoresis in sodium dodecyl sulfate-polyacrylamide gels were studied to set up a simple preparation procedure giving well-resolved bands in the electrophoresis gel. Hordein was extracted from single crushed seeds or flour...... by aqueous 50% propan-2-ol containing a Tris-borate buffer, pH 8.6. The presence of the buffer facilitates the consecutive complete reduction of the extracted protein in the alcohol. Reduction and alkylation in the buffer containing propan-2-ol give sharper bands in the electrophoresis than reduction...

  2. Determinants of protein elution rates from preparative ion-exchange adsorbents.

    Science.gov (United States)

    Angelo, James M; Lenhoff, Abraham M

    2016-04-01

    The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    Science.gov (United States)

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A novel approach to preparing magnetic protein microspheres with core-shell structure

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Wei, E-mail: climentjw@126.co [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Sun Zhendong; Li Fengsheng [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Chen Kai; Liu Tianyu; Liu Jialing [Department of Physics, Nanjing University of Science and Technology, Nanjing 210094 (China); Zhou Tianle [Department of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Guo Rui [Department of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2011-03-15

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

  5. Obtention and characterization of dried gels prepared with whey proteins, honey and hydrocolloids mixture.

    Science.gov (United States)

    Rodriguez, Ana C; Torrez Irigoyen, Martín R; Navarro, Alba S; Yamul, Diego K

    2017-11-01

    Large amounts of honey and liquid whey derived from the dairy industry are produced in Argentina. Honey is exported in bulk and whey is transformed into whey protein concentrates and isolates. The objective of this work was to investigate the effect of pH, composition and storage time on the properties of dried gels with honey, whey proteins and hydrocolloids. Color properties varied according to pH and composition. The fracture stress of dried gels prepared with corn starch was higher than that of gels prepared with guar gum in all conditions assayed. Young's modulus was higher at pH 7 for both compositions and increased with storage time. Rubbery characteristics were found in dried gels with guar gum, while both corn starch and guar gum made the microstructure rougher. Multivariate analysis showed that samples could be grouped by pH. Panelists preferred pH 7 products over acidic ones, and no significant differences in sensory properties were found using either corn starch or guar gum in the formulation. The results demonstrated that it is possible to generate a new product, which may open new applications for honey and whey in food formulations. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  6. A novel approach to preparing magnetic protein microspheres with core-shell structure

    International Nuclear Information System (INIS)

    Jiang Wei; Sun Zhendong; Li Fengsheng; Chen Kai; Liu Tianyu; Liu Jialing; Zhou Tianle; Guo Rui

    2011-01-01

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: → Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method.→ The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA).→ 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

  7. A mixture design approach to optimizing low cholesterol mayonnaise formulation prepared with wheat germ protein isolate.

    Science.gov (United States)

    Rahbari, Mahshid; Aalami, Mehran; Kashaninejad, Mahdi; Maghsoudlou, Yahya; Aghdaei, Seid Soheil Amiri

    2015-06-01

    The aim of this study was to optimize the mixture proportion of low cholesterol mayonnaise containing wheat germ protein isolate (WGPI) and xanthan gum (XG), as emulsifying agents in mayonnaise preparation. The mayonnaise prepared with different combinations of WGPI, egg yolk (0-9 % of each component) and XG (0-0.5 %). The optimized mixture proportions of low cholesterol mayonnaise were determined by applying the optimal mixture design method to acquire the mayonnaise with proper stability, texture, rheological properties and sensory scores. Optimum values of WGPI, XG and egg yolk in the mixture were found to be 7.87 %, 0.2 % and 0.93 %, respectively (of 9 % egg yolk). The WGPI, due to unique functional properties, had the greatest effect on properties of mayonnaise samples. Moreover, combination of XG and WGPI, improved the stability, heat stability, viscosity, consistency coefficient and textural properties of product. However, the overall acceptance was maximum in a mixture contained high amount of WGPI and XG and low amount of egg yolk. The results of this research showed the feasibility of preparation a low cholesterol mayonnaise by application a desirable combination of WGPI, XG, and egg yolk, with comparable properties those of the conventional mayonnaise.

  8. Preparation of canine C-reactive protein serum reference material: A feasibility study.

    Science.gov (United States)

    Canalias, Francesca; Piñeiro, Matilde; Pato, Raquel; Peña, Raquel; Bosch, Lluís; Soler, Lourdes; García, Natalia; Lampreave, Fermín; Saco, Yolanda; Bassols, Anna

    2018-03-01

    The availability of a species-specific reference material is essential for the harmonization of results obtained in different laboratories by different methods. We describe the preparation of a canine C-reactive protein (cCRP) serum reference material containing purified cCRP stabilized in a serum matrix. The material can be used by manufacturers to assign values to their calibrator and control materials. The serum matrix was obtained using blood collected from healthy dogs, stabilized and submitted for a delipidation process. The reference material was prepared by diluting purified cCRP in the serum matrix containing 1.0 mol/L HEPES buffer, 3.0 mmol/L calcium chloride, 80,000 kUI/L aprotinin, and 1.0 mmol/L benzamidine hydrochloride monohydrate at a pH of 7.2, and dispensing (0.5 mL) the matrix into vials that were then frozen. The pilot batch of 200 vials was shown to be homogeneous and stable after a stability study at various temperatures and over a total time of 110 days. The prepared material was submitted to an assignment value study. Eight laboratories from different European countries participated by using the same reagents for an immunoturbidimetric method adapted for different analyzers. The obtained cCRP concentration in the reference material was 78.5 mg/L with an expanded uncertainty (k = 2) of 4.2 mg/L. Canine C-reactive protein serum reference material has been produced that allows harmonization of results obtained by different methods and different laboratories, thus reducing the possibility of errors and misunderstandings. © 2018 American Society for Veterinary Clinical Pathology.

  9. Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method

    Science.gov (United States)

    Using a homogenization-evaporation method, beta-carotene (BC) loaded nano-particles were prepared with different ratios of food-grade sodium caseinate (SC), whey protein isolate (WPI), or soy protein isolate (SPI) to BC and evaluated for their physiochemical stability, in vitro cytotoxicity, and cel...

  10. New insights into respirable protein powder preparation using a nano spray dryer.

    Science.gov (United States)

    Bürki, K; Jeon, I; Arpagaus, C; Betz, G

    2011-04-15

    In this study the Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Flawil, Switzerland) was evaluated with regard to the drying of proteins and the preparation of respirable powders in the size range of 1-5 μm. β-galactosidase was chosen as a model protein and trehalose was added as a stabilizer. The influence of inlet temperature, hole size of the spray cap membrane and ethanol concentration in the spray solution was studied using a 3³ full factorial design. The investigated responses were enzyme activity, particle size, span, yield and shelf life. Furthermore, the particle morphology was examined. The inlet temperature as well as the interaction of inlet temperature and spray cap size significantly influenced the enzyme activity. Full activity was retained with the optimized process. The particle size was affected by the hole size of the spray cap membrane and the ethanol content. The smallest cap led to a monodisperse particle size distribution and the greatest yield of particles of respirable size. Higher product recovery was achieved with lower inlet temperatures, higher ethanol contents and smaller cap sizes. Particle morphology differed depending on the cap size. The protein exhibited higher storage stability when spray dried without ethanol and when a larger spray cap size was used. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese.

    Science.gov (United States)

    Wagh, Y R; Pushpadass, Heartwin A; Emerald, F Magdaline Eljeeva; Nath, B Surendra

    2014-12-01

    Casein and whey protein concentrate (WPC) films, plasticized with glycerol and sorbitol independently, were prepared by casting. The film thickness, water vapour and oxygen permeation and tensile and moisture sorption properties of the films were determined. The tensile strength (TS), tensile strain (TE) and elastic modulus (EM) of the films ranged from 0.71 to 4.58 MPa, 19.22 to 66.63 % and 2.05 to 6.93 MPa, respectively. The film properties were influenced by the type of biopolymer (casein and whey protein concentrate), plasticizer and its concentration. Increasing the plasticizer concentration, increased the film thickness, TE and water vapour permeability (WVP), but decreased the TS and EM. As the concentration of plasticizer increased to the highest level, the film thickness increased from 0.168 to 0.305 mm for glycerol-plasticized films and from 0.251 to 0.326 mm for sorbitol-plasticized films. The film thickness increased because the amount of plasticizer in the film network increased and the amount of biopolymer remained same. Casein films showed superior tensile properties as compared to WPC films. The WVP of both casein and WPC films lied between 3.87 and 13.97 g.mm./(m(2).h.kPa). The moisture sorption isotherms of both films were typical of high-protein material, and were adequately described by the GAB model. The oxygen permeability of casein films was relatively lower than that of WPC films, regardless of the plasticizer used. The sensory data revealed that the organoleptic quality of Cheddar cheese was unaffected by milk-protein film packaging.

  12. A novel approach to preparing magnetic protein microspheres with core-shell structure

    Science.gov (United States)

    Jiang, Wei; Sun, Zhendong; Li, Fengsheng; Chen, Kai; Liu, Tianyu; Liu, Jialing; Zhou, Tianle; Guo, Rui

    2011-03-01

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail.

  13. Antifreeze glycopeptides: from structure and activity studies to current approaches in chemical synthesis.

    Science.gov (United States)

    Urbańczyk, Małgorzata; Góra, Jerzy; Latajka, Rafał; Sewald, Norbert

    2017-02-01

    Antifreeze glycopeptides (AFGPs) are a class of biological antifreeze agents found predominantly in Arctic and Antarctic species of fish. They possess the ability to regulate ice nucleation and ice crystal growth, thus creating viable life conditions at temperatures below the freezing point of body fluids. AFGPs usually consist of 4-55 repetitions of the tripeptide unit Ala-Ala-Thr that is O-glycosylated at the threonine side chains with β-D-galactosyl-(1 → 3)-α-N-acetyl-D-galactosamine. Due to their interesting properties and high antifreeze activity, they have many potential applications, e.g., in food industry and medicine. Current research is focused towards understanding the relationship between the structural preferences and the activity of the AFGPs, as well as developing time and cost efficient ways of synthesis of this class of molecules. Recent computational studies in conjunction with experimental results from NMR and THz spectroscopies were a possible breakthrough in understanding the mechanism of action of AFGPs. At the moment, as a result of these findings, the focus of research is shifted towards the analysis of behaviour of the hydration shell around AFGPs and the impact of water-dynamics retardation caused by AFGPs on ice crystal growth. In the field of organic synthesis of AFGP analogues, most of the novel protocols are centered around solid-phase peptide synthesis and multiple efforts are made to optimize this approach. In this review, we present the current state of knowledge regarding the structure and activity of AFGPs, as well as approaches to organic synthesis of these molecules with focus on the most recent developments.

  14. STUDY ON PROPERTIES OF SKID RESISTANCE ON FREEZING PAVEMENTS AND QUANTITATIVE EVALUATION METHOD OF ANTIFREEZING EFFECTS

    Science.gov (United States)

    Tanaka, Shunsuke; Takeichi, Kiyoshi; Masuyama, Yukiei; Takahashi, Naoto

    Snow and ice control in winter roads trends to be controlled by the skid friction coefficients in North America and North European countries at present, but the measurements are not necessarily easy. We studied on a simplified measurement method based on the relationship between skid friction coefficients and the bare pavement ratio (BPR) in the laboratory tests and field tests. The factors of BPR, surface textures and antifreezing materials which affect the skid friction coefficient are reviewed by a multiple linear regression analysis and a spectrum analysis, considering different freezing surfaces. These studies indicate that conclusions induced by laboratory tests could be applied to roads in service.

  15. Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.

    Science.gov (United States)

    Rodrigues, Silas Pessini; Ventura, José Aires; Zingali, R B; Fernandes, P M B

    2009-01-01

    A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

  16. Lactobacillus casei Encapsulated in Soy Protein Isolate and Alginate Microparticles Prepared by Spray Drying

    Directory of Open Access Journals (Sweden)

    Jasmina Hadzieva

    2017-01-01

    Full Text Available This article presents a novel formulation for preparation of Lactobacillus casei 01 encapsulated in soy protein isolate and alginate microparticles using spray drying method. A response surface methodology was used to optimise the formulation and the central composite face-centered design was applied to study the effects of critical material attributes and process parameters on viability of the probiotic after microencapsulation and in simulated gastrointestinal conditions. Spherical microparticles were produced in high yield (64 %, narrow size distribution (d50=9.7 µm, span=0.47 and favourable mucoadhesive properties, with viability of the probiotic of 11.67, 10.05, 9.47 and 9.20 log CFU/g after microencapsulation, 3 h in simulated gastric and intestinal conditions and four-month cold storage, respectively. Fourier-transform infrared spectroscopy confirmed the probiotic stability after microencapsulation, while differential scanning calorimetry and thermogravimetry pointed to high thermal stability of the soy protein isolate-alginate microparticles with encapsulated probiotic. These favourable properties of the probiotic microparticles make them suitable for incorporation into functional food or pharmaceutical products.

  17. Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

    Science.gov (United States)

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V.; Hong, Mei

    2016-01-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  18. Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum.

    Science.gov (United States)

    Liu, Jinxiang; Deng, Qiliang; Tao, Dingyin; Yang, Kaiguang; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-06-30

    Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g(-1). And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

  19. Strongly stretched protein resistant poly(ethylene glycol) brushes prepared by grafting-to.

    Science.gov (United States)

    Emilsson, Gustav; Schoch, Rafael L; Feuz, Laurent; Höök, Fredrik; Lim, Roderick Y H; Dahlin, Andreas B

    2015-04-15

    We present a new grafting-to method for resistant "non-fouling" poly(ethylene glycol) brushes, which is based on grafting of polymers with reactive end groups in 0.9 M Na2SO4 at room temperature. The grafting process, the resulting brushes, and the resistance toward biomolecular adsorption are investigated by surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy. We determine both grafting density and thickness independently and use narrow molecular weight distributions which result in well-defined brushes. High density (e.g., 0.4 coils per nm(2) for 10 kDa) and thick (40 nm for 20 kDa) brushes are readily achieved that suppress adsorption from complete serum (10× dilution, exposure for 50 min) by up to 99% on gold (down to 4 ng/cm(2) protein coverage). The brushes outperform oligo(ethylene glycol) monolayers prepared on the same surfaces and analyzed in the same manner. The brush heights are in agreement with calculations based on a simple model similar to the de Gennes "strongly stretched" brush, where the height is proportional to molecular weight. This result has so far generally been considered to be possible only for brushes prepared by grafting-from. Our results are consistent with the theory that the brushes act as kinetic barriers rather than efficient prevention of adsorption at equilibrium. We suggest that the free energy barrier for passing the brush depends on both monomer concentration and thickness. The extraordinary simplicity of the method and good inert properties of the brushes should make our results widely applicable in biointerface science.

  20. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    Science.gov (United States)

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2013-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α-AMP-activated protein kinase phosphorylation in mice sacrificed by cervical dislocation. Sacrifice of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation and similar levels of phosphorylation were observed compared to mice sacrificed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw-induced increase in α-AMP-activated protein kinase phosphorylation, but phosphorylation was higher in mice sacrificed by cervical dislocation compared to mice sacrificed by focused microwave irradiation. These results demonstrate that α-AMP-activated protein kinase phosphorylation is dependent on method of sacrifice and tissue preparation and that α-AMP-activated protein kinase phosphorylation can increase in a manner that does not reflect biological alterations. PMID:18088373

  1. Solubilization of proteins in extracted oil bodies by SDS: a simple and efficient protein sample preparation method for Tricine-SDS-PAGE.

    Science.gov (United States)

    Ying, Yusang; Zhao, Luping; Kong, Lingzhi; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-08-15

    A simple and efficient method for preparing Tricine-SDS-PAGE protein sample of extracted oil bodies (OBs) was supplied: OB suspension was vortexed with SDS buffer (pH 6.8) for 2 min at room temperature with SDS/protein of 1.52/1(w/w), which could be analyzed by Tricine-SDS-PAGE after simple treatments (dilution and 2-mercaptoethanol). At SDS/protein of 1.52/1, about 95% of proteins in soybean OB suspension were solubilized, whereas residual 5% of proteins were weakly bound to SDS-destroyed OBs; proteins in destroyed OBs might be further solubilized by SDS in the gel and cathode buffer of Tricine-SDS-PAGE, causing about 99% of proteins in soybean OB suspension recover on Tricine-SDS-PAGE gel, which was better than acetone (89%) and diethyl ether (96%) harvested protein samples. Higher or lower SDS/protein was unbeneficial for protein solubilization from OBs. Additionally, the above method was also better than organic solvent method for peanut, sesame, and rapeseed OB suspensions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.

    Science.gov (United States)

    Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

    2016-09-20

    Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include

  3. Preparation of salted meat products, e.g. cured bacon - by injecting liquid comprising meat proteins hydrolysed with enzymes

    DEFF Research Database (Denmark)

    1997-01-01

    Preparation of salted meat products comprises the following:(1) meat is chopped into fine pieces and mixed with water to form a slurry; (2) enzymes hydrolyse proteins in the meat; (3) adding a culture to the resulting medium, which comprises short peptide chains or amino acids; (4) forming...... flavourings as the culture is growing, and (5) injecting the liquid into pieces of meat....

  4. Lack of immunogenicity of ice structuring protein type III HPLC12 preparation administered by the oral route to human volunteers

    DEFF Research Database (Denmark)

    Crevel, R W R; Cooper, K J; Poulsen, Lars K.

    2007-01-01

    Before a novel protein can be used in foods, its potential allergenicity must be assessed. In this study, healthy volunteers consumed ice structuring protein (ISP) Type III preparation or a control material 5 days a week for a total of 8 weeks. General measures of health were recorded during...... background against which to interpret the results. Nevertheless, the absence of an immune response using a protocol which could have been expected to result in a response with a strongly immunogenic protein, confirms the conclusions of earlier published work, and attests to the lack of allergenicity of ISP...

  5. Antibody preparation and identification of the Cashmere goat c-kit protein in the testes.

    Science.gov (United States)

    Wu, S C L; Luo, F H; Kong, Q F; Wu, Y J

    2014-07-02

    The c-kit protein plays a major role in the regulation of germ cell development. Its expression and distribution in rodent testes have been widely reported. However, research regarding c-kit expression in domestic animals is scarce, and the expression pattern and distribution of c-kit in germ cells have not been clearly defined. In this study, a specific antigenic region for goat c-kit was designed, and a c-kit polyclonal antibody was prepared. This antibody was then applied in a study evaluating c-kit expression in Cashmere goat tissues. A Western blot analysis showed that three forms of c-kit were expressed in goat testes: precursor, mature, and soluble c-kit. Fluorescent immunohistochemical analyses showed that c-kit was primarily expressed in the spermatogonia and spermatocytes of goat testes. These results not only clarify the expression and localization of c-kit in the goat testis, but also accelerate further research regarding the function of c-kit in goat spermatogenesis.

  6. Preparation of C-terminally modified chemokines by expressed protein ligation.

    Science.gov (United States)

    Baumann, Lars; Steinhagen, Max; Beck-Sickinger, Annette G

    2013-01-01

    In order to link structural features on a molecular level to the function of chemokines, site-specific modification strategies are strongly required. These can be used to incorporate fluorescent dyes and/or physical probes to allow investigations in a wide range of biological and physical techniques, e.g., nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, fluorescence resonance energy transfer (FRET), or fluorescence correlation spectroscopy (FCS). Only a limited number of functional groups within the 20 canonical amino acids allow ligation strategies that can be helpful to introduce novel functionalities, which in turn expand the scope of chemoselective and orthogonal reactivity of (semi)synthetic chemokines. In the present chapter we mainly focus on the fabulous history of native chemical ligation (NCL) and provide a general protocol for the preparation of C-terminally modified SDF-1α including tips and tricks for practical work. We believe that this protocol can be easily adapted to other chemokines and many proteins in general.

  7. Preparation and properties of fast temperature-responsive soy protein/PNIPAAm IPN hydrogels

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2014-01-01

    Full Text Available The interpenetrating polymer network of fast temperature-responsive hydrogels based on soy protein and poly(N-isopropylacrylamide were successfully prepared using the sodium bicarbonate (NaHCO3 solutions as the reaction medium. The structure and properties of the hydrogels were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, differential scanning calorimetry and thermal gravimetric analysis. The swelling and deswelling kinetics were also investigated in detail. The results have shown that the proposed hydrogels had high porous structure, good miscibility and thermal stability, and fast temperature responsivity. The presence of NaHCO3 had little effect on the volume phase transition temperature (VPTT of the hydrogels, and the VPTTs were at about 32°C. Compared with the traditional hydrogels, the proposed hydrogels had much faster swelling and deswelling rate. The swelling mechanism of the hydrogels was the non-Fickian diffusion. This fast temperature-responsive hydrogels may have potential applications in the field of biomedical materials.

  8. Design and Preparation of Nano-Lignin Peroxidase (NanoLiP by Protein Block Copolymerization Approach

    Directory of Open Access Journals (Sweden)

    Turgay Tay

    2016-06-01

    Full Text Available This study describes the preparation of nanoprotein particles having lignin peroxidase (LiP using a photosensitive microemulsion polymerization technique. The protein-based nano block polymer was synthesized by cross-linking of ligninase enzyme with ruthenium-based aminoacid monomers. This type polymerization process brought stability in different reaction conditions, reusability and functionality to the protein-based nano block polymer system when compared the traditional methods. After characterization of the prepared LiP copolymer nanoparticles, enzymatic activity studies of the nanoenzymes were carried out using tetramethylbenzidine (TMB as the substrate. The parameters such as pH, temperature and initial enzyme concentration that affect the activity, were investigated by using prepared nanoLip particles and compared to free LiP. The reusability of the nano-LiP particles was also investigated and the obtained results showed that the nano-LiP particles exhibited admirable potential as a reusable catalyst.

  9. Comparison of the amino acid and peptide composition and postprandial response of beef, hydrolyzed chicken, and whey protein nutritional preparations

    Directory of Open Access Journals (Sweden)

    Christopher J. Detzel

    2016-10-01

    Full Text Available Background: Increasing dietary protein intake synergistically improves the effect of exercise to stimulate muscle protein synthesis. The purpose of this study was to evaluate the plasma amino acid response of two novel protein nutritional preparations, beef protein isolate (BeefISO™ and hydrolyzed chicken protein isolate (MyoCHX™. Methods: The postprandial plasma amino acid response over 3 hours was monitored in young adults (n=6 following consumption of 23 grams of WPC, BeefISO™, or MyoCHX™. Amino acid compositional analysis and molecular weight distributions of each protein were performed by HPLC. Statistical analyses were performed using one-way or two-way ANOVA where appropriate and corrected for multiple comparisons to account for the cross-over design. Results: Compositional evaluations revealed similar levels of essential and branched-chain amino acids for WPC and MyoCHX™. While the results of this study predictably demonstrated plasma amino acids levels increased following consumption of the different proteins, the kinetics of the postprandial response was unique to each protein source. WPC and MyoCHX™ were rapidly absorbed with maximum plasma amino acid concentrations observed at 30 and 15 min, respectively. The slightly faster absorption of MyoCHX™ was associated with the increased peptide content of MyoCHX™ (greater than 76% of protein is <2kDa. BeefISO™ exhibited sustained release characteristics as evidenced by increased post prandial amino acid concentrations after 3 hours. Conclusions: The protein preparations studied each had different amino acid profiles and absorption kinetics. WPC and MyoCHX™ contained a higher essential amino acid content and were rapidly absorbed with plasma amino acid concentrations peaking within 30 minutes following consumption. BeefISO™ contained a higher proportion of conditionally essential amino acids that steadily increased in plasma over 3 hours, indicating a sustained release

  10. [Gene cloning, expression and polyclonal antibody preparation and application of translocated intimin receptor-cytoskeleton coupling protein].

    Science.gov (United States)

    Yang, Qin; Cao, Jun-hao; Ding, Jin-ya; Huang, Qian-chuan

    2012-12-01

    To prepare translocated intimin receptor-cytoskeleton coupling protein (TccP) of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and its polyclonal antibody. TccP was amplified from the genome of EHEC O157:H7 Sakai strain by PCR and used to construct the recombinant prokaryotic expression vector pET28a-TccP. The recombinant vector was transformed into E.coli BL21( DE3) to express the protein in the bacteria under the induction of isopropy-D-thiogalactoside (IPTG). After purification, the protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by ELISA and Western blotting for its sensitivity and specificity. The rabbit anti-TccP polyclonal antibody was then applied in the study on the localization of TccP within the host cells adhered by EHEC O157:H7. The sequence of TccP cDNA we amplified was the same as reported by GenBank. The recombinant prokaryotic expression vector pET28a-TccP was constructed successfully. Western blotting revealed that M(r); of the target protein expressed in E.coli BL21(DE3) was 37 000 and the rabbit anti-TccP polyclonal antibody had a specific reaction with the target protein, which demonstrated that the recombinant protein and its polyclonal antibody were prepared successfully. Immunofluorescence detection using rabbit anti-TccP polyclonal antibody showed that TccP aggregated in the cell membrane of the host cell adhered by EHEC O157:H7. We successfully prepared the recombinant vector pET28a-TccP and the anti-TccP polyclonal antibody and applied the antibody to confirm the localization of TccP in EHEC O157:H7 adhesion host cells.

  11. Isoflavone aglycone content and the thermal, functional, and structural properties of soy protein isolates prepared from hydrothermally treated soybeans.

    Science.gov (United States)

    Wally-Vallim, Ana Paula; Vanier, Nathan Levien; Zavareze, Elessandra da Rosa; Zambiazi, Rui Carlos; de Castro, Luis Antônio Suita; Schirmer, Manoel Artigas; Elias, Moacir Cardoso

    2014-07-01

    Soybeans were hydrothermally treated at 2 different temperatures (40 °C and 60 °C) and for 4 different hydration times (4, 8, 12, and 16 h) to (i) increase the isoflavone aglycone content in a soy protein isolate and (ii) evaluate the changes in thermal, functional, and structural properties of a soy protein isolate as a function of hydrothermal treatment conditions. Our study is the first to evaluate aglycone content, extraction yield, β-glucosidase activity, differential scanning calorimetry, protein digestibility, scanning electron microscopy, water absorption capacity (WAC), foaming capacity (FC), and foaming stability of soy protein isolates prepared from hydrothermally treated soybeans. For aglycone enhancement and the extraction yield maintenance of soy protein isolates, the condition of 40 °C for 12 h was the best soybean hydrothermal treatment. The structural rearrangement of proteins that occurred with the hydrothermal treatment most likely promoted the capacity of proteins to bind to aglycone. Moreover, the structure shape and size of soy protein isolates verified by scanning electron microscopy appears to be related to the formation of hydrophobic surfaces and hydrophobic zones at 40 °C and 60 °C, respectively, affecting the protein digestibility, WAC, and FC of soy protein isolates. The aglycone content in the soy protein isolate can be improved with the hydrothermal treatment of soybeans. The temperature and time used for hydrothermal treatment must be selected in order to achieve a soy protein isolate with high aglycone content, extraction yield, and functionality. This technology is suitable for providing healthier soy protein isolates for food industry with improved functional and structural properties. © 2014 Institute of Food Technologists®

  12. In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

    Full Text Available Abstract Background Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. Results Glutathione was conjugated to low molecular weight poly(ethylene glycol diacrylate (PEGDA via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH homogenates, we were able to purify a glutathione S-transferase (GST green fluorescent protein (GFP fusion protein (GST-GFP from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. Conclusions GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

  13. Preparation and thermo-reversible gelling properties of protein isolate from defatted Antarctic krill (Euphausia superba) byproducts.

    Science.gov (United States)

    Wang, Yanchao; Wang, Ruo; Chang, Yaoguang; Gao, Ying; Li, Zhaojie; Xue, Changhu

    2015-12-01

    Protein isolate was prepared from defatted Antarctic krill (Euphausia superba) byproducts by 0.1 M NaOH extraction. Maximum yield of krill protein isolate reached 28.66% by precipitation at pH 4.6. Krill protein isolate demonstrated its excellent nutritional values through amino acid composition and in vitro digestibility. Thermal transition of krill protein isolate was determined by differential scanning calorimetry measurement. Extrapolated values of glass transition temperature (Tg) and denaturation temperature (Td) of krill protein isolate were 33.8 °C and 80.3 °C when the heating rate was 2 °C/min. Dispersions of krill protein isolate generated self-supported gels at concentrations above 100 g/L without the addition of salt or other additives. A noticeable enhancement of gel strength was induced through cooling. Gels with krill protein isolate displayed a thermo-reversible behavior under repeating heating/cooling cycles, which was primarily due to the formation and disruption of hydrogen bonds. Network strength of protein gels was strongly dependent on protein concentrations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Numerical prediction of micro-channel LD heat sink operated with antifreeze based on CFD method

    Science.gov (United States)

    Liu, Gang; Liu, Yang; Wang, Chao; Wang, Wentao; Wang, Gang; Tang, Xiaojun

    2014-12-01

    To theoretically study the feasibility of antifreeze coolants applied as cooling fluids for high power LD heat sink, detailed Computational Fluid Dynamics (CFD) analysis of liquid cooled micro-channels heat sinks is presented. The performance operated with antifreeze coolant (ethylene glycol aqueous solution) compared with pure water are numerical calculated for the heat sinks with the same micro-channels structures. The maximum thermal resistance, total pressure loss (flow resistance), thermal resistance vs. flow-rate, and pressure loss vs. flow-rate etc. characteristics are numerical calculated. The results indicate that the type and temperature of coolants plays an important role on the performance of heat sinks. The whole thermal resistance and pressure loss of heat sinks increase significantly with antifreeze coolants compared with pure water mainly due to its relatively lower thermal conductivity and higher fluid viscosity. The thermal resistance and pressure loss are functions of the flow rate and operation temperature. Increasing of the coolant flow rate can reduce the thermal resistance of heat sinks; meanwhile increase the pressure loss significantly. The thermal resistance tends to a limit with increasing flow rate, while the pressure loss tends to increase exponentially with increasing flow rate. Low operation temperature chiefly increases the pressure loss rather than thermal resistance due to the remarkable increasing of fluid viscosity. The actual working point of the cooling circulation system can be determined on the basis of the pressure drop vs. flow rate curve for the micro-channel heat sink and that for the circulation system. In the same system, if the type or/and temperature of the coolant is changed, the working point is accordingly influenced, that is, working flow rate and pressure is changed simultaneously, due to which the heat sink performance is influenced. According to the numerical simulation results, if ethylene glycol aqueous

  15. Model to predict inhomogeneous protein-sugar distribution in powders prepared by spray drying

    NARCIS (Netherlands)

    Grasmeijer, Niels; Frijlink, Henderik W.; Hinrichs, Wouter L. J.

    2016-01-01

    A protein can be stabilized by spray drying an aqueous solution of the protein and a sugar, thereby incorporating the protein into a glassy sugar matrix. For optimal stability, the protein should be homogeneously distributed inside the sugar matrix. The aim of this study was to develop a model that

  16. Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

    Directory of Open Access Journals (Sweden)

    Zheng Xiaojuan

    2009-10-01

    Full Text Available Abstract Background Two-dimensional gel electrophoresis (2-DE is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF, a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results An extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAPS, 50 mM dithiothreitol (DTT, 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF, 20 U/ml Deoxyribonuclease I (DNase I, and 0.25 mg/ml Ribonuclease A (RNase A, combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF. When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found. Conclusion These results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

  17. Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications

    Directory of Open Access Journals (Sweden)

    Gorbatiuk O. B.

    2015-04-01

    Full Text Available Aim. Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys for preparation of affinity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affinity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purified and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affinity chromatography stationary phase was 10–12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purification procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affinity chromatography stationary phase and formation of the bioselective element of immunosensor.

  18. Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants.

    Science.gov (United States)

    Lee, Hyelee; Liu, Shih-Yuan

    2017-03-25

    We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

  19. Nanomaterials for efficiently lowering the freezing point of anti-freeze coolants.

    Science.gov (United States)

    Hong, Haiping; Zheng, Yingsong; Roy, Walter

    2007-09-01

    In this paper, we report, for the first time, the effect of the lowered freezing point in a 50% water/50% anti-freeze coolant (PAC) or 50% water/50% ethylene glycol (EG) solution by the addition of carbon nanotubes and other particles. The experimental results indicated that the nano materials are much more efficient (hundreds fold) in lowering the freezing point than the regular ionic materials (e.g., NaCl). The possible explanation for this interesting phenomenon is the colligative property of fluid and relative small size of nano material. It is quite certain that the carbon nanotubes and metal oxide nano particles could be a wonderful candidate for the nano coolant application because they could not only increase the thermal conductivity, but also efficiently lower the freezing point of traditional coolants.

  20. Preparation and characterization of baru (Dipteryx alata Vog) nut protein isolate and comparison of its physico-chemical properties with commercial animal and plant protein isolates.

    Science.gov (United States)

    Nunes, Ângela A; Favaro, Simone P; Miranda, Cesar H B; Neves, Valdir A

    2017-01-01

    The Brazilian leguminous tree locally known in the Cerrado Biome as baru (Dipteryx alata Vog), provides a healthy edible oil source. The proteinaceous cake remaining after oil extraction could be transformed into new products to foodstuff development, such as protein concentrates and isolates, adding value to the production chain. In this study, it is described the preparation and characterization of baru nut protein isolate (BPI) from deffated baru flour, and measurements of its functional, nutritional, and thermal properties, in comparison to the more common vegetable (soybeans) and animal (casein and albumin) protein sources of the food industry. BPI presented higher protein content than soybean, casein and albumin commercial protein isolates, despite losses of albumins and low molecular weight globulins during the isolation procedure. Thermodynamics studies suggested that BPI has a well-conserved protein arrangement and lower thermostability than the other protein sources. BPI showed high in vitro digestibility and suitable and desirable functional properties such as water and oil absorption capacity, emulsifying activity, and foam formation and stability at mild and neutral pH. BPI could be used either as a substitute ingredient in oily food formulations or in the development of new products of its own. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  1. Preparation of Low Allergenic Protein Concentrated Natural Rubber Latex Using Suitable Low Molecular Weight Cellulose Derivatives Induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Siri-Upathum, Chyagrit; Boonyawat, Jariya

    2007-08-01

    Full text: Low molecular weight carboxy methyl cellulose (CMC), hydroxyl ethyl cellulose (HEC), hydroxyl propyl cellulose (HPC) and methyl cellulose (MC) prepared by radiation-induced degradation were added into diluted natural concentrated latex prior to centrifuge for a purpose of reducing allergenic rubber protein in the latex. Optimum molecular weight (Mv) of CMC and HEC for such a purpose was found to be 17-18 kDa which decreased allergenic rubber protein (14-94 kDa) to an undetectable amount as determined by SDS PAGE method

  2. Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

    Science.gov (United States)

    Chow-Shi-Yée, Mélanie; Briard, Jennie G; Grondin, Mélanie; Averill-Bates, Diana A; Ben, Robert N; Ouellet, François

    2016-05-01

    Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells. © 2016 The Protein Society.

  3. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    International Nuclear Information System (INIS)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

    2007-01-01

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

  4. Kinetic Effects on Self-Assembly and Function of Protein-Polymer Bioconjugates in Thin Films Prepared by Flow Coating

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Dongsook [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA; Huang, Aaron [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA; Olsen, Bradley D. [Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave Cambridge MA 02142 USA

    2016-11-04

    The self-assembly of nanostructured globular protein arrays in thin films is demonstrated using protein–polymer block copolymers based on a model protein mCherry and the polymer poly(oligoethylene glycol acrylate) (POEGA). Conjugates are flow coated into thin films on a poly(ethylene oxide) grafted Si surface, forming self-assembled cylindrical nanostructures with POEGA domains selectively segregating to the air–film interface. Long-range order and preferential arrangement of parallel cylinders templated by selective surfaces are demonstrated by controlling relative humidity. Long-range order increases with coating speed when the film thicknesses are kept constant, due to reduced nucleation per unit area of drying film. Fluorescence emission spectra of mCherry in films prepared at <25% relative humidity shows a small shift suggesting that proteins are more perturbed at low humidity than high humidity or the solution state.

  5. Preparation of gluten-free bread using a meso-structured whey protein particle system

    NARCIS (Netherlands)

    Riemsdijk, van L.E.; Goot, van der A.J.; Hamer, R.J.; Boom, R.M.

    2011-01-01

    This article presents a novel method for making gluten-free bread using mesoscopically structured whey protein. The use of the meso-structured protein is based on the hypothesis that the gluten structure present in a developed wheat dough features a particle structure on a mesoscopic length scale

  6. The Protein Data Bank at 40: Reflecting on the Past to Prepare for the Future

    OpenAIRE

    Berman, Helen M.; Kleywegt, Gerard J.; Nakamura, Haruki; Markley, John L.

    2012-01-01

    A symposium celebrating the 40th anniversary of the Protein Data Bank archive (PDB), organized by the Worldwide Protein Data Bank, was held at Cold Spring Harbor Laboratory (CSHL) October 28–30, 2011. PDB40’s distinguished speakers highlighted four decades of innovation in structural biology, from the early era of structural determination to future directions for the field.

  7. Preparation, composition and functional properties of pennycress (Thlaspi arvense L.) seed protein isolates

    Science.gov (United States)

    This study evaluated two methods, saline extraction (SE) and conventional acid precipitation (AP), to recover proteins from pennycress (Thlaspi arvense L.) seed meal. SE was done using 0.1 M NaCl at 50ºC while AP involved alkaline extraction (pH 10) first followed by protein precipitation at pH 4. C...

  8. Detecting seasonal variation of antifreeze protein distribution in Rhagium mordax using immunofluorescence and high resolution microscopy

    DEFF Research Database (Denmark)

    Buch, Johannes Lørup; Ramløv, Hans

    2017-01-01

    with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than...

  9. Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation

    Directory of Open Access Journals (Sweden)

    Qie Kuang

    2015-01-01

    Full Text Available Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.

  10. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Gao, Xiong-Zhuo; Li, Lan-Fen; Su, Xiao-Dong; Zhao, XiaoJun; Liang, Yu-He

    2007-01-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°

  11. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiong-Zhuo [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Li, Lan-Fen; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, XiaoJun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

    2007-10-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.

  12. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins: A case study of a bacterially expressed recombinant biopharmaceutical protein.

    Science.gov (United States)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per; Holmbjerg, Anne Fich; Grimstrup, Marie; Mørtz, Ejvind; Kofoed, Thomas; Højrup, Peter

    2018-07-01

    Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Huihui; Qian, Bin; Zhang, Wei [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); Lan, Minbo, E-mail: minbolan@ecust.edu.cn [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2016-02-15

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU–PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU–PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU–PVP (6.0 h) film reduced greatly to 0.08 μg/cm{sup 2}, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  14. The in vitro antioxidant properties of alcalase hydrolysate prepared from silkie fowl (Gallus gallus) blood protein.

    Science.gov (United States)

    Cheng, Fu-Yuan; Lai, I-Chun; Lin, Liang-Chuan; Sakata, Ryoichi

    2016-07-01

    Two types of proteins including blood plasma protein and blood cell protein were isolated from silkie fowl (Gallus gallus) blood and hydrolyzed using alcalase for 0, 2, 4 and 6 h. The blood plasma protein hydrolysate (BPH) and blood cell protein hydrolysate (BCH) were analyzed for pH value, peptide content and antioxidative properties. The significantly higher peptide contents were observed in BPH than that of BCH, which showed that blood plasma protein was more suitable to hydrolysis by alcalase than blood cell protein. Both BPH and BCH showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and Fe(2+) chelating ability. BPH at 4 h of hydrolysis (BPH4) demonstrated significantly higher antioxidant capacity than those treated by alcalase in most of the assays. The BPH4 was separated using ultra-filtration and assessment of the fractions and indicated that low molecular weight of peptides (< 3 kDa) possessed greater DPPH scavenging activity, Fe(2+) chelating ability and inhibitory activity of lipid peroxidation. These results show that BPH has the potential to be ingredients in the food industry as a replacement of synthetic antioxidants. © 2015 Japanese Society of Animal Science.

  15. Effect of Different Tumbling Marination Methods and Time on the Water Status and Protein Properties of Prepared Pork Chops

    Directory of Open Access Journals (Sweden)

    Tian Gao

    2015-07-01

    Full Text Available The combined effect of tumbling marination methods (vacuum continuous tumbling marination, CT; vacuum intermittent tumbling marination, IT and effective tumbling time (4, 6, 8, and 10 h on the water status and protein properties of prepared pork chops was investigated. Results showed that regardless of tumbling time, CT method significantly decreased the muscle fiber diameter (MD and significantly increased the total moisture content, product yield, salt soluble proteins (SSP solubility, immobilized water component (p<0.05 compared with IT method. With the effective tumbling time increased from 4 h to 10 h, the fat content and the MD were significantly decreased (p<0.05, whereas the SSP solubility of prepared pork chops increased firstly and then decreased. Besides, an interactive effect between CT method and effective tumbling time was also observed for the chemical composition and proportion of immobilized water (p<0.05. These results demonstrated that CT method of 8 h was the most beneficial for improving the muscle structure and water distribution status, increasing the water-binding capacity and accelerating the marinade efficiency of pork chops; and thus, it should be chosen as the most optimal treatment method for the processing production of prepared pork chops.

  16. imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation.

    Science.gov (United States)

    Zhao, Qun; Fang, Fei; Wu, Ci; Wu, Qi; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-03-17

    An integrated sample preparation method, termed "imFASP", which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50-500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Food emulsion type oil in water prepared with high-protein from shrimp (Penaeus vannamei heads flour – SHF

    Directory of Open Access Journals (Sweden)

    Yuli Cano

    2017-09-01

    Full Text Available The use of flour from shrimp (Penaeus vannamei heads with a high content of protein (SHF to stabilize food emulsions type oil in water (o/w is an alternative to take advantage of the by-products of the shrimp industry. The aim of this work was to prepare food emulsion type oil in water (o/w using the SHF due to the high percentage in proteins; for this procedure a physicochemical and bromatological characterization of flour of shrimps (Penaeus vannamei heads has been done, in which a percentage of protein 51 %, moisture of 11,82 %, fat 8,52 % and 22,23 % of ash has been obtained. The base emulsions may be used in food products such as salad dressing, mayonnaise, spreads, dressings and other products. The different emulsions with adequate rheological and microstructural characteristics were prepared using different concentrations of palm oil (20, 30 and 40%w/w and different concentrate of SHF (0,5, 1 and 2 % w/w. Therefore, we have obtained a food emulsion stable type oil in water (O/W with 2 % w/w of SHF, which presented a behavior non-Newtonian fluid type shear-thinning and homogeneous distribution of droplets.

  18. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.

    Science.gov (United States)

    Feist, Peter; Hummon, Amanda B

    2015-02-05

    Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

  19. Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

    Science.gov (United States)

    Feist, Peter; Hummon, Amanda B.

    2015-01-01

    Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed. PMID:25664860

  20. Systematic study on the preparation of a food grade soybean protein

    Energy Technology Data Exchange (ETDEWEB)

    Bazinet, L.; Labrecque, R.; Toupin, R. [Institut de Recherche d`Hydro-Quebec, Varennes, PQ (Canada); Boulet, M.; Ippersiel, D.; Lamarche, F. [Food Research and Development Centre, St. Hyacinthe, PQ (Canada)

    1996-05-01

    A new application of electrodialysis for the precipitation and separation of soy proteins, called electro-acidification, was evaluated. The compositional properties of electro-acidified proteins were compared to those of commercial standards. Since protein solutions have physico-chemical properties that make their behaviour difficult to predict, the limiting current, and the factors which influence them, were determined. These factors included KCl concentration, protein concentration, the recirculation rate in the stack, the concentration of added salt and the temperature of the protein solution. Three approaches to electro-acidification were developed, i. e., bipolar-membrane (most rapid with the lowest energy consumption), alternating anionic and cationic membranes (most time-consuming), and anodic proton generation (intermediate between the other two). The compositional quality of electro-acidified samples was shown to be superior or equal to that of commercial standards as confirmed by the solubility profile of the products. It was therefore concluded that using electrodialysis to produce protein isolates was technologically feasible and energy efficient. 76 refs., 4 tabs., 33 figs.

  1. The impact of particle preparation methods and polymorphic stability of lipid excipients on protein distribution in microparticles

    DEFF Research Database (Denmark)

    Liu, Jingying; Christophersen, Philip C; Yang, Mingshi

    2017-01-01

    into SLM prepared with different excipients, i.e. trimyristin (TG14), glyceryl distearate (GDS), and glyceryl monostearate (GMS), by water-oil-water (w/o/w) or solid-oil-water (s/o/w) method. The distribution of lysozyme in SLM and the release of the protein from SLM were evaluated by confocal laser...... formulated by the w/o/w method or evenly distributed in TG14 SLM prepared by the s/o/w method. Stability study at 37 °C revealed that only TG14 SLM made by the w/o/w method was able to maintain the lysozyme amount both on the particle surface and released from the SLM. Elevated storage temperature induced...

  2. Preparation and characterization of protein isolate from Yellowfin tuna Thunnus albacares roe by isoelectric solubilization/precipitation process

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-05-01

    Full Text Available Abstract Isoelectric solubilization/precipitation (ISP processing allows selective, pH-induced water solubility of proteins with concurrent separation of lipids and removal of materials not intended for human consumption such as bone, scales, skin, etc. Recovered proteins retain functional properties and nutritional value. Four roe protein isolates (RPIs from yellowfin tuna roe were prepared under different solubilization and precipitation condition (pH 11/4.5, pH 11/5.5, pH 12/4.5 and pH 12/5.5. RPIs contained 2.3–5.0 % moisture, 79.1–87.8 % protein, 5.6–7.4 % lipid and 3.0–3.8 % ash. Protein content of RPI-1 and RPI-2 precipitated at pH 4.5 and 5.5 after alkaline solubilization at pH 11, was higher than those of RPI-3 and RPI-4 after alkaline solubilization at pH 12 (P < 0.05. Lipid content (5.6–7.4 % of RPIs was lower than that of freeze-dried concentrate (10.6 %. And leucine and lysine of RPIs were the most abundant amino acids (8.8–9.4 and 8.5–8.9 g/100 g protein, respectively. S, Na, P, K as minerals were the major elements in RPIs. SDS-PAGE of RPIs showed bands at 100, 45, 25 and 15 K. Moisture and protein contents of process water as a 2’nd byproduct were 98.9–99.0 and 1.3–1.8 %, respectively. Therefore, yellowfin tuna roe isolate could be a promising source of valuable nutrients for human food and animal feeds.

  3. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

    Science.gov (United States)

    Mackin, Robert B

    2014-01-01

    The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.

  4. Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".

    Science.gov (United States)

    Wiśniewski, Jacek R

    2017-01-01

    Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the "Total Protein Approach."

  5. Influence of Bovine Whey Protein Concentrate and Hydrolysate Preparation Methods on Motility in the Isolated Rat Distal Colon

    Science.gov (United States)

    Dalziel, Julie E.; Anderson, Rachel C.; Bassett, Shalome A.; Lloyd-West, Catherine M.; Haggarty, Neill W.; Roy, Nicole C.

    2016-01-01

    Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response. PMID:27983629

  6. Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes.

    Science.gov (United States)

    Near, Thomas J; Dornburg, Alex; Kuhn, Kristen L; Eastman, Joseph T; Pennington, Jillian N; Patarnello, Tomaso; Zane, Lorenzo; Fernández, Daniel A; Jones, Christopher D

    2012-02-28

    The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6-5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

  7. Effects of seed preparation and oil pressing on milkweed (Asclepias spp.) protein functional properties

    Science.gov (United States)

    The effects of seed cooking and oil processing conditions on functional properties of milkweed seed proteins were determined to identify potential value-added uses for the meal. Milkweed seeds were flaked and then cooked in the seed conditioner at 82°C for 30, 60 or 90 min. Oil was extracted by scre...

  8. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-01-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

  9. A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

    Directory of Open Access Journals (Sweden)

    Matthew P. Padula

    2017-04-01

    Full Text Available Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE. This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

  10. Encapsulation of flaxseed oil using a benchtop spray dryer for legume protein-maltodextrin microcapsule preparation.

    Science.gov (United States)

    Can Karaca, Asli; Low, Nicholas; Nickerson, Michael

    2013-05-29

    Flaxseed oil was microencapsulated employing a wall material matrix of either chickpea (CPI) or lentil protein isolate (LPI) and maltodextrin using a benchtop spray dryer. Effects of emulsion formulation (oil, protein and maltodextrin levels) and protein source (CPI vs LPI) on the physicochemical characteristics, oxidative stability, and release properties of the resulting capsules were investigated. Microcapsule formulations containing higher oil levels (20% oil, 20% protein, 60% maltodextrin) were found to have higher surface oil and lower encapsulation efficiencies. Overall, LPI-maltodextrin capsules gave higher flaxseed oil encapsulation efficiencies (∼88.0%) relative to CPI-maltodextrin matrices (∼86.3%). However, both designs were found to provide encapsulated flaxseed oil protection against oxidation over a 25 d room temperature storage study relative to free oil. Overall, ∼37.6% of encapsulated flaxseed oil was released after 2 h under simulated gastric fluid, followed by the release of an additional ∼46.6% over a 3 h period under simulated intestinal fluid conditions.

  11. Fibrous scaffolds loaded with protein prepared by blend or coaxial electrospinning.

    NARCIS (Netherlands)

    Ji, W.; Yang, F.; Beucken, J.J.J.P van den; Bian, Z.; Fan, M.; Chen, Z.; Jansen, J.A.

    2010-01-01

    The aim of the present study was to fabricate polycaprolactone-based nanofibrous scaffolds with incorporated protein via either the blend or coaxial electrospinning technique. Both techniques were compared with respect to processing set-up and scaffold characteristics as well as the release kinetics

  12. Applicability of analytical and preparative monolithic columns to the separation and isolation of major whey proteins.

    Science.gov (United States)

    Albreht, Alen; Vovk, Irena

    2012-03-02

    The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of β-lactoglobulin variant A and B (β-LgA and β-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of β-LgA and β-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5-3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC-MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified β-LgA and β-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein-ligand binding studies. The developed methods

  13. Design and characterization of controlled-release edible packaging films prepared with synergistic whey-protein polysaccharide complexes.

    Science.gov (United States)

    Liu, Fei; Jiang, Yanfeng; Du, Bingjian; Chai, Zhi; Jiao, Tong; Zhang, Chunyue; Ren, Fazheng; Leng, Xiaojing

    2013-06-19

    This paper describes an investigation into the properties of a doubly emulsified film incorporated with protein-polysaccharide microcapsules, which serves as a multifunctional food packaging film prepared using common edible materials in place of petroleum--based plastics. The relationships between the microstructural properties and controlled release features of a series of water-in-oil-in-water (W/O/W) microcapsulated edible films prepared in thermodynamically incompatible conditions were analyzed. The hydrophilic riboflavin (V(B2)) nano-droplets (13-50 nm) dispersed in α-tocopherol (V(E)) oil phase were embedded in whey protein-polysaccharide (WPs) microcapsules with a shell thickness of 20-56 nm. These microcapsules were then integrated in 103 μm thick WPs films. Different polysaccharides, including gum arabic (GA), low-methoxyl pectin (LMP), and κ-carrageenan (KCG), exhibited different in vitro synergistic effects on the ability of both films to effect enteric controlled release of both vitamins. GA, which showed a strong emulsifying ability, also showed better control of V(E) than other polysaccharides, and the highly charged KCG showed better control of V(B2) than GA did.

  14. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    DEFF Research Database (Denmark)

    Houen, G.; Olsen, D.T.; Hansen, P.R.

    2003-01-01

    of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...

  15. Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure

    Czech Academy of Sciences Publication Activity Database

    Mašek, J.; Bartheldyová, E.; Korvasová, Z.; Škrabalová, J.; Koudelka, Š.; Kulich, P.; Kratochvílová, Irena; Miller, A. D.; Ledvina, Miroslav; Raška, M.; Turánek, J.

    2011-01-01

    Roč. 408, č. 1 (2011), s. 95-104 ISSN 0003-2697 R&D Projects: GA ČR(CZ) GAP304/10/1951 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40550506 Keywords : liposome * proteoliposome * detergent removal method * recombinant protein * metallochelatation * AF microscopy * TEM * dynamic light scattering Subject RIV: CE - Biochemistry Impact factor: 2.996, year: 2011

  16. Antioxidant and antimicrobial activity of lecithin free egg yolk protein preparation hydrolysates obtained with digestive enzymes

    Directory of Open Access Journals (Sweden)

    Aleksandra Zambrowicz

    2012-12-01

    Full Text Available ABSTRACT:Several biological activities have now been associated with egg protein- derived peptides, including antihypertensive, antimicrobial, immunomodulatory, anticancer and antioxidantactivities, highlighting the importance of these biopeptides in human health, and disease prevention and treatment. Special attention has been given to peptides with antioxidant and antimicrobial activities as a new source of natural preservatives in food industry. In this study, the antioxidant properties of the egg-yolk protein by-product (YP hydrolysates were evaluated based on their radical scavenging capacity (DPPH, Fe2+chelating effect and ferric reducing power (FRAP. Furthermore, antimicrobial properties of obtained hydrolysates against Bacillus species were studied. The degrees (DHs of hydrolysis for 4h hydrolysates were: 19.1%, 13.5% and 13.0%, for pepsin, chymotrypsin and trypsin, respectively. Pepsin was the most effective in producing the free amino groups (1410.3 μmolGly/g. The RP-HPLC profiles of the protein hydrolysates showed differences in the hydrophobicity of the generated peptides.Trypsin hydrolysate obtained after 4h reaction demonstrated the strongest DPPH free radical scavenging activity (0.85 µmol Troloxeq/mg. Trypsin and chymotrypsin hydrolysates obtained after 4h reaction exhibited 4 times higher ferric reducing capacity than those treated bypepsin. The hydrolysis products obtained from YP exhibited significant chelating activity. The 4h trypsin hydrolysate exhibited weak antimicrobial activity against B. subtilis B3; B. cereus B512; B. cereus B 3p and B. laterosporum B6.

  17. Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging.

    Science.gov (United States)

    Nworu, Chinedu U; Krieg, Paul A; Gregorio, Carol C

    2014-04-01

    Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy. Copyright © 2013. Published by Elsevier Inc.

  18. Preparation of antioxidative corn protein hydrolysates, purification and evaluation of three novel corn antioxidant peptides.

    Science.gov (United States)

    Jin, Du-Xin; Liu, Xiao-Lan; Zheng, Xi-Qun; Wang, Xiao-Jie; He, Jun-Fang

    2016-08-01

    Corn gluten meal is a major co-product of corn wet milling. Corn gluten meal was hydrolyzed with Alcalase, Flavourzyme, Alcalase+Flavourzyme and Flavourzyme+Alcalase. At the substrate concentration of 10%, corn protein hydrolysate catalyzed by Alcalase had a degree of hydrolysis of 17.83%, which was higher than that by Flavourzyme (3.65%). The hydrolysate catalyzed by Alcalase+Flavourzyme exhibited better antioxidant activities and was further purified. Three novel antioxidant peptides were purified by a series of chromatographic techniques. Sequences of the three peptides were identified as Cys-Ser-Gln-Ala-Pro-Leu-Ala, Tyr-Pro-Lys-Leu-Ala-Pro-Asn-Glu and Tyr-Pro-Gln-Leu-Leu-Pro-Asn-Glu, respectively. Among the three peptides, Cys-Ser-Gln-Ala-Pro-Leu-Ala exhibited good reducing power and excellent scavenging capacities for DPPH radical and superoxide anion radical, with IC50 values of 0.116 and 0.39mg/ml, respectively. The results from our study indicate antioxidant potency of corn protein hydrolysates and peptides separated from corn gluten meal and can provide basic understanding for the application of corn protein hydrolysates as natural antioxidants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Replacement of eggs with soybean protein isolates and polysaccharides to prepare yellow cakes suitable for vegetarians.

    Science.gov (United States)

    Lin, Muyang; Tay, Siang Hong; Yang, Hongshun; Yang, Bao; Li, Hongliang

    2017-08-15

    To evaluate the feasibility of substituting eggs in yellow cake by a mixture of soybean proteins, plant polysaccharides, and emulsifiers, the batter properties, including specific gravity and viscosity; cake properties, including specific volume, texture, colour, moisture, microstructures, and structural properties of starch and glutens of the replaced cake and traditional cake containing egg, were evaluated. Replacing eggs with a soy protein isolate and 1% mono-, di-glycerides yielded a similar specific volume, specific gravity, firmness and moisture content (1.92 vs. 2.08cm 3 /g, 0.95 vs. 1.03, 319.8 vs. 376.1g, and 28.03% vs. 29.01%, respectively) compared with the traditional cakes baked with eggs. Structurally, this formulation comprised dominant gliadin aggregates in the size range of 100-200nm and glutenin networking structures containing fewer but larger porosities. The results suggest that a mixture of soybean proteins and emulsifier is a promising substitute for eggs in cakes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Determination of adsorption isotherm parameters for minor whey proteins by gradient elution preparative liquid chromatography.

    Science.gov (United States)

    Faraji, Naeimeh; Zhang, Yan; Ray, Ajay K

    2015-09-18

    Ion-Exchange Chromatography (IEC) techniques have been extensively investigated in protein purification processes, due to the more selective and milder separation steps. To date, existing studies of minor whey proteins fractionation in IEC have primarily been conducted as batch uptake studies, which require more experimental search space, time and materials. In this work, the selected resin's (SP Sepharose FF) equilibrium and dynamic binding capacity were first investigated. Next, adsorption of the pure binary mixture of lactoperoxidase and lactoferrin was studied to calibrate steric mass action (SMA) model using a simplified approach with data from single column experiments. The calibrated model was then verified by performing factorial-design based experiments for various process operating conditions assessing process performance on a larger bed height column. The model predicted results demonstrated a realistic agreement with the experiments providing reproducible column elution profile and reduced experimental work. Finally, whey protein isolate was used to evaluate model parameters in real conditions. Results obtained herein are suitable for future large scale applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Jeremy Potriquet

    Full Text Available To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate, providing processing times similar to FASP for the processing of up to 96 samples per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.

  2. Preparation of dichlorvos-protein complete antigen by Mannich-type reaction

    Science.gov (United States)

    Feng, Qianqian; Xu, Ying; Zhou, Youxiang; Lu, Liang; Chen, Fusheng; Wang, Xiaohong

    2010-08-01

    Dichlorvos (DDVP) residues have been linked to substantial adverse health effects on several organ systems. To ensure food safety, rapid and low-cost immunological methods must be applied to detect DDVP residues in food. In immunological methods, a key step is coupling DDVP to carrier proteins to obtain a complete antigen due to DDVP being hapten. In the current research, DDVP was coupled with cationized bovine serum albumin (cBSA) using a method based on Mannich-type reaction. A DDVP-cBSA conjugate, with a molar ratio of 40:1 DDVP to cBSA was synthesized. The cationized proteins and their conjugates were identified by UV-Vis and FT-IR spectra, which showed the characteristic bands of the ethylenediamine group and DDVP, respectively. BALB/c mice were immunized with DDVP-cBSA. One hybridoma cell line secreted anti-DDVP monoclonal antibody (Mab) that had high sensitivity and specificity for DDVP. Competitive ELISA identified an IC50 of 600 ng/mL and a limit of detection of 1 ng/mL in aqueous solution. The Mab had some cross-reactivity with phosmet, but no cross-reactivity with chlorothalonil and procymidone. We also detected a trace of DDVP in waste water. In conclusion the Mannich-type reaction couples DDVP to protein, yielding an antigen for the production of Mab to detect residual DDVP in the environment.

  3. Molecularly imprinted polymers prepared using protein-conjugated cleavable monomers followed by site-specific post-imprinting introduction of fluorescent reporter molecules.

    Science.gov (United States)

    Suga, Yusuke; Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2013-10-04

    Molecularly imprinted polymers were prepared using a protein-conjugated disulfide cleavable monomer. After removing the protein by disulfide reduction, a thiol-reactive fluorophore was introduced into the thiol residue located only inside the imprinted cavity, resulting in specific transduction of the binding events into fluorescence spectral change.

  4. [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

    Science.gov (United States)

    Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

    2011-08-01

    We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

  5. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

    /ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used as the first protein screening method in many laboratories because of its inherent simplicity, mass accuracy, sensitivity and relatively high sample throughput. We present a simplified sample preparation method for MALDI-MS that enables in-gel digestion...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...... of protein samples directly on the MALDI-MS metal probe. Removal of detergent and reagents as well as protein reduction and S-alkylation were performed prior to cutting of protein samples from the polyacrylamide gel slab. The general utility of this approach was demonstrated by on-probe digestion and MALDI...

  6. Influence of the liposomal preparation Butaintervite on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning

    OpenAIRE

    M. I. Hariv; B. V. Gutyj

    2016-01-01

    This article presents the results of research into the influence of the complex liposomal preparation Butaintervit on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning. Intramuscular injection of carbon tetrachloride into rats at a dose of 0.25 ml per100 gof body weight causes antigenic load on the body and leads to disruption of protein synthesis function in the liver. This is shown by reduction in blood levels of total protein and its fra...

  7. Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy.

    Science.gov (United States)

    Cameron Varano, A; Harafuji, Naoe; Dearnaley, William; Guay-Woodford, Lisa; Kelly, Deborah F

    2017-01-01

    Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

  8. Preparation of 125I-protein A usable for up to 10 months in immunoassays

    DEFF Research Database (Denmark)

    Dyrberg, T; Billestrup, Nils

    1984-01-01

    Chloramine-T iodination of protein A from Staphylococcus aureus and gel electrophoretic purification of the iodination mixture results in a stable tracer of high specific and functional activity. Following repeated gel electrophoresis of the tracer only a single component was observed. The specif...... weeks resulted in a moderate decrease in maximal binding to immunoglobulin (from 91% to 64%), in TCA precipitable radioactivity (from 97% to 80%) and an approx. 30% decrease in the ability to detect cell bound immunoglobulin. It is concluded that gel electrophoretic purification of 125I...

  9. Protein Compatible Polymer Brushes on Polymeric Substrates Prepared by Surface-Initiated Transfer Radica Polymerization

    DEFF Research Database (Denmark)

    Fristrup, Charlotte Juel; Eskimergen, Rüya; Burkrinsky, J.T.

    2008-01-01

    have been made with model systems of poly(ether ether ketone) (PEEK) films as they can easily be functionalized [1]. Moreover, the inert material polypropylene has successfully beel! activated using a photochemical method [2]. Different polymers including PEG-like matenals have been investigated...... when the PEEK films were modified. The surface roughness should either be unchanged or decreased as it 'will affect the protein adsorption [3]. 1. O. Noiset, C. Henneuse, Y.-J. Schneider, J. Marchand-Brynaert Macromolecules 30 (1997) 540-548 2. J. Huang, H. Murata, R.R. Koepsel, A.J. Russell, K...

  10. Preparation and Characterization of Chitosan/Soy Protein Isolate Nanocomposite Film Reinforced by Cu Nanoclusters

    Directory of Open Access Journals (Sweden)

    Kuang Li

    2017-06-01

    Full Text Available Soy protein isolate (SPI based films have received considerable attention for use in packaging materials. However, SPI-based films exhibit relatively poor mechanical properties and water resistance ability. To tackle these challenges, chitosan (CS and endogenous Cu nanoclusters (NCs capped with protein were proposed and designed to modify SPI-based films. Attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction patterns of composite films demonstrated that interactions, such as hydrogen bonds in the film forming process, promoted the cross-linking of composite films. The surface microstructure of CS/SPI films modified with Cu NCs was more uniform and transmission electron microscopy (TEM showed that uniform and discrete clusters were formed. Compared with untreated SPI films, the tensile strength and elongation at break of composite films were simultaneously improved by 118.78% and 74.93%, respectively. Moreover, these composite films also exhibited higher water contact angle and degradation temperature than that of pure SPI film. The water vapor permeation of the modified film also decreased. These improved properties of functional bio-polymers show great potential as food packaging materials.

  11. Preparation of mayonnaise from extracted plant protein isolates of chickpea, broad bean and lupin flour: chemical, physiochemical, nutritional and therapeutic properties.

    Science.gov (United States)

    Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Ereifej, Khalil; Gammoh, Sana; Kubow, Stan; Tawalbeh, Deia

    2017-05-01

    This investigation was aimed to study the molecular, physico-chemical, and biofunctional health properties of mayonnaise prepared using proteins isolated from broad bean, lupin and chickpea flour. Proteins were isolated from chickpea (CPPI), broad bean (BBPI) and lupin (LPPI) flour and assessed for molecular, physico-chemical, biofunctional, and protein yield. The highest water holding capacity, foaming stability, emulsion stability as well as protein yield and protein content of 44.0, 70.8, 37.5, 81.2, and 36.4, respectively were observed for BBPI. Mayonnaise prepared from the isolated plant proteins was evaluated for chemical composition, molecular properties of the protein subunits, and potential nutraceutical properties. Preparation of mayonnaise using BBPI or a mixture of either BBPI and CPPI or BBPI and LPPI showed superior values for lightness and lowered values for redness. Mayonnaise prepared from either BBPI or the BBPI and CPPI mixture showed the best antioxidant, antihypertensive and antidiabetic properties. The present study results indicated that the use of the BBPI and CPPI mixture can be a novel technological approach for the development of a mayonnaise with improved health promoting properties.

  12. Characterization of type IV antifreeze gene in Nile tilapia (Oreochromis niloticus) and influence of cold and hot weather on its expression and some immune-related genes.

    Science.gov (United States)

    Ammar, Asmma Y; El Nahas, Abeer F; Mahmoud, Shawky; Barakat, Mohamed E; Hassan, Asmaa M

    2018-04-01

    The aim of this work is to study the effect of the thermal stress of ambient temperature during winter and summer on the expression of type IV antifreeze gene (ANF IV) in different tissues of Nile tilapia (Oreochromis niloticus) as well as some immune-related genes. At first, genomic ANF IV gene was characterized from one fish; 124 amino acids were identified with 92.7% similarity with that on the gene bank. Expression of ANF IV and immune-related genes were done twice, once at the end of December (winter sample, temperature 14 °C) and the other at August (summer sample, temperature 36 °C). Assessment of ANF IV gene expression in different organs of fish was done; splenic mRNA was used for assessment of immune-related gene transcripts (CXCl2 chemokine, cc-chemokine, INF-3A, and MHC IIβ). Winter expression analysis of AFP IV in O. niloticus revealed significant upregulation of mRNA transcript levels in the intestine, gills, skin, spleen, liver, and brain with 324.03-, 170.06-, 107.63-, 97.61-, 94.35-, and 27.85-folds, respectively. Furthermore, upregulation in the gene was observed in some organs during summer: in the liver, gills, skin, intestine, and brain with lower levels compared with winter. The level of expression of immune-related genes in winter is significantly higher than summer in all assessed genes. Cc-chemokine gene expression was the most affected in both winter and summer. Variable expression profile of ANF IV in different organs and in different seasons together with its amino acid similarity of N-terminal and C-terminal with apolipoprotein (lipid binder) and form of high-density lipoprotein (HDL) suggests a different role for this protein which may be related to lipid metabolism.

  13. Depot injectable biodegradable nanoparticles loaded with recombinant human bone morphogenetic protein-2: preparation, characterization, and in vivo evaluation

    Directory of Open Access Journals (Sweden)

    Hassan AH

    2015-07-01

    Full Text Available Ali Habiballah Hassan,1 Khaled Mohamed Hosny,2,3 Zuahir A Murshid,1 Adel Alhadlaq,4 Ahmed Alyamani,5 Ghada Naguib6 1Department of Orthodontics, Faculty of Dentistry, 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt; 4Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh, 5Department of Oral Surgery, 6Department of Restorative Dentistry, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Objective: The aim of this study is to utilize the biocompatibility characteristics of biodegradable polymers, viz, poly lactide-co-glycolide (PLGA and polycaprolactone (PCL, to prepare sustained-release injectable nanoparticles (NPs of bone morphogenetic protein-2 (BMP-2 for the repair of alveolar bone defects in rabbits. The influence of formulation parameters on the functional characteristics of the prepared NPs was studied to develop a new noninvasive injectable recombinant human BMP-2 (rhBMP-2 containing grafting material for the repair of alveolar bone clefts.Materials and methods: BMP-2 NPs were prepared using a water-in-oil-in-water double-emulsion solvent evaporation/extraction method. The influence of molar ratio of PLGA to PCL on a suitable particle size, encapsulation efficiency, and sustained drug release was studied. Critical size alveolar defects were created in the maxilla of 24 New Zealand rabbits divided into three groups, one of them treated with 5 µg/kg of rhBMP-2 NP formulations.Results: The results found that NPs formula prepared using blend of PLGA and PCL in 4:2 (w/w ratio showed the best sustained-release pattern with lower initial burst, and showed up to 62.7% yield, 64.5% encapsulation efficiency, 127 nm size, and more than 90% in vitro release. So, this formula was selected for

  14. Preparation of ferrous chelate of hairtail (Trichiurus haumela) protein hydrolysate (Fe(II)-HPH) and its antibacterial activity

    Science.gov (United States)

    Lin, Huimin; Zhang, Bin; Yu, Tian; Deng, Shanggui

    The preparation of a ferrous chelate of hairtail (Trichiurus haumela) protein hydrolysate (Fe(II)-HPH) and its antibacterial activity were studied. The optimal conditions of hydrolysis by papain and ferrous chelation were obtained by single-factor experiments and orthogonal test, with the antibacterial activities as the index. In addition, the antibacterial activity of Fe(II)-HPH was evaluated using the Plackett-Burman design. The orthogonal test results showed that Fe(II)-HPH had an antibacterial activity of 98.3% under a temperature of 40 °C at pH 6.5 for an enzymolysis duration of eight hours in the presence of 20,000 U/g of enzyme. The Plackett-Burman design analysis showed that the three most significant factors (P < 0.05) influencing the antibacterial activity of Fe(II)-HPH were pH, the concentration (mg/mL), and presence of magnesium sulfate.

  15. Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins.

    Science.gov (United States)

    Schaffer, Miroslava; Mahamid, Julia; Engel, Benjamin D; Laugks, Tim; Baumeister, Wolfgang; Plitzko, Jürgen M

    2017-02-01

    While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Preparation and Characterization of Soluble Eggshell Membrane Protein/PLGA Electrospun Nanofibers for Guided Tissue Regeneration Membrane

    Directory of Open Access Journals (Sweden)

    Jun Jia

    2012-01-01

    Full Text Available Guided tissue regeneration (GTR is a widely used method in periodontal therapy, which involves the placement of a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells. The objective of this study is to prepare and evaluate a new type of soluble eggshell membrane protein (SEP/poly (lactic-co-glycolic acid (PLGA nanofibers using electrospinning method for GTR membrane application. SEP/PLGA nanofibers were successfully prepared with various blending ratios. The morphology, chemical composition, surface wettability, and mechanical properties of the nanofibers were characterized using scanning electron microscopy (SEM, contact angle measurement, Fourier transform-infrared spectroscopy (FTIR, and a universal testing machine. L-929 fibroblast cells were used to evaluate the biocompatibility of SEP/PLGA nanofibers and investigate the interaction between cells and nanofibers. Results showed that the SEP/PLGA electrospun membrane was composed of uniform, bead-free nanofibers, which formed an interconnected porous network structure. Mechanical property of SEP has been greatly improved by the addition of PLGA. The biological study results showed that SEP/PLGA nanofibers could enhance cell attachment, spreading, and proliferation. The study indicated the potential of SEP/PLGA nanofibers for GTR application and provided a basis for future optimization.

  17. Preparation and properties of BSA-loaded microspheres based on multi-(amino acid) copolymer for protein delivery

    Science.gov (United States)

    Chen, Xingtao; Lv, Guoyu; Zhang, Jue; Tang, Songchao; Yan, Yonggang; Wu, Zhaoying; Su, Jiacan; Wei, Jie

    2014-01-01

    A multi-(amino acid) copolymer (MAC) based on ω-aminocaproic acid, γ-aminobutyric acid, L-alanine, L-lysine, L-glutamate, and hydroxyproline was synthetized, and MAC microspheres encapsulating bovine serum albumin (BSA) were prepared by a double-emulsion solvent extraction method. The experimental results show that various preparation parameters including surfactant ratio of Tween 80 to Span 80, surfactant concentration, benzyl alcohol in the external water phase, and polymer concentration had obvious effects on the particle size, morphology, and encapsulation efficiency of the BSA-loaded microspheres. The sizes of BSA-loaded microspheres ranged from 60.2 μm to 79.7 μm, showing different degrees of porous structure. The encapsulation efficiency of BSA-loaded microspheres also ranged from 38.8% to 50.8%. BSA release from microspheres showed the classic biphasic profile, which was governed by diffusion and polymer erosion. The initial burst release of BSA from microspheres at the first week followed by constant slow release for the next 7 weeks were observed. BSA-loaded microspheres could degrade gradually in phosphate buffered saline buffer with pH value maintained at around 7.1 during 8 weeks incubation, suggesting that microsphere degradation did not cause a dramatic pH drop in phosphate buffered saline buffer because no acidic degradation products were released from the microspheres. Therefore, the MAC microspheres might have great potential as carriers for protein delivery. PMID:24855351

  18. Preparation of ceramic filler from reusing sewage sludge and application in biological aerated filter for soy protein secondary wastewater treatment.

    Science.gov (United States)

    Wu, Suqing; Qi, Yuanfeng; Yue, Qinyan; Gao, Baoyu; Gao, Yue; Fan, Chunzhen; He, Shengbing

    2015-01-01

    Dehydrated sewage sludge (DSS) and clay used as raw materials for preparation of novel media-sludge ceramic filler (SCF) and SCF employed in a lab-scale up-flow biological aerated filter (BAF) were investigated for soy protein secondary wastewater treatment. Single factor experiments were designed to investigate the preparation of SCF, and the characteristics (microstructure properties, toxic metal leaching property and other physical properties) of SCF prepared under the optimum conditions were examined. The influences of media height, hydraulic retention time (HRT) and air-liquid ratio (A/L) on chemical oxygen demand (CODcr) and ammonia nitrogen (NH4(+)-N) removal rate were studied. The results showed that the optimum addition of DSS was approximately 25.0 wt% according to the physical properties of SCF (expansion ratio of 53.0%, v/v, water absorption of 8.24 wt%, bulk density of 350.4 kg m(-3) and grain density of 931.5 kg m(-3)), and the optimum conditions of BAF system were media height of 75.0 cm, HRT of 10.0 h and A/L of 15:1 in terms of CODcr and NH4(+)-N removal rate (91.02% and 90.48%, respectively). Additionally, CODcr and NH4(+)-N (81.6 and 15.3 mg L(-1), respectively) in the final effluent of BAF system met the national standard (CODcr ≤ 100 mg L(-1), NH4(+)-N ≤ 25.0 mg L(-1), GB 18918-2002, secondary standard). Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Ice growth in supercooled solutions of a biological "antifreeze", AFGP 1-5: an explanation in terms of adsorption rate for the concentration dependence of the freezing point.

    Science.gov (United States)

    Knight, C A; DeVries, A L

    2009-07-21

    It is widely accepted, and we agree, that the lowering of the temperature at which ice can grow in a water solution of one of the biological antifreezes is a result of adsorption of the antifreeze molecules at the ice surface. However, how this can produce a well-defined "freezing point" that varies with the solution concentration has remained problematical. The results of a series of measurements of ice growing in supercooled solutions of an effective antifreeze are reported and interpreted in terms of this fundamental problem. It seemed that the solution of the problem would have to rely upon adsorption rate, because that appeared to be the only way for the concentration in solution to be so important. The crystal growth results are most unusual, and appear to confirm this. The growth rates over a wide range of antifreeze concentration in solution (about 0.05 to 9 mg ml(-1)) are zero from the thermodynamic freezing point down to the "non-equilibrium" freezing point, where there is a very sudden increase to a plateau value that then remains about constant as the supercooling is increased by about 2 degrees C. The plateau values of growth rate are faster than those from pure water at the lower-supercooling ends of the plateaus, but slower at higher supercooling, until the growth rate starts rising toward that from pure water. These plateau values of growth rate increase markedly with increasing concentration of the antifreeze in solution. Along with these changes there are complex changes in the growth orientations, from c-axis spicules in the plateaus to those more characteristic of growth from pure water at greater supercooling. We conclude that the non-equilibrium freezing point is determined by the adsorption rate. It is the warmest temperature at which the ice growth rate on the basal plane (where the antifreeze does not adsorb) is fast enough to prevent the area of basal face on a growing ice crystal from becoming too small to grow, which is determined in

  20. Thermal hysteresis proteins.

    Science.gov (United States)

    Barrett, J

    2001-02-01

    Extreme environments present a wealth of biochemical adaptations. Thermal hysteresis proteins (THPs) have been found in vertebrates, invertebrates, plants, bacteria and fungi and are able to depress the freezing point of water (in the presence of ice crystals) in a non-colligative manner by binding to the surface of nascent ice crystals. The THPs comprise a disparate group of proteins with a variety of tertiary structures and often no common sequence similarities or structural motifs. Different THPs bind to different faces of the ice crystal, and no single mechanism has been proposed to account for THP ice binding affinity and specificity. Experimentally THPs have been used in the cryopreservation of tissues and cells and to induce cold tolerance in freeze susceptible organisms. THPs represent a remarkable example of parallel and convergent evolution with different proteins being adapted for an anti-freeze role.

  1. Effects of anti-freeze concentration in the engine coolant on the cavitation temperature of a water pump

    International Nuclear Information System (INIS)

    David Huang, K.; Tzeng, S.-C.; Ma Weiping

    2004-01-01

    Improvements in engine-manufacturing technology have gradually increased the thermal efficiencies of engines as well as the burning temperature and pressure of fuels within the cylinders. Accordingly, greater heat dissipation are required. However, the volume of the radiators is constrained by the configuration of the engines, leading to excessive internal resistance in the engine-cooling system. Therefore, water pumps in engines are prone to cavitation, and air bubbles are likely to permeate into the anti-freeze, thereby severely reducing the performance, reliability and service life of the engines. Ethylene glycol (EG) is added to the radiator of some vehicles in cold areas to reduce the solidification point of the coolant and prevent freezing. This study probes the effects of the percentage of anti-freeze added to the cooling water in a water pump in an engine on the water-supply capability and cavitation temperature, whether air or burnt gas is present in the system. The results of this study have revealed that engines have a higher tolerance to air bubbles at lower rates of rotation. At a given fixed rotational speed, the tolerable cavitation temperature of an engine's water pump will fall slowly as the amount of air bubbles increases

  2. Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.

    Science.gov (United States)

    Rupp, R A; Sippel, A E

    1987-12-10

    The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity.

  3. Antioxidant activity of protein hydrolysates from whole anchovy sprat (Clupeonella engrauliformis) prepared using endogenous enzymes and commercial proteases.

    Science.gov (United States)

    Ovissipour, Mahmoudreza; Rasco, Barbara; Shiroodi, Setareh Ghorban; Modanlow, Maryam; Gholami, Sanaz; Nemati, Mahrokh

    2013-05-01

    The antioxidant activity and chemical properties of fish protein hydrolysates (FPHs) prepared from anchovy sprat (Clupeonella engrauliformis) using endogenous enzymes (autolysis) and commercial proteases were investigated. The highest degree of hydrolysis (DH) was observed with Alcalase and papain and the highest protein recovery (PR) with Alcalase and bromelain. FPH yield was highest with Alcalase (82.3%) and lowest with autolysis (63.64%). Increased DH resulted in increased FPH yield (R(2) = 0.77). The highest oil recovery was observed with bromelain (6.41%) and the lowest with autolysis (3.58%). Antioxidant activity determined by DPPH, reducing power and ferrous chelation assays was highest in bromelain, Promod and papain FPHs respectively. The highest ABTS activity was observed in Alcalase FPH, followed by Promod and Protamex™ FPHs. The lowest antioxidant activity was observed in autolysed and Flavourzyme FPHs (P > 0.05). Heavy metals (arsenic, lead and mercury) were recorded at levels below the regulatory limits established by the FDA. Anchovy sprat hydrolysates showed high antioxidant activities and amino acid contents and low heavy metal concentrations, indicating that they have high potential for use in human and animal diets. The high antioxidant activities are related to the high levels of hydrophobic amino acids found in this study. © 2012 Society of Chemical Industry.

  4. Preparation of anchovy (Engraulis japonicus) protein hydrolysates with high free radical-scavenging activity using endogenous and commercial enzymes.

    Science.gov (United States)

    He, Silian; Wang, Fanghua; Ning, Zhengxiang; Yang, Bo; Wang, Yonghua

    2014-12-01

    Anchovy protein hydrolysates with high free radical-scavenging activity were prepared by endogenous and commercial enzymes. Various hydrolytic factors (commercial protease composition, protease concentration, temperature, and reaction time) were optimized. Using a single-factor experiment, three commercial proteases (Protamex, Flavourzyme 500 MG, and Alcalase 2.4 L) were selected for further optimization using a simplex lattice design. The optimum composition of Protamex:Flavourzyme 500 MG:Alcalase 2.4 L was found to be 1.1:1.0:0.9. The hydrolytic conditions (commercial protease concentration, temperature, and reaction time) for the optimum protease composition were optimized using a Box-Behnken design. The optimum hydrolytic conditions were as follows: total commercial protease concentration of 3.27%, pH of 7.5, temperature of 55.4℃, and reaction time of 2.7 h. Under these conditions, hydrolysate with a 1, 1-diphenyl-2-picryhydrazyl scavenging activity of 84.7% was obtained. Meanwhile, a degree of hydrolysis of 33.2% and high protein nitrogen recovery of 87.5% were achieved. The amino acid composition of the hydrolysates demonstrated that they have high nutritional value, thereby suggesting that the hydrolysates have potential to be used as raw material for functional food. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  5. UTILIZATION OF MEMBRANE MICROFILTRATION IN PREPARATION OF HYDROLYZED VEGETABLE PROTEIN FROM FERMENTED RED BEAN (Phaseolus vulgaris L. EXTRACT AS FORTIFICATION AGENT

    Directory of Open Access Journals (Sweden)

    Sri Moerniati

    2010-06-01

    Full Text Available Preparation of Hydrolyzed Vegetable Protein (HVP as savory flavor from fermented red bean broth through stirred membrane cell using micro filtration membrane with pore size of 0.45 µm was performed to get fortified agent utilized in preparation of beans sauce. The objective of this work was to study an effect of pressure and kind of red bean broth extract on content of total protein, soluble protein and dry solid in the retentate and permeate as hydrolyzed vegetable protein used for fortified agent of red bean sauces. Preparation process of hydrolyzed vegetable protein was done using fixed rotary speed of 400 rpm, pressure of 20, 25 and 30 psi at room temperature. To investigate the effect of pressure on this separation, the feed were red bean broth extract fermented for 6, 8, 10 and 12 weeks, respectively. Fermentation process were conducted using salt fermentation with inoculum of Rhizopus-C1, salt and red bean ratios of 30:10:60%. The analysis of flux and contents of total protein, dissolved protein and dry solid in the retentate and permeate was carried out, and the result of experiment showed that interaction of Red bean broth extract with 6, 8, 10 and 12 weeks of fermentation and operation condition of microfiltration membrane separation tends to affect on flux and content of total protein, dissolved protein and dry solid in retentate and permeate. Red bean broth extract for 6 weeks fermentation resulted higher protein content in permeate as hydrolyzed vegetable protein than in retentate. Permeate at pressure of 25 psi gives flux value of 0.0217 mL/cm2.minute and contents of total protein of 1.31 %, dissolved protein of 6.9 mg/g, and dry solid of 2.6%, while retentate as hydrolyzed vegetable protein or fortified agent indicate contents of total protein of 1.52%, dissolved protein of 4.15 mg/g, and dry solid of 3.64%. It was found that micro filtration process was able to increase dissolved protein content of about 3 times.   Keywords

  6. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    Science.gov (United States)

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Preparation and rebinding properties of protein-imprinted polysiloxane using mesoporous calcium silicate grafted non-woven polypropylene as matrix.

    Science.gov (United States)

    Kan, Bohong; Feng, Lingzhi; Zhao, Kongyin; Wei, Junfu; Zhu, Dunwan; Zhang, Linhua; Ren, Qian

    2016-03-01

    Calcium silicate particle containing mesoporous SiO2 (CaSiO3@SiO2) was grafted on the surface of non-woven polypropylene. The PP non-woven grafted calcium silicate containing mesoporous SiO2 (PP-g-CaSiO3@SiO2) was used as the matrix to prepare bovine serum albumin (BSA) molecularly imprinted polysiloxane (MIP) by using silanes as the functional monomers and BSA as the template. PP non-woven grafted BSA-imprinted polysiloxane (PP-g-CaSiO3@SiO2 MIP) was characterized by scanning electron microscope (SEM), Fourier transform infrared spectometry (FTIR) and drilling string compensator (DSC). Influence factors on the rebinding capacity of the MIP were investigated, such as grafting degree, the pH in treating CaSiO3 and the type and proportion of silanes. The rebinding properties of BSA on PP-g-CaSiO3@SiO2 and MIP were investigated under different conditions. The results indicated that the rebinding capacity of MIP for BSA reached 56.32 mg/g, which was 2.65 times of NIP. The non-woven polypropylene grafted BSA-imprinted polysiloxane could recognize the template protein and the selectivity factor (β) was above 2.4 when using ovalbumin, hemoglobin and γ-globulin as control proteins. The PP-g-CaSiO3@SiO2 MIP has favorable reusability. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Safety evaluation of ice-structuring protein (ISP) type III HPLC 12 preparation. Lack of genotoxicity and subchronic toxicity.

    Science.gov (United States)

    Hall-Manning, T; Spurgeon, M; Wolfreys, A M; Baldrick, A P

    2004-02-01

    Ice-structuring proteins (ISPs) naturally occur in a range of species (including edible plants and fish) that need to protect themselves against freeze damage. ISPs have potential applications in a number of areas including cryopreservation and frozen foods manufacture. However, these materials are not currently generally available for commercial use. ISP type III HPLC 12 is of particular interest and although it is likely to be consumed naturally, its toxicological safety has not previously been assessed. This paper presents data from a set of in vitro and in vivo genotoxicity assays (bacterial mutation, chromosome aberration, mammalian cell gene mutation and rat bone marrow micronucleus) and a 3-month repeat-dose gavage study in the rat using high levels of ISP type III HPLC 12 preparation produced by recombinant baker's yeast. No evidence was seen of a genotoxic potential (using levels accepted as limit concentrations for the assays used) or notable subchronic toxicity following oral administration for 3 months in the rat at up to 580 mg ISP type III HPLC 12/kg/day, the highest dose tested (which was considered to be a NOAEL).

  9. Preparative expression and purification of a nacreous protein N16 and testing its effect on osteoporosis rat model.

    Science.gov (United States)

    Xu, Zhen-Yan; Liu, Yu-Ling; Lin, Jia-Bi; Cheng, Ke-Ling; Wang, Yong-Gang; Yao, Hong-Liang; Wei-Peng; Wu, Hoi-Yan; Su, Wei-Wei; Shaw, Pang-Chui; Li, Pei-Bo

    2018-05-01

    N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized. N16 was expressed in inclusion bodies. It was denatured and refolded in 8 M urea buffer and purified to 97% purity by passing through a gel filtration column. The glucocorticoid induced osteoporosis (GIO) rat model was used to investigate the anti-osteoporosis activity of N16 in vivo. Results showed that the decrease of the bone mineral density (BMD) and the ultimate load was significantly relieved after N16 treatment. N16 displayed dual regulatory effects by promoting osteogenesis as well as inhibiting bone resorption in vivo. Our work will contribute to further clinical studies on N16 for osteoporosis treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Morphology-Variable Aggregates Prepared from Cholesterol-Containing Amphiphilic Glycopolymers: Their Protein Recognition/Adsorption and Drug Delivery Applications

    Directory of Open Access Journals (Sweden)

    Zhao Wang

    2018-02-01

    Full Text Available In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose-b-poly(6-cholesteryloxyhexyl methacrylate (PMAgala-b-PMAChols, with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM and dynamic laser light scattering (DLS. In addition, the lectin (Ricinus communis agglutinin II, RCA120 recognition and bovine serum albumin (BSA adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120 and BSA. Spherical aggregates (d ≈ 92 nm possessed the highest RCA120 recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features.

  11. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

    Science.gov (United States)

    Yasmin, Nusrat; Saleem, Mahjabeen; Naz, Mamoona; Gul, Roquyya; Rehman, Hafiz Muzzammel

    2017-01-01

    A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli . Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus ; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  12. Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

    Directory of Open Access Journals (Sweden)

    Nusrat Yasmin

    2017-01-01

    Full Text Available A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+ and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

  13. The effect of addition of selected milk protein preparations on the growth of Lactobacillus acidophilus and physicochemical properties of fermented milk.

    Science.gov (United States)

    Gustaw, Waldemar; Kozioł, Justyna; Radzki, Wojciech; Skrzypczak, Katarzyna; Michalak-Majewska, Monika; Sołowiej, Bartosz; Sławińska, Aneta; Jabłońska-Ryś, Ewa

    2016-01-01

    The intake of fermented milk products, especially yoghurts, has been systematically increasing for a few decades. The purpose of this work was to obtain milk products fermented with a mix of bacterial cultures (yoghurt bacteria and Lactobacillus acidophillus LA-5) and enriched with selected milk protein preparations. Secondly, the aim of the work was to determine physiochemical and rheological properties of the obtained products. The following additives were applied in the experiment: whey protein concentrate (WPC 65), whey protein isolate (WPI), demineralised whey powder (SPD), caseinoglycomacropeptide (CGMP), α-lactalbumin (α-la), sodium caseinate (KNa) and calcium caseinate (KCa). Milk was fermented using probiotic strain Lactobacillus acidophillus LA-5 and a typical yoghurt culture. The products were analysed in terms of the survivability of bacterial cells during refrigerated storage, rheological properties and syneresis. Fermented milk products were obtained using blends of bacterial strains: ST-B01:Lb-12 (1:1), ST-B01:Lb-12:LA-5 (1:1:2). Milk beverages fermented with typical yoghurt bacteria and LA-5 strain showed intensive syneresis. The addition of LA-5 strain caused formation of harder acid gels, comparing to typical yoghurts. Milk products which were prepared from skimmed milk possessed higher values of hardness and consistency coefficient. The increase of concentrations of milk preparations (except of WPI) did not cause significant differences in the hardness of acidic gels obtained by fermentation of mixed culture with a probiotic strain. The applied preparations improved physiochemical properties of the milk beverages which were prepared with a probiotic strain. The increase of protein milk preparations concentration resulted in a gradual decrease of the secreted whey. Among the products that were made of full milk powder and were subjected to three weeks of refrigerated storage the highest survivability of Lb. acidophilus LA-5 was noticed in the

  14. Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

    Science.gov (United States)

    Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

    2015-05-01

    This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein.

    Science.gov (United States)

    Wang, Xiaohua; Liu, Haifeng; Liu, Yawei; Li, Yuting; Yan, Lei; Yuan, Xiaohuan; Zhang, Yufei; Wu, Yan; Liu, Jieting; Zhang, Chunlei; Chu, Yanhui

    2016-04-01

    Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni-NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris-HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36% of the soluble form by addition of isopropyl β-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni-NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95%. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTβRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 10(4) U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis.

  16. Protein release from poly(lactide-co-glycolide) implants prepared by hot-melt extrusion: thioester formation as a reason for incomplete release.

    Science.gov (United States)

    Ghalanbor, Zahra; Körber, Martin; Bodmeier, Roland

    2012-11-15

    The aim of this study was to characterize the protein release from PLGA-based implants prepared by hot-melt extrusion with special emphasis on identifying reasons for incomplete release. Biodegradable PLGA-implants loaded with BSA were prepared with a syringe-die extrusion device. A burst-free release was achieved up to 25% BSA loading by milling the protein prior to extrusion. The release was incomplete at 70% at loadings below the percolation threshold of the protein; higher protein loadings increased the release to 97%. However, an insoluble implant mass remained for over 180 days, which was attributed to the acylation of BSA by PLGA oligomers via a thioester bond. The incomplete protein release due to the formation of this covalent bond was overcome when increasing the porosity of the implant, which effectively reduced the contact between BSA and PLGA oligomers. Accordingly, melt-extrusion facilitated incorporating high loadings of BSA into burst-free biodegradable implants. Additionally, it enhanced complete protein release by a process- or formulation controlled increase of the implant porosity. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Surface protein imprinted core-shell particles for high selective lysozyme recognition prepared by reversible addition-fragmentation chain transfer strategy.

    Science.gov (United States)

    Li, Qinran; Yang, Kaiguang; Liang, Yu; Jiang, Bo; Liu, Jianxi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-12-24

    A novel kind of lysozyme (Lys) surface imprinted core-shell particles was synthesized by reversible addition-fragmentation chain transfer (RAFT) strategy. With controllable polymer shell chain length, such particles showed obviously improved selectivity for protein recognition. After the RAFT initial agent and template protein was absorbed on silica particles, the prepolymerization solution, with methacrylic acid and 2-hydroxyethyl methacrylate as the monomers, and N,N'-methylenebis(acrylamide) as the cross-linker, was mixed with the silica particles, and the polymerization was performed at 40 °C in aqueous phase through the oxidation-reduction initiation. Ater polymerization, with the template protein removal and destroying dithioester groups with hexylamine, the surface Lyz imprinted particles were obtained with controllable polymer chain length. The binding capacity of the Lys imprinted particles could reach 5.6 mg protein/g material, with the imprinting factor (IF) as 3.7, whereas the IF of the control material prepared without RAFT strategy was only 1.6. The absorption equilibrium could be achieved within 60 min. Moreover, Lys could be selectively recognized by the imprinted particles from both a four-proteins mixture and egg white sample. All these results demonstrated that these particles prepared by RAFT strategy are promising to achieve the protein recognition with high selectivity.

  18. Preparation and physicochemical properties of protein concentrate and isolate produced fromAcacia tortilis(Forssk.) Hayne ssp.raddiana.

    Science.gov (United States)

    Embaby, Hassan E; Swailam, Hesham M; Rayan, Ahmed M

    2018-02-01

    The composition and physicochemical properties of defatted acacia flour (DFAF), acacia protein concentrate (APC) and acacia protein isolate (API) were evaluated. The results indicated that API had lower, ash and fat content, than DFAF and APC. Also, significant difference in protein content was noticed among DFAF, APC and API (37.5, 63.7 and 91.8%, respectively). Acacia protein concentrate and isolates were good sources of essential amino acids except cystine and methionine. The physicochemical and functional properties of acacia protein improved with the processing of acacia into protein concentrate and protein isolate. The results of scanning electron micrographs showed that DFAF had a compact structure; protein concentrate were, flaky, and porous type, and protein isolate had intact flakes morphology.

  19. Preparation of nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x histidine-tagged proteins.

    Science.gov (United States)

    Liao, Yiqun; Cheng, Yangjian; Li, Qingge

    2007-03-02

    In this report, we describe the preparation of novel nitrilotriacetic acid/Co2+-linked, silica/boron-coated magnetite nanoparticles for purification of 6 x His-tagged proteins. The nanoparticles were approximately 200 nm in size and were stable against hydrochloric acid and had negligible non-specific binding for protein. Elimination of non-specific binding by nucleic acids was readily achieved by digestion of samples with DNase and RNase. The modified nanoparticles were used to purify two model proteins: one had a C-terminal 6 x His tag, and the other had an internal 6 x His tag. Both proteins were purified within one hour into single band purity on sodium dodecyl sulfate-polyacrylamide electrophoresis gel.

  20. A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody.

    Science.gov (United States)

    Ji, Qin C; Rodila, Ramona; El-Shourbagy, Tawakol A

    2007-03-01

    The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.

  1. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  2. Advanced fluidic handling and use of two-phase flow for high throughput structural investigation of proteins on a microfluidic sample preparation platform

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Møller, M.

    2010-01-01

    Research on the structure of proteins can bring forth a wealth of information about biological function and can be used to better understand the processes in living cells. This paper reports a new microfluidic sample preparation system for the structural investigation of proteins by Small Angle X......-ray Scattering (SAXS). The system includes hardware and software features for precise fluidic control, synchrotron beamline control, UV absorbance measurements and automated data analysis. The precise fluidic handling capabilities are used to transport and precisely position samples as small as 500 n...

  3. Influence of the liposomal preparation Butaintervite on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning

    Directory of Open Access Journals (Sweden)

    M. I. Hariv

    2016-09-01

    Full Text Available This article presents the results of research into the influence of the complex liposomal preparation Butaintervit on protein synthesis function in the livers of rats under the influence of carbon tetrachloride poisoning. Intramuscular injection of carbon tetrachloride into rats at a dose of 0.25 ml per100 gof body weight causes antigenic load on the body and leads to disruption of protein synthesis function in the liver. This is shown by reduction in blood levels of total protein and its fractions. Thus, the level of albumin in the serum of rats under the conditions of oxidative stress was 70% lower than in clinically healthy animals. However, the level of total protein in the serum was only 10% lower. This is because, along with the decrease of albumin content in the serum, the levels of globulin protein fraction increased by 8.8%. This has led to albumin/globulin disparities in the serum of sick animals. As a result, the value of A/G coefficient was 0.28 ± 0.03, compared to 0.52 ±0.02 inclinically healthy rats. For the normalization of functional state of the liver under oxidative stress it is advisable to apply the liposomal preparation Butaintervite, which in its structure contains butafosfan, interferon, thistle and vitamins A, D and E. Under conditions of oxidative stress and under the action of the liposomal preparation in the rats from the second experimental group we have found significant increase in the levels of total protein and albumins and a decrease in serum globulin in the animals on the fifth and tenth days of the experiment. On the fourteenth day of the experiment under the conditions of oxidative stress and under the action of the liposomal preparation in the rats from the second experimental group the normalization of protein synthesis function in the liver was observed. The level of indicators of total protein, albumin, globulin and the coefficient of albumin/globulin compared with the control group of animals were

  4. Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography

    NARCIS (Netherlands)

    Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

    2010-01-01

    An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late

  5. Preparation of antioxidant peptide from egg white protein and improvement of its activities assisted by high-intensity pulsed electric field.

    Science.gov (United States)

    Lin, Songyi; Guo, Yang; You, Qi; Yin, Yongguang; Liu, Jingbo

    2012-05-01

    Egg white protein powder was hydrolyzed by three proteases-alcalase, trypsin, and pepsin-to produce antioxidant peptides. Three kinds of hydrolysates were prepared under optimal enzymatic parameters that were obtained from the preliminary one-factor-at-a-time test and response surface methodology. Thereafter, three enzymatic hydrolysates were sequentially fractionated by ultrafiltration membranes in cut-off MW of 30, 10, and 1 kDa, and tested in terms of their reducing power (RP). Effects of high-intensity pulsed electric field (PEF) were further investigated on the antioxidant peptides to improve their activities. Alcalase hydrolysates possessed stronger RP ability than the other two hydrolysates, particularly for the fraction within alcalase could be regarded as the most appropriate enzyme for preparation of bioactive peptide from egg white protein, with the best antioxidant activity. Also, PEF showed some effects on the peptide and could be further applied to improve its antioxidant activity. Copyright © 2011 Society of Chemical Industry.

  6. Rheological properties of oil-in-water emulsions prepared with oil and protein isolates from sesame (Sesamum Indicum

    Directory of Open Access Journals (Sweden)

    David Ramirez BREWER

    2016-01-01

    Full Text Available In this study, food emulsions of oil in water from sesame (Sesamum indicum protein isolates and their oil were formulated and standardised. The effect of the concentrations of sesame (Sesamum indicum protein isolates and base oil and the speed of the emulsification process for the food emulsion stability was studied. The protein isolates were achieved from the defatted sesame flour (DSF, obtaining a percentage of 80% ± 0.05% of protein. Emulsions were formulated through a factorial design 23. The rheological behaviour of sesame (Sesamum indicum protein isolates-stabilised emulsions and microstructural composition were investigated. Stable emulsions with suitable rheological properties and microstructure were formulated at a concentration of 10% sesame oil and different concentrations of protein isolates, between 1.5% and 2.5%, with the best droplet distribution characteristics being shown for the 2.5% sesame protein isolates. The emulsions showed a non-Newtonian fluid behaviour, adjusting the Sisko model.

  7. Effects of gamma irradiation on physicochemical properties of heat-induced gel prepared with chicken salt-soluble proteins

    International Nuclear Information System (INIS)

    Choi, Yun-Sang; Kim, Hyun-Wook; Hwang, Ko-Eun; Song, Dong-Heon; Jeong, Tae-Jun; Seo, Kwang-Wook; Kim, Young-Boong; Kim, Cheon-Jei

    2015-01-01

    The technological effects of gamma irradiation (0, 3, 7, and 10 kGy) on chicken salt-soluble meat proteins in a model system were investigated. There were no significant differences in protein, fat, and ash content, and sarcoplasmic protein solubility among all samples. The samples with increasing gamma irradiation levels had higher pH, lightness, yellowness, and apparent viscosity, whereas moisture content, water holding capacity, redness, myofibrillar protein solubility, total protein solubility, hardness, springiness, cohesiveness, gumminess, and chewiness were the highest in the unirradiated control. The result from meat products using gamma irradiation was intended to provide a basic resource processing technology. - Highlights: • The effect of gamma irradiation on salt-soluble meat proteins was investigated. • Gelling properties of salt-soluble protein affected by gamma irradiation. • Gamma irradiation of meat products provides a basic resource processing technology

  8. Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

    Directory of Open Access Journals (Sweden)

    Zarei Saeed

    2011-08-01

    Full Text Available Abstract Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also

  9. Aqueous geochemistry in icy world interiors: Equilibrium fluid, rock, and gas compositions, and fate of antifreezes and radionuclides

    Science.gov (United States)

    Neveu, Marc; Desch, Steven J.; Castillo-Rogez, Julie C.

    2017-09-01

    The geophysical evolution of many icy moons and dwarf planets seems to have provided opportunities for interaction between liquid water and rock (silicate and organic solids). Here, we explore two ways by which water-rock interaction can feed back on geophysical evolution: the production or consumption of antifreeze compounds, which affect the persistence and abundance of cold liquid; and the potential leaching into the fluid of lithophile radionuclides, affecting the distribution of a long-term heat source. We compile, validate, and use a numerical model, implemented with the PHREEQC code, of the interaction of chondritic rock with pure water and with C, N, S-bearing cometary fluid, thought to be the materials initially accreted by icy worlds, and describe the resulting equilibrium fluid and rock assemblages at temperatures, pressures, and water-to-rock ratios of 0-200 ° C, 1-1000 bar, and 0.1-10 by mass, respectively. Our findings suggest that water-rock interaction can strongly alter the nature and amount of antifreezes, resulting in solutions rich in reduced nitrogen and carbon, and sometimes dissolved H2, with additional sodium, calcium, chlorine, and/or oxidized carbon. Such fluids can remain partially liquid down to 176 K if NH3 is present. The prominence of Cl in solution seems to hinge on its primordial supply in ices, which is unconstrained by the meteoritical record. Equilibrium assemblages, rich in serpentine and saponite clays, retain thorium and uranium radionuclides unless U-Cl or U-HCO3 complexing, which was not modeled, significantly enhances U solubility. However, the radionuclide 40 K can be leached at high water:rock ratio and/or low temperature at which K is exchanged with ammonium in minerals. We recommend the inclusion of these effects in future models of the geophysical evolution of ocean-bearing icy worlds. Our simulation products match observations of chloride salts on Europa and Enceladus; CI chondrites mineralogies; the observation of

  10. Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Snakenborg, Detlef; Nielsen, Søren Skou

    2011-01-01

    A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure...... control, UV absorbance measurements and automated data analysis. As little as 15 l of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis...... cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space...

  11. PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites.

    Science.gov (United States)

    Benhalevy, Daniel; McFarland, Hannah L; Sarshad, Aishe A; Hafner, Markus

    2017-04-15

    The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale. PAR-CLIP involves irreversible UV-mediated crosslinking of RNAs labeled with photoreactive nucleosides to interacting proteins, followed by stringent purification steps and the conversion of crosslinked RNA into small RNA cDNA libraries compatible with next-generation sequencing. The defining hallmark of PAR-CLIP is a diagnostic mutation at the crosslinking site that is introduced into cDNA during the library preparation process. This feature allows for efficient computational removal of contaminating sequences derived from non-crosslinked fragments of abundant cellular RNAs. In the following, we present two different step-by-step procedures for PAR-CLIP, which differ in the small RNA cDNA library preparation procedure: (1) Standard library preparation involving gel size selections after each enzymatic manipulation, and (2) A modified PAR-CLIP procedure ("on-beads" PAR-CLIP), where most RNA manipulations including the necessary adapter ligation steps are performed on the immobilized RNP. This streamlined procedure reduces the protocol preparation time by three days compared to the standard workflow. Copyright © 2016. Published by Elsevier Inc.

  12. A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

    Science.gov (United States)

    Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

    2013-05-01

    Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 k

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

    Science.gov (United States)

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

    1982-08-06

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

  14. Buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions: from protein buffer capacity prediction to bioprocess applications.

    Science.gov (United States)

    Bahrenburg, Sven; Karow, Anne R; Garidel, Patrick

    2015-04-01

    Protein therapeutics, including monoclonal antibodies (mAbs), have significant buffering capacity, particularly at concentrations>50 mg/mL. This report addresses pH-related issues critical to adoption of self-buffered monoclonal antibody formulations. We evaluated solution conditions with protein concentrations ranging from 50 to 250 mg/mL. Samples were both buffer-free and conventionally buffered with citrate. Samples were non-isotonic or adjusted for isotonicity with NaCl or trehalose. Studies included accelerated temperature stability tests, shaking stability studies, and pH changes in infusion media as protein concentrate is added. We present averaged buffering slopes of capacity that can be applied to any mAb and present a general method for calculating buffering capacity of buffer-free, highly concentrated antibody liquid formulations. In temperature stability tests, neither buffer-free nor conventionally buffered solution conditions showed significant pH changes. Conventionally buffered solutions showed significantly higher opalescence than buffer-free ones. In general, buffer-free solution conditions showed less aggregation than conventionally buffered solutions. Shaking stability tests showed no differences between buffer-free and conventionally buffered solutions. "In-use" preparation experiments showed that pH in infusion bag medium can rapidly approximate that of self-buffered protein concentrate as concentrate is added. In summary, the buffer capacity of proteins can be predicted and buffer-free therapeutic antibody preparations provide a viable alternative to conventionally buffered solutions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Preparation of magnetic chitosan and graphene oxide-functional guanidinium ionic liquid composite for the solid-phase extraction of protein

    International Nuclear Information System (INIS)

    Ding, Xueqin; Wang, Yuzhi; Wang, Ying; Pan, Qi; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2015-01-01

    Highlights: • A strategy for the solid-phase extraction of protein based on magnetic chitosan and graphene oxide-functional guanidinium ionic liquids. • Trypsin, lysozyme, ovalbumin and bovine serum albumin were used as the analyst. • The possibility of reusability and regeneration has been evaluated. - Abstract: A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized, and then magnetic chitosan graphene oxide (MCGO) composite has been prepared and coated with these functional guanidinium ionic liquids to extract protein by magnetic solid-phase extraction. MCGO-functional guanidinium ionic liquid has been characterized by vibrating sample magnetometer, field emission scanning electron microscopy, X-ray diffraction spectrometer and Fourier transform infrared spectrometer. After extraction, the concentrations of protein were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer. The advantages of MCGO-functional guanidinium ionic liquid in protein extraction were compared with magnetic chitosan, graphene oxide, MCGO and MCGO-ordinary imidazolium ionic liquid. The proposed method has been applied to extract trypsin, lysozyme, ovalbumin and bovine serum albumin. A comprehensive study of the adsorption conditions such as the concentration of protein, the amount of MCGO-functional guanidinium ionic liquid, the pH, the temperature and the extraction time were also presented. Moreover, the MCGO-functional guanidinium ionic liquid can be easily regenerated, and the extraction capacity was about 94% of the initial one after being used three times

  16. Serve Size and Estimated Energy and Protein Contents of Meals Prepared by ‘Meals on Wheels’ South Australia Inc.: Findings from a Meal Audit Study

    Science.gov (United States)

    Arjuna, Tony; Miller, Michelle; Soenen, Stijn; Chapman, Ian; Visvanathan, Renuka; Luscombe-Marsh, Natalie D

    2018-01-01

    An audit of ‘standard’ (STD) and ‘energy and protein fortified’ (HEHP) meals from Meals on Wheels (MOW) South Australia’s summer menu was conducted to evaluate the consistency, and serve size and nutrient contents, of their menu items. Twenty soups, 20 mains and 20 desserts from each of the STD and HEHP menus were prepared at the MOW South Australia’s kitchen and delivered to three ‘sham(dummy)-clients’ over a 5-week period. Each meal component was weighed in triplicate, to the nearest gram, the variation within the serve weight was calculated, and the overall energy and protein content of each meal was determined using FoodWorks (Xyris Software, Highgate Hill, Queensland, Australia). On average, the variability for soups and mains was ≤6% and for desserts was ≤10% and although the measured serve sizes of the MOW meals were consistently smaller than prescribed serve size, the differences were minor. As a percentage of recommended daily intakes (RDIs) for adults aged over 60 years, we calculated that the STD meals contained 21–39% for energy and 42–63% for protein while the HEHP meals contained 29–55% for energy and 46–69% for protein. These findings demonstrate that MOW meals currently meet the voluntary meal guidelines for energy and protein. PMID:29461476

  17. Serve Size and Estimated Energy and Protein Contents of Meals Prepared by 'Meals on Wheels' South Australia Inc.: Findings from a Meal Audit Study.

    Science.gov (United States)

    Arjuna, Tony; Miller, Michelle; Soenen, Stijn; Chapman, Ian; Visvanathan, Renuka; Luscombe-Marsh, Natalie D

    2018-02-20

    An audit of 'standard' (STD) and 'energy and protein fortified' (HEHP) meals from Meals on Wheels (MOW) South Australia's summer menu was conducted to evaluate the consistency, and serve size and nutrient contents, of their menu items. Twenty soups, 20 mains and 20 desserts from each of the STD and HEHP menus were prepared at the MOW South Australia's kitchen and delivered to three 'sham(dummy)-clients' over a 5-week period. Each meal component was weighed in triplicate, to the nearest gram, the variation within the serve weight was calculated, and the overall energy and protein content of each meal was determined using FoodWorks (Xyris Software, Highgate Hill, Queensland, Australia). On average, the variability for soups and mains was ≤6% and for desserts was ≤10% and although the measured serve sizes of the MOW meals were consistently smaller than prescribed serve size, the differences were minor. As a percentage of recommended daily intakes (RDIs) for adults aged over 60 years, we calculated that the STD meals contained 21-39% for energy and 42-63% for protein while the HEHP meals contained 29-55% for energy and 46-69% for protein. These findings demonstrate that MOW meals currently meet the voluntary meal guidelines for energy and protein.

  18. Preparation of magnetic chitosan and graphene oxide-functional guanidinium ionic liquid composite for the solid-phase extraction of protein

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Xueqin; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Wang, Ying; Pan, Qi; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2015-02-25

    Highlights: • A strategy for the solid-phase extraction of protein based on magnetic chitosan and graphene oxide-functional guanidinium ionic liquids. • Trypsin, lysozyme, ovalbumin and bovine serum albumin were used as the analyst. • The possibility of reusability and regeneration has been evaluated. - Abstract: A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized, and then magnetic chitosan graphene oxide (MCGO) composite has been prepared and coated with these functional guanidinium ionic liquids to extract protein by magnetic solid-phase extraction. MCGO-functional guanidinium ionic liquid has been characterized by vibrating sample magnetometer, field emission scanning electron microscopy, X-ray diffraction spectrometer and Fourier transform infrared spectrometer. After extraction, the concentrations of protein were determined by measuring the absorbance at 278 nm using an ultra violet visible spectrophotometer. The advantages of MCGO-functional guanidinium ionic liquid in protein extraction were compared with magnetic chitosan, graphene oxide, MCGO and MCGO-ordinary imidazolium ionic liquid. The proposed method has been applied to extract trypsin, lysozyme, ovalbumin and bovine serum albumin. A comprehensive study of the adsorption conditions such as the concentration of protein, the amount of MCGO-functional guanidinium ionic liquid, the pH, the temperature and the extraction time were also presented. Moreover, the MCGO-functional guanidinium ionic liquid can be easily regenerated, and the extraction capacity was about 94% of the initial one after being used three times.

  19. Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins

    DEFF Research Database (Denmark)

    Heissel, Søren; Bunkenborg, Jakob; Kristiansen, Max Per

    2018-01-01

    the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced...

  20. [Preparation and the biological effect of fusion protein GLP-1-exendin-4/ IgG4(Fc) fusion protein as long acting GLP-1 receptor agonist].

    Science.gov (United States)

    Zheng, Yun-cheng

    2015-12-01

    GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity

  1. Palladium Mediated Rapid Deprotection of N-Terminal Cysteine under Native Chemical Ligation Conditions for the Efficient Preparation of Synthetically Challenging Proteins.

    Science.gov (United States)

    Jbara, Muhammad; Maity, Suman Kumar; Seenaiah, Mallikanti; Brik, Ashraf

    2016-04-20

    Facilitating the process of chemical protein synthesis is an important goal in order to enable the efficient preparation of large and novel protein analogues. Native chemical ligation, which is widely used in the synthesis and semisynthesis of proteins, has been going through several developments to expedite the synthetic process and to obtain the target protein in high yield. A key aspect of this approach is the utilization of protecting groups for the N-terminal Cys in the middle fragments, which bear simultaneously the two reactive groups, i.e., N-terminal Cys and C-terminal thioester. Despite important progress in this area, as has been demonstrated in the use of thiazolidine protecting group in the synthesis of over 100 proteins, finding optimal protecting group(s) remains a challenge. For example, the thiazolidine removal step is very slow (>8 h), and in some cases the applied conditions lead to undesired side reactions. Here we show that water-soluble palladium(II) complexes are excellent reagents for the effective unmasking of thiazolidine, enabling its complete removal within 15 min under native chemical ligation conditions. Moreover, palladium is also able to rapidly remove propargyloxycarbonyl-protecting group from the N-terminal Cys in a similar efficiency. The utility of the new removal conditions for both protecting groups is exemplified in the rapid and efficient synthesis of Lys34-ubiquitinated H2B and for the first time neddlyated peptides derived from cullin1. The current approach expands the use of palladium in protein chemistry and should significantly facilitate the chemical and semisynthesis of synthetically challenging proteins from multiple fragments.

  2. Ethylene glycol assisted preparation of Ti(4+)-modified polydopamine coated magnetic particles with rough surface for capture of phosphorylated proteins.

    Science.gov (United States)

    Ma, Xiangdong; Ding, Chun; Yao, Xin; Jia, Li

    2016-07-27

    The reversible protein phosphorylation is very important in regulating almost all aspects of cell life, while the enrichment of phosphorylated proteins still remains a technical challenge. In this work, polydopamine (PDA) modified magnetic particles with rough surface (rPDA@Fe3O4) were synthesized by introduction of ethylene glycol in aqueous solution. The PDA coating possessing a wealth of catechol hydroxyl groups could serve as an active medium to immobilize titanium ions through the metal-catechol chelation, which makes the fabrication of titanium ions modified rPDA@Fe3O4 particles (Ti(4+)-rPDA@Fe3O4) simple and very convenient. The spherical Ti(4+)-rPDA@Fe3O4 particles have a surface area of 37.7 m(2) g(-1) and superparamagnetism with a saturation magnetization value of 38.4 emu g(-1). The amount of Ti element in the particle was measured to be 3.93%. And the particles demonstrated good water dispersibility. The particles were used as adsorbents for capture of phosphorylated proteins and they demonstrated affinity and specificity for phosphorylated proteins due to the specific binding sites (Ti(4+)). Factors affecting the adsorption of phosphorylated proteins on Ti(4+)-rPDA@Fe3O4 particles were investigated. The adsorption capacity of Ti(4+)-rPDA@Fe3O4 particles for κ-casein was 1105.6 mg g(-1). Furthermore, the particles were successfully applied to isolate phosphorylated proteins in milk samples, which demonstrated that Ti(4+)-rPDA@Fe3O4 particles had potential application in selective separation of phosphorylated proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Soybean protein fraction digested with neutral protease preparation, "Peptidase R", produced by Rhizopus oryzae, stimulates innate cellular immune system in mouse.

    Science.gov (United States)

    Egusa, Shintaro; Otani, Hajime

    2009-07-01

    A soybean protein fraction was prepared from defatted soybean seed flour and digested with 29 kinds of commercially available protease originating from preparations of animals, plants, and microorganisms. Some digests, in particular, Ro-digest prepared using a Rhizopus oryzae neutral protease preparation (Peptidase R), displayed strong mitogenic activity toward C3H/HeN mouse spleen cells. The number of spleen CD11b+, CD49b+, interleukin (IL)-12+CD11b+, and interferon (IFN)-gamma+CD49b+ cells significantly increased when cultured with Ro-digest. Similarly, the number of spleen IFN-gamma+CD4+ cells significantly increased in the presence of Ro-digest while that of spleen IL-4+CD4+ cells was largely unchanged. Additionally, 5-week-old male C3H/HeN mice were given diets consisting of ovalbumin (OVA) alone (control diet) or a mixture of OVA and Ro-digest (Ro-digest-added diet) as a protein source for 5 weeks, and the immune properties of the mice were investigated. The number of IL-12+CD11b+ cells was greater in spleens from mice given the Ro-digest-added diet than in those given the control diet. The cytotoxic activity of spleen cells toward the human erythroleukemia cell line, K562, was significantly higher in mice given the Ro-digest-added diet than in those given the control diet. Furthermore, in a microarray analysis of mRNAs extracted from mice Peyer's patch cells, gene expression related to innate immune responses was increased in mice given the Ro-digest-added diet. These results indicate that the Ro-digest might stimulate cellular immune systems, in particular, an innate immunity in mice.

  4. Preparation of Monoclonal Antibodies Against SpiC Protein Secreted by T3SS-2 of Salmonella spp.

    Science.gov (United States)

    Geng, Shizhong; Qian, Shanshan; Pan, Zhiming; Sun, Lin; Chen, Xiang; Jiao, Xinan

    2015-12-01

    SpiC protein, a member of Salmonella spp. type III secretion system (T3SS)-2, is necessary for the survival of Salmonella within macrophages, and it plays a vital role in Salmonella pathogenesis. To develop and test monoclonal antibodies (MAbs) against SpiC protein, two recombinant proteins, rHis-SpiC and rGST-SpiC, were expressed in vitro in the prokaryotic expression vectors pET-30(a) and pGEX-6p-1, respectively, and rHis-SpiC protein used to immunize mice. Hybridomas were generated from the splenocytes of these mice and the monoclonal antibodies produced by these cells were assessed using indirect enzyme-linked immunosorbent assay (ELISA) with rGST-SpiC as the coating antigen. An immunoblotting analysis indicated that all seven of the MAbs developed in this study could specifically recognize the SpiC protein. These MAbs will be very useful in the study of SpiC function and for use in the immunodiagnosis of Salmonella infection.

  5. Enzymolysis kinetics and activities of ACE inhibitory peptides from wheat germ protein prepared with SFP ultrasound-assisted processing.

    Science.gov (United States)

    Qu, Wenjuan; Ma, Haile; Jia, Junqiang; He, Ronghai; Luo, Lin; Pan, Zhongli

    2012-09-01

    There is a great demand for developing efficient enzymolysis methods in order to increase the enzymolysis efficiencies and activities of angiotensin converting enzyme (ACE) inhibitory peptides from wheat germ protein. The enzymolysis kinetics, ACE inhibitory activity of peptide and conversion rate of protein were studied using sweep frequency and pulsed (SFP) ultrasound-assisted enzymolysis and the results were compared with traditional enzymolysis. The studied factors were enzymolysis time and substrate concentration. By considering the activity of ACE inhibitory peptide and operation cost, the recommended conditions of SFP ultrasound-assisted enzymolysis were enzymolysis time of 120 min and substrate concentration of 24.0 g/L, which gave high conversion rates of protein (60.7%) and ACE inhibitory activity of peptide (65.9%). Compared to traditional enzymolysis, SFP ultrasound-assisted enzymolysis significantly increased the initial reaction rate (V) by 60.0% at substrate concentration of 24.0 g/L, increased the apparent breakdown rate constant (k(A)) by 66.7%, decreased the apparent constant (K(M)) by 6.9%, and raised the conversion rate of protein by 35.5% and ACE inhibitory activity of peptides by 35.6% under the recommended conditions. It has been concluded that SFP ultrasound can remarkably raise the enzymolysis efficiency and activity of ACE inhibitory peptides from wheat germ protein. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    International Nuclear Information System (INIS)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He; Su, Xiao-Dong

    2007-01-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  7. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  8. Preparation and In Vitro Release of Drug-Loaded Microparticles for Oral Delivery Using Wholegrain Sorghum Kafirin Protein

    Directory of Open Access Journals (Sweden)

    Esther T. L. Lau

    2015-01-01

    Full Text Available Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content, in vitro protein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released in in vitro conditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

  9. Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins.

    Science.gov (United States)

    Oh-Ishi, M; Satoh, M; Maeda, T

    2000-05-01

    A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

  10. Preparation and characterization of poly (methyl methacrylate) and sulfonated poly (ether ether ketone) blend ultrafiltration membranes for protein separation applications

    International Nuclear Information System (INIS)

    Arthanareeswaran, G.; Thanikaivelan, P.; Raajenthiren, M.

    2009-01-01

    Poly (methyl methacrylate) (PMMA) and poly (methyl methacrylate)/sulfonated poly (ether ether ketone) (SPEEK) blend membranes were prepared by phase inversion technique in various composition using N,N'-dimethylformamide as solvent. The prepared membranes were characterized in terms of pure water flux, water content, porosity and thermal stability. The addition of SPEEK to the casting solution resulted in membranes with high pure water flux, water content, porosity and slightly low thermal stability. The cross sectional views of the blend membranes under electron microscope confirm the porosity and water flux results. The effect of the addition of SPEEK into the PMMA matrix on the extent of bovine serum albumin (BSA) separation was studied. It was found that the permeate flux increased significantly while the rejection of BSA from aqueous solution reduced moderately during ultrafiltration (UF) process. The effect was attributed to the increase in porosity and charge of the membrane due to the addition of SPEEK into the PMMA blend solution

  11. Developing immunological methods for detecting Macrobrachium rosenbergii nodavirus and extra small virus using a recombinant protein preparation.

    Science.gov (United States)

    Wang, C-S; Chang, C-Y; Wen, C-M

    2016-06-01

    Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) have been identified as the causative agents for white tail disease (WTD) of M. rosenbergii. In this study, the gene sequences encoding MrNV and XSV capsid proteins were separately ligated into the pGEX-4T-3 expression vector and transformed into Escherichia coli. After induction, glutathione-S-transferase (GST)-tagged MrNV and XSV fusion proteins were obtained with molecular masses of 68 and 43 kDa, respectively. Specific polyclonal antibodies for MrNV and XSV against viral recombinant proteins and infected prawn tissues were verified using Western blotting. According to immunodot blot assay results, the detection sensitivities of antibodies were approximately 5 ng μL(-1) for both recombinant proteins GST-MrNV and GST-XSV. In additional, MrNV and XSV were detected at dilution levels of 1:2560 and 1:640 in the infected prawn tissues, respectively. No cross-reactions with white spot syndrome virus or grouper nervous necrosis virus were observed using immunodot blot assays. MrNV and XSV in infected muscle tissues were detected using immunohistochemistry. Although the detection limit of the immunodot blot assay was lower than that of nested reverse transcription polymerase chain reaction, these polyclonal antibodies can be useful for confirming MrNV and XSV infections in field tests. © 2015 John Wiley & Sons Ltd.

  12. Filter-aided sample preparation with dimethyl labeling to identify and quantify milk fat globule membrane proteins.

    NARCIS (Netherlands)

    Lu, J.; Boeren, J.A.; Vries, de S.C.; Valenberg, van H.J.F.; Vervoort, J.J.M.; Hettinga, K.A.

    2011-01-01

    Bovine milk is a major nutrient source in many countries and it is produced at an industrial scale. Milk is a complex mixture of proteins, fats, carbohydrates, vitamins and minerals. The composition of the bovine milk samples can vary depending on the genetic makeup of the bovine species as well as

  13. Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis.

    Science.gov (United States)

    Jozefowicz, Anna M; Matros, Andrea; Witzel, Katja; Mock, Hans-Peter

    2018-01-01

    Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

  14. Effects of Preparation Conditions on Morphology of Polyacrylonitrile Micro/Ultrafiltration Membrane and Its Application in Protein and Fat Separation from Milk

    Directory of Open Access Journals (Sweden)

    Seyed Ali Alavi

    2014-04-01

    Full Text Available Polyacrylonitrile (PAN micro/ultrafiltration membranes were prepared by phase inversion method. The effects of various preparation conditions including polymeric solution concentration, evaporation time, temperature, composition and residence time of the coagulation bath were investigated. Various important membrane characteristics such as pore size, bulk porosity, and mechanical and morphological properties were taken into the consideration. The characterizations were performed by measuring the bubble point, water flux, tensile strength and scanning electron microscopy (SEM analyses. The results showed that by increasing the polymeric solution concentration from 13 to 17 wt%, the porosity and water flux were decreased. Moreover, the membrane skin layer was considerably thickened with a very significant decrease in its pore sizes which was achieved in ultrafiltration region. By increasing the evaporation time at atmospheric pressure, membrane skin layer was thickened and the pore sizes were decreased. Low coagulation bath temperatures (below 30°C resulted in lower pore size, water flux, and an increase in membrane mechanical strength. Introduction of isopropanol (IPA into the water coagulation bath led to lower coagulation rate and consequently, the formation of smaller pores became possible by using pure isopropanol as coagulation bath. Furthermore, by increasing the residence time in coagulation bath, a more porous structure with more uniform pore sizes were formed that showed better mechanical properties. Finally, the so-called ultrafiltration membranes were applied in concentration process of protein and milk fat. A protein rejection more than 93% was attained while a complete removal of milk fat was achieved.

  15. Antigenicity of fractions of Helicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies.

    Science.gov (United States)

    Stacey, A R; Hawtin, P R; Newell, D G

    1990-10-01

    The antigenicity of Helicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well defined Helicobacter pylori status. The antigenic material of Helicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding of Helicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91-100% specific, however fractions 16 to 19 showed a reduced sensitivity (78%) compared with the acid extract (95%). The urease fractions were 91% sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83% sensitive and 93.3% specific.

  16. In-line Fourier-transform infrared spectroscopy as a versatile process analytical technology for preparative protein chromatography.

    Science.gov (United States)

    Großhans, Steffen; Rüdt, Matthias; Sanden, Adrian; Brestrich, Nina; Morgenstern, Josefine; Heissler, Stefan; Hubbuch, Jürgen

    2018-03-05

    Fourier-transform infrared spectroscopy (FTIR) is a well-established spectroscopic method in the analysis of small molecules and protein secondary structure. However, FTIR is not commonly applied for in-line monitoring of protein chromatography. Here, the potential of in-line FTIR as a process analytical technology (PAT) in downstream processing was investigated in three case studies addressing the limits of currently applied spectroscopic PAT methods. A first case study exploited the secondary structural differences of monoclonal antibodies (mAbs) and lysozyme to selectively quantify the two proteins with partial least squares regression (PLS) giving root mean square errors of cross validation (RMSECV) of 2.42 g/l and 1.67 g/l, respectively. The corresponding Q 2 values are 0.92 and, respectively, 0.99, indicating robust models in the calibration range. Second, a process separating lysozyme and PEGylated lysozyme species was monitored giving an estimate of the PEGylation degree of currently eluting species with RMSECV of 2.35 g/l for lysozyme and 1.24 g/l for PEG with Q 2 of 0.96 and 0.94, respectively. Finally, Triton X-100 was added to a feed of lysozyme as a typical process-related impurity. It was shown that the species could be selectively quantified from the FTIR 3D field without PLS calibration. In summary, the proposed PAT tool has the potential to be used as a versatile option for monitoring protein chromatography. It may help to achieve a more complete implementation of the PAT initiative by mitigating limitations of currently used techniques. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  17. [Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein].

    Science.gov (United States)

    Huang, Shigao; Yin, Yuting; Xiong, Chunhui; Wang, Caihong; Lü, Jianxin; Gao, Jimin

    2013-01-01

    In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

  18. Preparation of Sulfobetaine-Grafted PVDF Hollow Fiber Membranes with a Stably Anti-Protein-Fouling Performance

    Directory of Open Access Journals (Sweden)

    Qian Li

    2014-04-01

    Full Text Available Based on a two-step polymerization method, two sulfobetaine-based zwitterionic monomers, including 3-(methacryloylamino propyl-dimethyl-(3-sulfopropyl ammonium hydroxide (MPDSAH and 2-(methacryloyloxyethyl ethyl-dimethyl-(3-sulfopropyl ammonium (MEDSA, were successfully grafted from poly(vinylidene fluoride (PVDF hollow fiber membrane surfaces in the presence of N,N′-methylene bisacrylamide (MBAA as a cross-linking agent. The mechanical properties of the PVDF membrane were improved by the zwitterionic surface layers. The surface hydrophilicity of PVDF membranes was significantly enhanced and the polyMPDSAH-g-PVDF membrane showed a higher hydrophilicity due to the higher grafting amount. Compared to the polyMEDSA-g-PVDF membrane, the polyMPDSAH-g-PVDF membrane showed excellent significantly better anti-protein-fouling performance with a flux recovery ratio (RFR higher than 90% during the cyclic filtration of a bovine serum albumin (BSA solution. The polyMPDSAH-g-PVDF membrane showed an obvious electrolyte-responsive behavior and its protein-fouling-resistance performance was improved further during the filtration of the protein solution with 100 mmol/L of NaCl. After cleaned with a membrane cleaning solution for 16 days, the grafted MPDSAH layer on the PVDF membrane could be maintain without any chang; however, the polyMEDSA-g-PVDF membrane lost the grafted MEDSA layer after this treatment. Therefore, the amide group of sulfobetaine, which contributed significantly to the higher hydrophilicity and stability, was shown to be imperative in modifying the PVDF membrane for a stable anti-protein-fouling performance via the two-step polymerization method.

  19. Preparation, crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC1692 from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Chin, Ko-Hsin; Huang, Zhao-Wei; Wei, Kun-Chou; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A conserved hypothetical protein XC1692 from X. campestris pv. campestris has been overexpressed in E. coli. The purified recombinant protein crystallized in a variety of forms and diffracted to a resolution of at least 1.45 Å. Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 Å resolution. They are hexagonal and belong to space group P6 3 , with unit-cell parameters a = b = 56.9, c = 71.0 Å. They contain one molecule per asymmetric unit

  20. Optimization of the formulation for preparing Lactobacillus casei loaded whey protein-Ca-alginate microparticles using full-factorial design.

    Science.gov (United States)

    Smilkov, Katarina; Petreska Ivanovska, Tanja; Petrushevska Tozi, Lidija; Petkovska, Rumenka; Hadjieva, Jasmina; Popovski, Emil; Stafilov, Trajce; Grozdanov, Anita; Mladenovska, Kristina

    2014-01-01

    This article presents specific approach for microencapsulation of Lactobacillus casei using emulsion method followed by additional coating with whey protein. Experimental design was employed using polynomial regression model at 2nd level with three independent variables, concentrations of alginate, whey protein and CaCl2. Physicochemical, biopharmaceutical and biological properties were investigated. In 11 series generated, negatively charged microparticles were obtained, with size 6.99-9.88 µm, Ca-content 0.29-0.47 mg per 10 mg microparticles, and viability of the probiotic 9.30-10.87 log10CFU/g. The viability after 24 hours in simulated gastrointestinal conditions was between 3.60 and 8.32 log10CFU/g. Optimal formulation of the microparticles that ensures survival of the probiotic and achieves controlled delivery was determined: 2.5% (w/w) alginate, 3% (w/w) CaCl2 and 3% (w/w) whey protein. The advantageous properties of the L. casei-loaded microparticles make them suitable for incorporation in functional food and/or pharmaceutical products.

  1. Protein valves prepared by click reaction grafting of poly(N-isopropylacrylamide) to electrospun poly(vinyl chloride) fibrous membranes

    Science.gov (United States)

    Guo, Jian-Wei; Lin, Zhen-Yu; Chang, Chi-Jung; Lu, Chien-Hsing; Chen, Jem-Kun

    2018-05-01

    In this study, poly(vinyl chloride) (PVC) was electrospun into fibrous membranes and then reacted with NaN3 to generate azido-terminated PVC fibrous membranes. A propargyl-terminated poly(N-isopropylacrylamide) (PNIPAAm) was also synthesized and then grafted, through click reactions, onto the azido-terminated PVC fiber surface. Protrusion-, scale-, and joint-like structures of the PNIPAAm grafts on the PVC fibers were formed upon increasing the molecular weight of the PNIPAAm grafts. The PNIPAAm-grafted PVC fibrous mats exhibited completely wetted surfaces at 25 °C because of their high roughness. The static water contact angle of the PNIPAAm-grafted PVC fibrous mats reached 140° when the temperature was increased to 45 °C. This thermoresponsive behavior was significantly greater than that of the PNIPAAm grafted on a flat surface. Temperature-responsive membranes were constructed having a pore size of 1.38 μm and applied as protein valves to block and release an antibody (fluorescein-conjugated AffiniPure goat anti-rabbit IgG). At 25 °C, the collection efficiency remained at 94% for antibody concentrations up to 60 ng/L. As the temperature increased to 45 °C, the collection efficiency decreased abruptly, to 4%, when the antibody concentration was greater than 20 ng/L. Accordingly, this system of PNIPAAm-grafted PVC fibers functioned as a protein valve allowing the capture and concentration of proteins.

  2. Protein adsorption characteristics of porous and tentacle anion-exchange membrane prepared by radiation-induced graft polymerization

    Science.gov (United States)

    Tsuneda, Satoshi; Saito, Kyoichi; Sugo, Takanobu; Makuuchi, Keizo

    1995-08-01

    A polymer chain containing a diethylamino group was grafted onto the pore surface of a porous hollow-fiber membrane by radiation-induced graft polymerization. Dependence of the protein binding capacity of the membrane on environmental parameters such as salt concentration, pH and temperature was investigated. Saturation capacity of protein bound onto the graft chain containing ion-exchange group was governed by the conformation of the graft chain and the intensity of ion-exchange interaction. The conformation of the graft chain was investigated based on the pore radius of the membrane estimated from the permeation flux of a buffer solution through the membrane. By sufficiently permeating a bovine serum albumin (BSA) solution within the concentration range of 0.2-10 mg-BSA/ml through the membrane, the BSA binding capacity was determined. With increasing salt concentration or pH of the protein buffer solution, the graft chain shrank and BSA binding capacity decreased. On the other hand, the BSA binding capacity slightly increased with increasing temperature, and the conformation of the graft chain was insensitive to temperature in the range from 278 to 303 K. The bound BSA could be quantitatively eluted by permeating a buffer solution containing 0.5 M NaCl, and no deterioration in the BSA binding capacity was observed during five cycles of adsorption, elution and conditioning.

  3. Preparation of denatured protein bone sterilized with gamma radiation; Preparacion de hueso desproteinizado esterilizado con radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Luna Z, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: dlz@nuclear.inin.mx

    2005-07-01

    The bone is one of the tissues more transplanted in the entire world by that the bone necessity for transplant every day becomes bigger. In the Bank of tissues Radio sterilized of the ININ the amnion and the pig skin are routinely processed. The tissue with which will be continued is with bone. Due to that in our country it doesn't have enough bone of human origin for the necessities required in the bone transplant, an option is the bone of bovine. Of this bone one can obtain denatured protein bone, with the same characteristics of the denatured protein human bone, the one which has been proven that it has good acceptance and incorporation in the human body when is transplanted. The method for the obtaining of the denatured protein bone of bovine, with the confirmation of the final product by means of X-ray diffraction is described. The radiosterilization of this bone with gamma rays and the determination of the lead content. (Author)

  4. The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Cheng Xu

    2014-08-01

    Full Text Available Follicle-stimulating hormone receptor (FSHR, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star TM (DE3 and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

  5. [Preparation of monoclonal antibodies against flagellin core protein of Vibrio cholerae and its application in establishing double-antibody sandwich ELISA for testing Vibrio cholerae from food products].

    Science.gov (United States)

    Cheng, Jinxia; Zeng, Jing; Zhang, Lei; Zhang, Lin; Zhang, Haiyu; Liu, Xuesong; Cao, Dong

    2013-11-01

    To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio cholerae and establish the double-antibody sandwich ELISA method for testing Vibrio cholerae from food products. BALB/c mice were immunized with flagellin extracted from Vibrio cholerae Vc75 by differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer in serum reached 1:32 000. The hybridoma cell lines were obtained by regular subcloning and used to generate ascites. And mAbs reacting to Vibrio cholerae flagellin were achieved by purified from the ascites. Six hybridoma cell lines stably secreting mAbs against Vibrio cholerae flagellin were taken and named VcNo.1-VcNo.6. The mAb titer in serum by indirect ELISA was 1:2 × 10(6). SDS-PAGE showed that the flagellin protein molecular weight (Mr) was 44 000 and the purity was high. Double-antibody sandwich ELISA method was set up using VcNo.6 antibody for detecting Vibrio cholerae. The sensitivity reached 10(3) CFU/mL. The ELISA method showed high specificity to Vibrio cholerae through testing 100 Vibrio cholerae (100% positive) and 101 non-Vibrio cholerae strains (100% negative). The detection limit was 1 CFU/g sample in artificial contaminated samples. The mAbs against flagellin core protein of Vibrio cholerae was successfully prepared and used to set up the double-antibody sandwich ELISA. The mAb of VcNo.6 was highly specific to Vibrio cholerae. The sensitivity of the established ELISA was as high as 10(3) CFU/mL. Moreover, it did not react to non-Vibrio cholerae strains. Therefore, the mAbs of VcNo.6 could be widely used in Vibrio cholerae detection from food samples as well as clinical samples.

  6. Optimal condition to remove mercury in yellowfin tuna protein isolates and ACE-inhibitory property of peptide prepared using commercial proteases

    Directory of Open Access Journals (Sweden)

    Hathaigan Kokkaew

    2016-08-01

    Full Text Available Response surface methodology (RSM was performed to maximize the mercury (Hg reduction from yellowfin tuna (Thunnus albacare by products protein isolates (YBPI. The optimal condition of Hg reduction (89.3% was 10.5 mM CaCl 2 and a Water:YB of 12.9:1, while other variables were fixed at 5 mM citric acid, 60 min incubation, pH 11 and 8,000 x g for 15 min of centrifugation. At these conditions, the significant protein recovery (80.1% was obtained. Hydrolysates sequentially hydrolyzed with G6 followed by GN exhibited the highest angiotensin I-converting enzyme (ACE inhibitory activity than other enzyme preparations. Fractionated yellowfin tuna by products protein hydrolysate (YBPH increased in ACE-inhibitory activity when peptide size decreased. In-vitro gastrointestinal (GI digestion significantly increased bioactive property. ACEinhibitory activity of YBPH with and without simulated GI digestion significantly increased after incubating against ACE, demonstrating pro-drug type peptides.

  7. Effect of PSK, a protein-bound polysaccharide preparation, on liver tumors of Syrian hamsters induced by Thorotrast injection

    Energy Technology Data Exchange (ETDEWEB)

    Shiga, Junji (Teikyo Univ., Tokyo (Japan). Faculty of Medicine); Maruyama, Takashi; Takahashi, Hisahide; Irie, Hiroshi; Mori, Takesaburo

    1993-09-01

    The contrast medium Thorotrast, an agent well known to be carcinogenic, was injected into 400 congeneic Syrian hamsters. The resulting incidence of malignant hepatic tumors such as cholangiocarcinoma, hepatocellular carcinoma and hemangiosarcoma, was significantly higher in the male experimental group than in the control group, and the 50% survival period in the male group was shortened by about 100 days (P<0.01). However administration of the antitumor drug PSK (Polysaccharide Kureha), a protein bound-polysaccharide extracted from basidiomycete fungi, prevented this carcinogenic effect. The incidence of malignant hepatic tumors in the experimental group was 22.5% compared with 2.8% in the control group (P<0.01) and 10.5% in the PSK-treated group (P<0.01). PSK also increased the 50% survival period by 61 days (P<0.01). (author).

  8. Effect of PSK, a protein-bound polysaccharide preparation, on liver tumors of Syrian hamsters induced by Thorotrast injection.

    Science.gov (United States)

    Shiga, J; Maruyama, T; Takahashi, H; Irie, H; Mori, T

    1993-09-01

    The contrast medium Thorotrast, an agent well known to be carcinogenic, was injected into 400 congeneic Syrian hamsters. The resulting incidence of malignant hepatic tumors such as cholangiocarcinoma, hepatocellular carcinoma and hemangiosarcoma, was significantly higher in the male experimental group than in the control group, and the 50% survival period in the male group was shortened by about 100 days (P Kureha), a protein bound-polysaccharide extracted from basidiomycete fungi, prevented this carcinogenic effect. The incidence of malignant hepatic tumors in the experimental group was 22.5% compared with 2.8% in the control group (P < 0.01) and 10.5% in the PSK-treated group (P < 0.01). PSK also increased the 50% survival period by 61 days (P < 0.01).

  9. A CTAB based method for the preparation of total protein extract of wine spoilage microrganisms for proteomic analysis.

    Science.gov (United States)

    Polati, Rita; Zapparoli, Giacomo; Giudici, Paolo; Bossi, Alessandra

    2009-04-01

    Mapping the proteome of microrganisms by 2D-electrophoresis is often a hard task, because many contaminants, e.g. polysaccharides of the cell wall and nucleic acid, can obstruct the pores of the IEF gel resulting in streaks and smears. A protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed. The cellulose-producing strain Gluconoacetobacter hansenii AAB0248 was resolved on 7cm Minigels in over 500 protein spots (a hundred more than with protocols reported in literature). The method was further employed for mapping the proteome of some acid adapted, wine spoilage microrganisms e.g. acetic acid bacteria and a yeast.

  10. Preparation of a multiple emulsion based on pectin-whey protein complex for encapsulation of saffron extract nanodroplets.

    Science.gov (United States)

    Faridi Esfanjani, Afshin; Jafari, Seid Mahdi; Assadpour, Elham

    2017-04-15

    The present study illustrates a simple and practical way to produce an adequate delivery system of bioactive compounds of saffron by protein-polysaccharide complex. Frist, crocin, safranal, and picrocrocin were loaded in nanodroplets (WPC)-maltodextrin or WPC-pectin-maltodextrin through water in oil in water (W/O/W) multiple emulsions. The stability and release of loaded crocin, safranal, and picrocrocin in multiple emulsions were investigated during 22days storage. The produced multiple emulsion by WPC-pectin-maltodextrin along with 5% inner aqueous phase showed a high stability and low release of encapsulated compounds over time. This emulsion also provided a high protection of crocin, safranal, and picrocrocin in the gastric condition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: Application to microcrystalline and membrane protein preparations

    Science.gov (United States)

    Gopinath, T.; Veglia, Gianluigi

    2015-04-01

    Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POE allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this perspective, we describe the first generation of POE, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic nuclear polarization (DNP), to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes.

  12. Multiple acquisition of magic angle spinning solid-state NMR experiments using one receiver: application to microcrystalline and membrane protein preparations.

    Science.gov (United States)

    Gopinath, T; Veglia, Gianluigi

    2015-04-01

    Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. Relatively low-sensitivity and poor resolution of protein samples require long acquisition times for multidimensional NMR experiments. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments, all while using conventional probes with spectrometers equipped with one receiver. POE allow the concatenation of multiple 2D or 3D pulse sequences into a single experiment, thus potentially combining all of the aforementioned advances, boosting the capability of ssNMR spectrometers at least two-fold without the addition of any hardware. In this perspective, we describe the first generation of POE, such as dual acquisition MAS (or DUMAS) methods, and then illustrate the evolution of these experiments into MEIOSIS, a method that enables the simultaneous acquisition of multiple 2D and 3D spectra. Using these new pulse schemes for the solid-state NMR investigation of biopolymers makes it possible to obtain sequential resonance assignments, as well as distance restraints, in about half the experimental time. While designed for acquisition of heteronuclei, these new experiments can be easily implemented for proton detection and coupled with other recent advancements, such as dynamic nuclear polarization (DNP), to improve signal to noise. Finally, we illustrate the application of these methods to microcrystalline protein preparations as well as single and multi-span membrane proteins reconstituted in lipid membranes. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. EFFECTS OF STATINS AND OTHER BIOLOGICAL PREPARATIONS UPON ACTIVATION OF MITOGEN-ACTIVATED PROTEIN KINASES IN PATIENTS WITH RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    I. V. Shirinsky

    2009-01-01

    Full Text Available Abstract. In this study, we evaluated effects of statins and other biological preparations upon spontaneous and stimulated activation of МАРК p38 and ERK1/2 in monocytes from the patients with rheumatoid arthritis (RA. We used peripheral blood mononuclear cells (PBMC from RA patients and healthy donors. PBMC were cultured in presence of 0, 0.1, 1 or 10 мM mevastatin, 10 мg/ml IL-1 receptor antagonist (IL-1Ra, 5 мg/ml infliximab, and 5 мg/ml soluble pegylated p55 TNF-receptor (r-met-Hu-sTNF-RI. To study the mechanisms of mevastatin effects upon МАРК p38 and ERK1/2 activities, L-mevalonate was added to the cultures. The cells were stained with anti-phospho-MAPK p38, or anti-phospho-ERK1/2, and analyzed with flow cytometry. We have shown that IL-1Ra and r-met-Hu-sTNF-RI inhibited spontaneous MAPK р38 activation. Mevastatin reduced spontaneous MAPK p38 and ERK1/2 phosphorylation. Mevastatininduced suppression of MAPK p38 and ERK1/2 activation was not dose-dependent. L-mevalonate completely prevented mevastatin-induced reduction of MAPK р38 phosphorylation and partially reversed inhibition of МАРК ERK1/2. In conclusion, decrease in MAPK activation represents a common mechanism of anti-inflammatory effects exerted by statins and some other biologicals.

  14. Method of preparing structural components of proteins and polysaccharides labelled with 14C radioisotope by photoautotrophic microorganisms

    International Nuclear Information System (INIS)

    Kleinmann, I.; Dvorakova, J.; Nejedly, Z.; Entlicher, G.; Kolina, J.; Filip, J.; Svoboda, V.

    1987-01-01

    The biomass labelled with radioisotope 14 C (e.g., blue-green Synechococcus elongatus algae) from which lipids and nucleic acids had previously been removed is subjected to enzyme hydrolysis using pronase. Another enzyme hydrolysis using for instance lysozyme and glycoamylase will produce a mixture which contains L-amino acid fractions and sugar compounds, such as D-[U- 14 C]glucose, D-[U- 14 C]glucosamine, etc. The mixture of L-[U- 14 C]amino acids separated using a polystyrene cation exchanger is divided into separate amino acids using the sorbent Ostion LGKS 0803 and a lithium citrate eluent. Desalination of the obtained fractions of L-[U- 14 C]amino acids is implemented in 0.01 to 4 M formic acid or acetic acid which contains 0 to 0.5% of thiodiglycol and 0 to 5% of ethanol. Desalination is implemented on ethyleneglycol methacrylate ion exchangers with phosphate, carboxyl or sulphone exchange groups. The advantage of the procedure is the perfect desalination of amino acids, higher yields of pure L-amino acids and of the protein hydrolysate and higher chemical purity of products. (E.S)

  15. Silk fibroin protein-based nonwoven mats incorporating baicalein Chinese herbal extract: preparation, characterizations, and in vivo evaluation.

    Science.gov (United States)

    Chan, Wing P; Huang, Kuan-Chen; Bai, Meng-Yi

    2017-02-01

    In this study, we demonstrated a natural silk fibroin protein (SFP) that was blended with a Chinese herbal extract (baicalein, BAI) to obtain an effective combination for producing electrospun nonwoven mats with anti-inflammatory and antibacterial functions. A series of SFP-based electrospun nonwoven mats with additives of varying compositions were produced and investigated. Performance comparisons showed that the SFP/polyvinylpyrrolidone (PVP)/BAI nonwoven mat is the optimal one. In vitro, SFP/PVP/BAI nonwoven mat is effective in inhibiting the formation of nitrite in lipopolysaccharide (LPS)-induced nitrite formation in Raw 264.7 macrophages model and the growth of Staphylococcus aureus (S. aureus). Especially in the case of SFP/PVP/BAI nonwoven mat, Bai has been proved to reach their maximum amount of releases of approximately 64.8% within 24 h of contact with water-based environment as compared to the SFP/BAI nonwoven mat (only 30.1% of release within 24 h). For in vivo experiments, a 1.2 cm × 1.2 cm wound area was created on the back of mice and seeded with 1 × 10 7 CFU/mL of S. aureus to induce an infected wound model. The experimental results show significant acceleration of the wound closure process in mice treated with SFP/PVP/BAI nonwoven mat (4 days of reduction as compared to the untreated group), reduction in infiltration of neutrophils, nitrite formation, and inhibition of growth of wound bacteria. Histological images of the group treated with SFP/PVP/BAI nonwoven mat showed a compete repair of skin hierarchy, increasing production of collagen fibers, and enhancement of angiogenesis. This may bring a better recovery of skin appearance after treatment. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 420-430, 2017. © 2015 Wiley Periodicals, Inc.

  16. Effect of surface microstructure and wettability on plasma protein adsorption to ZnO thin films prepared at different RF powers.

    Science.gov (United States)

    Huang, Zhan-Yun; Chen, Min; Pan, Shi-Rong; Chen, Di-Hu

    2010-10-01

    In this paper, the adsorption behavior of plasma proteins on the surface of ZnO thin films prepared by radio frequency (RF) sputtering under different sputtering powers was studied. The microstructures and surface properties of the ZnO thin films were investigated by x-ray diffraction (XRD), scanning electron microscopy (SEM), UV-visible optical absorption spectroscopy and contact angle techniques. The results show that the ZnO thin films have better orientation of the (0 0 2) peak with increasing RF power, especially at around 160 W, and the optical band gap of the ZnO films varies from 3.2 to 3.4 eV. The contact angle test carried out by the sessile drop technique denoted a hydrophobic surface of the ZnO films, and the surface energy and adhesive work of the ZnO thin films decreased with increasing sputtering power. The amounts of human fibrinogen (HFG) and human serum albumin (HSA) adsorbing on the ZnO films and reference samples were determined by using enzyme-linked immunosorbent assay (ELISA). The results show that fewer plasma proteins and a smaller HFG/HSA ratio adsorb on the ZnO thin films' surface.

  17. Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation.

    Science.gov (United States)

    Schieltz, David M; McGrath, Sara C; McWilliams, Lisa G; Rees, Jon; Bowen, Michael D; Kools, John J; Dauphin, Leslie A; Gomez-Saladin, Eduardo; Newton, Bruce N; Stang, Heather L; Vick, Michael J; Thomas, Jerry; Pirkle, James L; Barr, John R

    2011-06-15

    In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin. Published by Elsevier Ireland Ltd.

  18. Investigation of in-situ poly(lactic acid)/soy protein concentrate composites: Composite preparation, properties and foam application development

    Science.gov (United States)

    Liu, Bo

    2011-12-01

    In this study, soy protein (SP), the residue of oil crushing, was used for preparation of value-added thermoplastics. Novel poly(lactic acid) (PLA)/soy protein concentrate (SPC) blends were investigated and foaming of the resulting blends was developed. PLA/SPC blends were prepared by twin-screw extrusion and test specimens by injection molding. Unlike the practice elsewhere SP was used as a filler in mixing with other polymers, SPC was processed as a plastic component in blending process in this work. Processing SPC as plastic component, water played an important role in terms of the deformability and the morphology of SP thus the properties of the blends. Plasticization of SP, compatibilization of the blends and structure-property relationship of the PLA/SPC blends were studied. In the literature water and glycerol were often used together in preparing SP plastics or plastic blends, but this study found that this traditional combination did not provide the best results in terms of morphology and mechanical properties. Water is only recommended in plasticizing SP in the blends. This study showed water as a plasticizer was a domain factor on control of morphology and properties of PLA/SPC blends. The due to the evaporation of water after extrusion, SP domain lost its deformability thus resulted in in-situ composites. Interconnected SPC phase structure was achieved by control water content in the pre-formulated SPC and SPC content in the blends. A novel dual compatibilization method was developed to improve the properties of PLA/SPC blends. Poly(2-ethyl-2-oxazoline) was used to improve the dispersion of SPC in the blending stage, and polymeric methylene diphenyl diisocyanate was used to improve the interfacial adhesion between SPC and PLA in the subsequent processing. The result showed excellent mechanical properties and improved thermal properties of PLA/SPC blends. Using processing aids is an effective way to decrease processing temperature and thermal degradation

  19. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

    Science.gov (United States)

    Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

    2016-01-05

    A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

  20. A two-step, one-pot enzymatic method for preparation of duck egg white protein hydrolysates with high antioxidant activity.

    Science.gov (United States)

    Ren, Yao; Wu, Hui; Li, Xiaofeng; Lai, Furao; Zhao, Guanglei; Xiao, Xinglong

    2014-02-01

    Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8-10% and soluble peptide yield (SEP) of 60.5-70.2% in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2% and 90.4%, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21% gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis.

  1. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    International Nuclear Information System (INIS)

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-01-01

    Arsenic trioxide (arsenite, As III ) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As III on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As III on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As III -mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As III were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As III than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As III in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As III -mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As III cytotoxicity between these cells. -- Highlights: ► Examination of effect of As III on primary cultured chorion (C) and amnion (A) cells. ► Dose-dependent As III -mediated cytotoxicity in C

  2. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Yuta [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Yuan, Bo, E-mail: yuanbo@toyaku.ac.jp [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Kaise, Toshikazu [Laboratory of Environmental Chemodynamics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Takeichi, Makoto [Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065 (Japan); Tanaka, Sachiko; Hirano, Toshihiko [Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Kroetz, Deanna L. [Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 1550 4th St, RH584E Box 2911 San Francisco, CA 94158-2911 (United States); Toyoda, Hiroo [Department of Clinical Molecular Genetics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  3. Ice cream structure modification by ice-binding proteins.

    Science.gov (United States)

    Kaleda, Aleksei; Tsanev, Robert; Klesment, Tiina; Vilu, Raivo; Laos, Katrin

    2018-04-25

    Ice-binding proteins (IBPs), also known as antifreeze proteins, were added to ice cream to investigate their effect on structure and texture. Ice recrystallization inhibition was assessed in the ice cream mixes using a novel accelerated microscope assay and the ice cream microstructure was studied using an ice crystal dispersion method. It was found that adding recombinantly produced fish type III IBPs at a concentration 3 mg·L -1 made ice cream hard and crystalline with improved shape preservation during melting. Ice creams made with IBPs (both from winter rye, and type III IBP) had aggregates of ice crystals that entrapped pockets of the ice cream mixture in a rigid network. Larger individual ice crystals and no entrapment in control ice creams was observed. Based on these results a model of ice crystals aggregates formation in the presence of IBPs was proposed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Branaa, P; Naar, J; Chinain, M; Pauillac, S

    1999-01-01

    With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate

  5. Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel.

    Science.gov (United States)

    Tomanee, Panarat; Hsu, James T; Ito, Yoichiro

    2006-01-01

    Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

  6. Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

    Science.gov (United States)

    de Jesus, Jemmyson Romário; da Silva Fernandes, Rafael; de Souza Pessôa, Gustavo; Raimundo, Ivo Milton; Arruda, Marco Aurélio Zezzi

    2017-08-01

    The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL -1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL -1 . Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Dataset on preparation of the phosphorylated counterparts of a Momordica charantia protein for studying antifungal activities against susceptible dose-dependent C. albicans to antimycotics.

    Science.gov (United States)

    Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

    2017-12-01

    The data presented here are related to a research article entitled "Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein" (Qiao et al., 2017) [1]. The data set includes three portions: (1) a relationship between reaction velocities of protein phosphorylation as a function of the substrate concentrations, determined in enzymatic reactions in aid of protein kinases; (2) a result of antifungal susceptibility testing of C. albicans after it is selected in antimycotics; and (3) a comparison of protein expression in the susceptible dose-dependent fungus relative to the wild C. albicans . In the first portion, the relationship of reaction velocities and substrate concentrations is expressed as an output from the inverse variation model. All data and analyses are made publicly available and citied in the research article using a style for the Data in Brief.

  8. Effects of rice bran fiber on heat-induced gel prepared with pork salt-soluble meat proteins in model system.

    Science.gov (United States)

    Choi, Yun-Sang; Choi, Ji-Hun; Han, Doo-Jeong; Kim, Hack-Youn; Lee, Mi-Ai; Kim, Hyun-Wook; Jeong, Jong-Youn; Kim, Cheon-Jei

    2011-05-01

    The technological effects of rice bran fiber on pork salt-soluble meat proteins in a model system were investigated. Rice bran fiber at levels of 0% (control), 0.1%, 0.5%, 1%, and 2% was added at the same time as salt-soluble meat protein to maintain similar moisture levels in all samples. Samples with increasing amounts of added rice bran fiber had higher pH, yellowness, sarcoplasmic and total protein solubilities. The moisture content, myofibrillar protein solubility and water holding capacity were the highest in the treatments containing with 1% rice bran fiber. However, the lightness and redness, textural properties decreased with increasing rice bran fiber levels. SDS gel electrophoresis did not reveal any changes in proteins regardless different rice bran fiber levels. The apparent viscosity indicated that improvements in water holding capacity and decreased texture due to added rice bran fiber. Copyright © 2010 The American Meat Science Association. All rights reserved.

  9. Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps.

    Science.gov (United States)

    Jin, Ya; Zhang, Jun; Yuan, Qi; Manabe, Takashi; Tan, Wen

    2015-08-01

    Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [1]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6% to 1 × 10(-5) % of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with a defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2D gel. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Graphite Supported Preparation (GSP) of α-Cyano-4-Hydroxycinnamic Acid (CHCA) for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) for Peptides and Proteins

    Science.gov (United States)

    Gorka, Jan; Bahr, Ute; Karas, Michael

    2012-11-01

    Graphite as MALDI matrix or in combination with other substances has been reported in recent years. Here, we demonstrate that graphite can be used as target coating supporting the crystallization of the α-cyano-4-hydroxycinnamic acid matrix. A conventional dried-droplet preparation of matrix and analyte solution on a graphite-coated metal target leads to a thin, uniform layer of cubic crystals with about 1 μm edge length. Commercially available graphite powder of 1-2 μm particle size is gently wiped over the target using a cotton Q-tip, leading to an ultra-thin, not-visible film. This surface modification considerably improves analysis of peptides and proteins for MALDI MS using conventional dried-droplet preparation. Compared with untreated targets, the signal intensities of standard peptides are up to eight times higher when using the graphite supported crystallization. The relative standard deviation in peak area of angiotensin II for sample amounts between 1 and 50 fmol is reduced to about 15 % compared with 45 % for untreated sample holders. For a quantification of 1 fmol of the peptide using an internal standard the coefficient of variation is reduced to 3.5 % from 8 %. The new graphite supported preparation (GSP) protocol is very simple and does not require any technical nor manual skills. All standard solvents for peptides and proteins can be used.

  11. Genetic Enhancement of an Anti-Freeze Protein for use as a Substitute for Ethylene Glycol for Aircraft Anti-icing

    Science.gov (United States)

    2001-10-01

    freezing point depression is called a " colligative property", denoting "depending on the collection". 2 ...BACKGROUND: Traditional anti-icing agents are either propylene or ethylene glycol. Glycols are effective in lowering the freezing point of water...mixtures by the phenomenon of freezing point depression based solely on the molal concentration.

  12. Theoretical and experimental study of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, Irena; Golan, Martin; Pomeisl, Karel; Richter, Jan; Sedláková, Silvia; Šebera, Jakub; Mičová, Júlia; Falk, Martin; Falková, Iva; Řeha, David; Elliott, J.R.; Varga, K.; Follett, S.E.; Šimek, Daniel

    2017-01-01

    Roč. 7, č. 1 (2017), s. 352-360 ISSN 2046-2069 R&D Projects: GA ČR(CZ) GA15-05095S; GA MŠk LO1409; GA ČR(CZ) GA14-10279S; GA ČR(CZ) GA16-12454S; GA ČR GBP302/12/G157; GA MZd NV16-29835A Grant - others:FUNBIO(XE) CZ.2.16/3.1.00/21568 Institutional support: RVO:68378271 ; RVO:61388971 ; RVO:68081707 Keywords : X-ray diffraction * DFT * Raman spectroscopy * DSC * cryoprotectants Subject RIV: BO - Biophysics; EE - Microbiology, Virology (MBU-M) OBOR OECD: Biophysics Impact factor: 3.108, year: 2016

  13. [An improved method of preparing protein and peptide probes in mass spectrometry with ionization of division fragments by californium-252 (TOF-PDMS)].

    Science.gov (United States)

    Chivanov, V D; Zubarev, R A; Aksenov, S A; Bordunova, O G; Eremenko, V I; Kabanets, V M; Tatarinova, V I; Mishnev, A K; Kuraev, V V; Knysh, A N; Eremenko, I A

    1996-08-01

    The addition of organic acids (picric, oxalic, citric, or tartaric) to peptide and protein samples was found to significantly increase the yield of their quasi-molecular ions (QMI) in time-of-flight 252Cf plasma desorption mass spectrometry. The yield of the ions depended on the pKa of the acid added.

  14. A comparative in vitro study of the digestibility of heat- and high pressure-induced gels prepared from industrial milk whey proteins

    Science.gov (United States)

    He, Jin-Song; Mu, Tai-Hua; Wang, Juan

    2013-06-01

    We undertook this study to compare the digestibility of heat- and high pressure-induced gels produced from whey protein isolate (WPI). To simulate in vivo gastrointestinal digestion of WPI gels, a pepsin-trypsin digestion system was used. The in vitro protein digestibility of WPI gels induced by high pressure (400 MPa and 30 min; P-gel) and those induced by heat (80°C and 30 min; H-gel) was compared using a protein concentration of 0.14 g mL-1. The in vitro protein digestibility of P-gels was significantly greater than that of H-gels (p<0.05). The size-exclusion chromatography profiles of the hydrolysates showed that the P-gel generated more and smaller peptides than natural WPI and H-gels. Furthermore, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed some soluble disulfide-mediated aggregation in the P-gel, while there was more insoluble aggregation in the H-gel than the P-gel. The P-gel was more sensitive to proteinase than the H-gel, which was related to the content of S-S bonds, and this in turn could be attributed to the differences in the gelation mechanism between the H-gel and P-gel.

  15. Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

    Science.gov (United States)

    Shi, Lei; Chen, Huai; Zhang, Si-Yu; Chu, Ting-Ting; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2017-06-01

    Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  16. Preparation, radioiodination and in vitro evaluation of a nido-carborane-dextran conjugate, a potential residualizing label for tumor targeting proteins and peptides

    International Nuclear Information System (INIS)

    Tolmachev, V.; Bruskin, A.; Uppsala University; Sjoeberg, S.; Carlsson, J.; Lundqvist, H.

    2004-01-01

    Polysaccharides are not degradable by proteolytic enzymes in lysosomes and do not diffuse through cellular membranes. Thus, attached to an internalizing, targeting protein, such polysaccharide linkers, will remain intracellularly after protein degradation. They can be labeled with halogens and provide then a so called residualizing label. Such an approach improves tumor-to-non-tumor radioactivity ratio and, consequently, the results of radionuclide diagnostics and therapy. A new approach to obtain a stable halogenation of the polysaccharide dextran using 7-(3-amino-propyl)-7,8-dicarba-nido-undecaborate (-) (ANC) is presented. Dextran T10 was partially oxidized by metaperiodate, and ANC was coupled to dextran by reductive amination. The conjugate was then labeled with 125 I using either Chloramine-T or IodoGen as oxidants. Labeling efficiency was 69-85%. Stability of the label was evaluated in rat liver homogenates. Under these conditions, the ANC-dextran conjugate was found to be more stable than labeled albumin, which was used as a control protein. (author)

  17. Dipentaerythritol penta-/hexa-acrylate based-highly cross-linked hybrid monolithic column: Preparation and its applications for ultrahigh efficiency separation of proteins.

    Science.gov (United States)

    Ma, Junqian; Dai, Qian; Li, Xueyun; Zhu, Xianghui; Ma, Teng; Qiao, Xiaoqiang; Shen, Shigang; Liu, Xiuhua

    2017-04-22

    In this study, multi-acrylate based dipentaerythritol penta-/hexa-acrylate (DPEPA) was exploited for fabrication of highly cross-linked hybrid monolithic column by copolymerization with polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) via a "one-pot" method. The new DPEPA-POSS hybrid monolithic column was respectively characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, scanning electron microscopy and nitrogen adsorption/desorption measurement. When it was used for the separation of amides, thioureas and positional isomers of phenols, ultrahigh column efficiency separation (up to 511,000 N m -1 ) was achieved with excellent selectivity. Moreover, intact protein standards could be efficiently separated with minimum tailing peaks, outperforming the commercially available silica-based C8 column. Furthermore, successful separation of complex egg white proteins and expressed BARD1 BRCT domains protein sample was also achieved with good chromatographic performance. In the future work, the DPEPA-POSS hybrid monolithic column will be further exploited and applied in capillary electrochromatography as well as the top-down based proteome research. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Preparation, crystallization and preliminary X-ray analysis of XC2382, an ApaG protein of unknown structure from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A putative ApaG gene product from X. campestris pv. campestris was overexpressed in E. coli, purified and crystallized. The crystals diffracted to a resolution of at least 2.3 Å. Xanthomonas campestris pv. campestris is the causative agent of black rot, one of the major worldwide diseases of cruciferous crops. Its genome encodes approximately 4500 proteins, roughly one third of which have unknown function. XC2382 is one such protein, with a MW of 14.2 kDa. Based on a bioinformatics study, it was annotated as an ApaG gene product that serves multiple functions. The ApaG protein has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of at least 2.30 Å. They are tetragonal and belong to space group P4 1/3 , with unit-cell parameters a = b = 57.6, c = 122.9 Å. There are two, three or four molecules in the asymmetric unit

  19. Nanocomposites based on graphene oxide and mesoporous silica nanoparticles: Preparation, characterization and nanobiointeractions with red blood cells and human plasma proteins

    Science.gov (United States)

    Fonseca, Leandro C.; de Araújo, Maciel M.; de Moraes, Ana Carolina M.; da Silva, Douglas S.; Ferreira, Ariane G.; Franqui, Lidiane S.; Martinez, Diego Stéfani T.; Alves, Oswaldo L.

    2018-04-01

    The current work refers to the development of a novel nanocomposite based on graphene oxide (GO) and mesoporous amino silica nanoparticles (H2N-MSNs) and its biological interaction with red blood cells (RBCs) and human blood plasma toward the investigation of nanobiointeractions. Silica nanoparticles and several graphene oxide-based materials are, separately, known for their high hemolytic potential and strong interaction with human plasma proteins. In this context, the GO-MSN interaction and its influence in minimizing the reported effects were investigated. The materials were synthesized by covalently attaching H2N-MSNs onto the surface of GO through an amidation reaction. GO-MSN nanocomposites were obtained by varying the mass of H2N-MSNs and were characterized by FTIR, NMR, XRD, TGA, zeta potential and TEM. The characterization results confirm that nanocomposites were obtained, suggest covalent bond attachment mostly by amine-epoxy reactions and evidence an unexpected reduction reaction of GO by H2N-MSNs, whose mechanism is proposed. Biological assays showed a decrease of hemolysis (RBC lysis) and a minimization of the interaction with human plasma proteins (protein corona formation). These are important findings toward achieving in vivo biocompatibility and understanding the nanobiointeractions. Finally, this work opens possibilities for new nanomedicine applications of GO-MSN nanocomposites, such as drug delivery system.

  20. Solution preparation

    International Nuclear Information System (INIS)

    Seitz, M.G.

    1982-01-01

    Reviewed in this statement are methods of preparing solutions to be used in laboratory experiments to examine technical issues related to the safe disposal of nuclear waste from power generation. Each approach currently used to prepare solutions has advantages and any one approach may be preferred over the others in particular situations, depending upon the goals of the experimental program. These advantages are highlighted herein for three approaches to solution preparation that are currently used most in studies of nuclear waste disposal. Discussion of the disadvantages of each approach is presented to help a user select a preparation method for his particular studies. Also presented in this statement are general observations regarding solution preparation. These observations are used as examples of the types of concerns that need to be addressed regarding solution preparation. As shown by these examples, prior to experimentation or chemical analyses, laboratory techniques based on scientific knowledge of solutions can be applied to solutions, often resulting in great improvement in the usefulness of results

  1. Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat1.

    Science.gov (United States)

    Zhao, X; Xing, T; Chen, X; Han, M-Y; Li, X; Xu, X-L; Zhou, G-H

    2017-05-01

    Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G΄) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G΄ was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH. © 2016

  2. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

  3. Preparation and characterization of room temperature ionic liquid/single-walled carbon nanotube nanocomposites and their application to the direct electrochemistry of heme-containing proteins/enzymes

    International Nuclear Information System (INIS)

    Du, Pan; Liu, Shuna; Wu, Ping; Cai, Chenxin

    2007-01-01

    This work describes the formation and possible electrochemical application of a novel nanocomposite based on single-walled carbon nanotubes (SWNTs) and imidazolium-based room-temperature ionic liquids (RTILs) of 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF 4 , a hydrophilic RTIL) and 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF 6 , a hydrophobic RTIL). The nanocomposites ([bmim]BF 4 -SWNTs, and [bmim]PF 6 -SWNTs) were formed by simply grinding the SWNTs with the respective RTIL. The results of the X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy indicated that the nanocomposites were formed by adsorption of an imidazolium ion on the surface of SWNTs via the 'cation-π' interaction. SEM images showed that [bmim]BF 4 -SWNTs (or [bmim]PF 6 -SWNTs) nanocomposites could uniformly cover the surface of a glassy carbon (GC) electrode resulting in a RTILs-SWNTs/GC modified electrode with a high stability. The RTILs-SWNTs composite could be readily used as a matrix to immobilize heme-containing proteins/enzymes (myoglobin, cytochrome c, and horseradish peroxidase) without undergoing denaturation, as was verified by UV-vis and circular dichroic (CD) spectroscopic results. The voltammetric results showed that heme-containing proteins/enzymes entrapped in RTILs-SWNTs composites displayed a pair of well-defined, stable redox peaks, which were ascribed to their direct electron-transfer reactions. The results of controlled experiments showed that the positive charged imidazolium ion played a significant effect on the electrochemical parameters, such as the redox peak separation and the value of the formal potentials, etc., of the electron-transfer reaction of non-neutral species dissolved in solution or immobilized on the electrode surface. Further results demonstrated that the heme-containing proteins/enzymes entrapped in RTILs-SWNTs composites could still retain their bioelectrocatalytic activity toward the reduction of oxygen and hydrogen

  4. Improvement of Interfacial Adhesion by Bio-Inspired Catechol-Functionalized Soy Protein with Versatile Reactivity: Preparation of Fully Utilizable Soy-Based Film

    Directory of Open Access Journals (Sweden)

    Zhong Wang

    2017-03-01

    Full Text Available The development of materials based on renewable resources with enhanced mechanical and physicochemical properties is hampered by the abundance of hydrophilic groups because of their structural instability. Bio-inspired from the strong adhesion ability of mussel proteins, renewable and robust soy-based composite films were fabricated from two soybean-derived industrial materials: soluble soybean polysaccharide (SSPS and catechol-functionalized soy protein isolate (SPI-CH. The conjugation of SPI with multiple catechol moieties as a versatile adhesive component for SSPS matrix efficiently improved the interfacial adhesion between each segment of biopolymer. The biomimetic adherent catechol moieties were successfully bonded in the polymeric network based on catechol crosslinking chemistry through simple oxidative coupling and/or coordinative interaction. A combination of H-bonding, strong adhesion between the SPI-CH conjugation and SSPS matrix resulted in remarkable enhancements for mechanical properties. It was found that the tensile strength and Young’s modulus was improved from 2.80 and 17.24 MPa of unmodified SP film to 4.04 and 97.22 MPa of modified one, respectively. More importantly, the resultant films exhibited favorable water resistance and gas (water vapor barrier performances. The results suggested that the promising way improved the phase adhesion of graft copolymers using catechol-functionalized polymers as versatile adhesive components.

  5. Changes in growth hormone-binding protein in girls with central precocious puberty treated with a depot preparation of luteinizing hormone-releasing hormone analogue.

    Science.gov (United States)

    Oliveira, S B; Donnadieu, M; Chaussain, J L

    1993-01-01

    Growth hormone-binding protein (GHBP) was studied in 11 girls with true precocious puberty, aged 7.3 +/- 0.2 years (mean +/- SE), before and after the first 6 months of treatment with luteinizing hormone-releasing hormone analogue D-Trp6-LHRH. The 125I-human GH was incubated with 150 microliters of serum, bound and free GH were separated by gel filtration. The levels of GHBP increased significantly from 24.2 +/- 1.3 to 28.1 +/- 1.9% (p pubertal spurt is mediated by a sex-steroid-induced rise in GH concentration, and they suggest that the levels of GHBP may be related to the GH secretion and its variation with treatment.

  6. Proteomics applied on plant abiotic stresses: role of heat shock proteins (HSP).

    Science.gov (United States)

    Timperio, Anna Maria; Egidi, Maria Giulia; Zolla, Lello

    2008-10-07

    The most crucial function of plant cell is to respond against stress induced for self-defence. This defence is brought about by alteration in the pattern of gene expression: qualitative and quantitative changes in proteins are the result, leading to modulation of certain metabolic and defensive pathways. Abiotic stresses usually cause protein dysfunction. They have an ability to alter the levels of a number of proteins which may be soluble or structural in nature. Nowadays, in higher plants high-throughput protein identification has been made possible along with improved protein extraction, purification protocols and the development of genomic sequence databases for peptide mass matches. Thus, recent proteome analysis performed in the vegetal Kingdom has provided new dimensions to assess the changes in protein types and their expression levels under abiotic stress. As reported in this review, specific and novel proteins, protein-protein interactions and post-translational modifications have been identified, which play a role in signal transduction, anti-oxidative defence, anti-freezing, heat shock, metal binding etc. However, beside specific proteins production, plants respond to various stresses in a similar manner by producing heat shock proteins (HSPs), indicating a similarity in the plant's adaptive mechanisms; in plants, more than in animals, HSPs protect cells against many stresses. A relationship between ROS and HSP also seems to exist, corroborating the hypothesis that during the course of evolution, plants were able to achieve a high degree of control over ROS toxicity and are now using ROS as signalling molecules to induce HSPs.

  7. Ganglioside contained in the neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) fraction prepared from the detergent-resistant membrane microdomain of rat brain inhibits the phosphatase activity of calcineurin.

    Science.gov (United States)

    Kobayashi, Yuumi; da Silva, Ronan; Kumanogoh, Haruko; Miyata, Shinji; Sato, Chihiro; Kitajima, Ken; Nakamura, Shun; Morita, Mistuhiro; Hayashi, Fumio; Maekawa, Shohei

    2015-09-01

    Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). Neuronal tissue-enriched acidic protein of 22 kDa (NAP-22) is one of the major protein components of neuronal DRM. To determine the cellular function of NAP-22, interacting proteins were screened with an immunoprecipitation assay, and calcineurin (CaN) was detected. Further studies with NAP-22 prepared from DRM and CaN expressed in bacteria showed the binding of these proteins and a dose-dependent inhibitory effect of the NAP-22 fraction on the phosphatase activity of CaN. On the other hand, NAP-22 expressed in bacteria showed low binding to CaN and a weak inhibitory effect on phosphatase activity. To solve this discrepancy, identification of a nonprotein component that modulates CaN activity in the DRM-derived NAP-22 fraction was attempted. After lyophilization, a lipid fraction was extracted with chloroform/methanol. The lipid fraction showed an inhibitory effect on CaN without NAP-22, and further fractionation of the extract with thin-layer chromatography showed the presence of several lipid bands having an inhibitory effect on CaN. The mobility of these bands coincided with that of authentic ganglioside (GM1a, GD1a, GD1b, and GT1b), and authentic ganglioside showed an inhibitory effect on CaN. Treatment of lipid with endoglycoceramidase, which degrades ganglioside to glycochain and ceramide, caused a diminution of the inhibitory effect. These results show that DRM-derived NAP-22 binds several lipids, including ganglioside, and that ganglioside inhibits the phosphatase activity of CaN. © 2015 Wiley Periodicals, Inc.

  8. Sample preparation

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Sample preparation prior to HPLC analysis is certainly one of the most important steps to consider in trace or ultratrace analysis. For many years scientists have tried to simplify the sample preparation process. It is rarely possible to inject a neat liquid sample or a sample where preparation may not be any more complex than dissolution of the sample in a given solvent. The last process alone can remove insoluble materials, which is especially helpful with the samples in complex matrices if other interactions do not affect extraction. Here, it is very likely a large number of components will not dissolve and are, therefore, eliminated by a simple filtration process. In most cases, the process of sample preparation is not as simple as dissolution of the component interest. At times, enrichment is necessary, that is, the component of interest is present in very large volume or mass of material. It needs to be concentrated in some manner so a small volume of the concentrated or enriched sample can be injected into HPLC. 88 refs

  9. Preparo de hidrolisados protéicos para a análise de aminoácidos Preparation of protein hydrolysates for amino acid analysis

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Bernardi

    2003-12-01

    Full Text Available Amostras de milho, farelo de soja e hidrolisado ácido de caseína foram submetidas à hidrólise ácida em estufa (110 ± 5°C, durante 22horas com ácido clorídrico 6 N, em ampolas de vidro seladas sob vácuo. Os hidrolisados foram preparados por quatro métodos: 1 evaporação em evaporador rotativo sob vácuo; 2 evaporação em câmaras a vácuo em presença de pastilhas de NaOH; 3 evaporação em bloco de aquecimento sob fluxo de argônio; 4 neutralização com solução padrão de citrato/NaOH. Os aminoácidos foram separados e quantificados em analisador específico Beckman System 6300 com derivatização pós-coluna por ninidrina. O método de neutralização dos reagentes da hidrólise apresentou os melhores resultados considerando todos os aminoácidos. Além disso, foi mais rápido e prático no preparo de um grande número de amostras. Quanto aos métodos de evaporação, o uso de evaporador rotativo apresentou perdas significativas apenas para treonina e serina na amostra farelo de soja; sob fluxo de argônio observou-se baixos teores para treonina, fenilalanina e lisina; em câmara sob vácuo verificou-se perdas significativas para treonina, serina e tirosina.Samples of corn, soybean corn and casein (acid hydrolysate were submitted to acid hydrolysis in oven (110 ± 5°C, for 22 hours with 6 N HCl in sealed ampoules under vacuum. Four methods of hydrolysate preparation were used: 1 evaporation to dryness in rotary evaporator under vacuum, 2 evacuated desiccator under vacuum and NaOH, 3 flushing the open vial with argon and 4 neutralisation with standard citrate/NaOH solution. Amino acids were isolated and quantified in specific analyzer Beckman System 6300 with ninhidrin post-column derivatization. The neutralization method presented the best result considering all analysed amino acids. Besides, it was more practical for rapid processing of a large number of samples. With regard to the evaporation methods in the rotary evaporator

  10. Radioprotective preparation

    International Nuclear Information System (INIS)

    Stefanova, D.; Frattadochi, A.; Gattavecchia, E.; Ferri, E.; Tonnelli, D.

    1988-01-01

    The invention is intended for radiation injuries prophylaxis in mammals. It has an well expressed radioprotective effect against acute gamma irradiation on cellular level as well as a prolonged action when applied up to 48 hours before the acute irradiation. The preparation is a coprecipitate of the natural tripeptide glutathione (reduced form) and polyvinyl pyrrolidone (pvp) in ratio 30-60/70-40. It is obtained by incubation method with subsequent lyophilization from water solution of the initial components. The molecular mass of the pvp is 20 till 360.10 3 . 2 claims

  11. Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins Preparação e escalonamento de um hidrolisado enzimático de proteínas do soro de leite bovino

    Directory of Open Access Journals (Sweden)

    Marilisa Guimarães Lara

    2005-12-01

    Full Text Available We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5% acetic acid at 60 liters h-1 at 25ºC. The product, recovered in 37% yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and Foi descrita a preparação de um hidrolisado de proteínas do soro de leite bovino com enzimas pancreáticas, contendo baixos níveis de aminoácidos aromáticos. Quando utilizadas pancreatina e tripsina/quimotripsina, por 27h a 37±2ºC, foram liberados 74% de Phe, 100% de Tyr e 100% de Trp como aminoácidos livres. A maioria dos aminoácidos aromáticos livres, presentes em dois quilos de hidrolisado (15 litros, foi separada dos peptídeos e outros aminoácidos remanescentes por filtração em coluna de gel de Sephadex G-25C eluída com ácido acético 5%, fluxo de 60 litros por hora a 25ºC. O produto, recuperado com 37% de rendimento, continha 0,70 mmol de Phe, 0,41 mmol de Tyr e <0,01 mmol de Trp/100 mmol de aminoácidos recuperados. A composição em aminoácidos do hidrolisado foi similar às proteínas do soro com as quais foi preparado. Após adição de aminoácidos aromáticos apropriados, ele pode ser usado como fonte de nitrogênio para pacientes com fenilcetonuria ou tirosinemia.

  12. Preparation and Characterization of Myosin Proteins.

    Science.gov (United States)

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  13. SHORT COMMUNICATION SOLVENT FREE PREPARATION OF N ...

    African Journals Online (AJOL)

    Preferred Customer

    carboxy, 4-bromo, 4- hydroxy ... which are an important class of substrates for biological as chemical probes of protein structure. [1], as a .... Table 1. Mixing time, melting point, yield% and elemental analysis of prepared N-substituted maleanilic acids.

  14. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  15. Preparation of cultured skin for transplantation using insulin-like growth factor I in conjunction with insulin-like growth factor binding protein 5, epidermal growth factor, and vitronectin.

    Science.gov (United States)

    Dawson, Rebecca A; Upton, Zee; Malda, Jos; Harkin, Damien G

    2006-06-27

    Cultured skin for transplantation is routinely prepared by growing patient keratinocytes in the presence of semidefined sources of growth factors including serum and feeder cells, but these materials require substantial risk remediation and can contribute to transplant rejection. We have therefore investigated the potential of a novel combination of recombinant and purified growth factors to replace serum and feeder cells in cultures of human keratinocytes suitable for clinical application. Our technique was investigated with respect to culture establishment, serial propagation, colony-forming efficiency, immunocytochemistry, epidermal reconstruction, and suitability to support transplantation by aerosolization. We demonstrate that insulin-like growth factor (IGF)-I--used in conjunction with epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-5 and vitronectin--supports growth in the absence of serum. Moreover, a threefold greater number of cells are generated within 7 days compared to those grown under current best practice conditions using serum (P<0.05). The resulting test cultures are suitable for epidermal reconstruction and support the option for delivery in the form of an aerosolized cell suspension. Serial propagation, with the view to producing confluent sheets for extensive injuries, was achieved but with less consistency and this result correlated with a significant decline in colony-forming efficiency compared to controls. IGF-I used in conjunction with IGFBP-5, EGF, and vitronectin provides a superior alternative to serum for the rapid expansion and transplantation of cultured keratinocytes within the first week of treatment. Nevertheless, further optimization is required with respect to elimination of feeder cells and serial expansion of cultures for treatment of extensive injuries.

  16. Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands

    Directory of Open Access Journals (Sweden)

    Tchankouo-Nguetcheu Stephane

    2012-11-01

    Full Text Available Abstract Background Arthropod-borne viral infections cause several emerging and resurging infectious diseases. Among the diseases caused by arboviruses, chikungunya is responsible for a high level of severe human disease worldwide. The salivary glands of mosquitoes are the last barrier before pathogen transmission. Methods We undertook a proteomic approach to characterize the key virus/vector interactions and host protein modifications that occur in the salivary glands that could be responsible for viral transmission by using quantitative two-dimensional electrophoresis. Results We defined the protein modulations in the salivary glands of Aedes aegypti that were triggered 3 and 5 days after an oral infection (3 and 5 DPI with chikungunya virus (CHIKV. Gel profile comparisons showed that CHIKV at 3 DPI modulated the level of 13 proteins, and at 5 DPI 20 proteins. The amount of 10 putatively secreted proteins was regulated at both time points. These proteins were implicated in blood-feeding or in immunity, but many have no known function. CHIKV also modulated the quantity of proteins involved in several metabolic pathways and in cell signalling. Conclusion Our study constitutes the first analysis of the protein response of Aedes aegypti salivary glands infected with CHIKV. We found that the differentially regulated proteins in response to viral infection include structural proteins and enzymes for several metabolic pathways. Some may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by arboviruses. For example, proteins involved in blood-feeding such as the short D7, an adenosine deaminase and inosine-uridine preferring nucleoside hydrolase, may favour virus transmission by exerting an increased anti-inflammatory effect. This would allow the vector to bite without the bite being detected. Other proteins, like the anti-freeze protein, may support vector protection.

  17. Batter and method for preparing a pasta

    NARCIS (Netherlands)

    Wind, P.; Linden, van der E.

    2011-01-01

    This invention describes a batter that is suitable for preparing a pasta. The batter comprises water, a starch and a protein, whereby the weight ratio between the protein and the total amount of starch in the batter is represented by the symbol y and whereby the weight percentage of the total amount

  18. Preparation and characterization of antigenic properties of ...

    African Journals Online (AJOL)

    A rapid, simple and low cost procedure for preparing hapten-protein conjugates was developed using gramicidin A (GA) and two other water-soluble proteins, keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). GA was a kind of antimicrobial peptides. Two lysines and a cysteine were linked to amino- terminus and ...

  19. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  20. Preparing for Surgery

    Science.gov (United States)

    ... FAQs Preparing for Surgery Page Navigation ▼ ACOG Pregnancy Book Preparing for Surgery Patient Education FAQs Preparing for ... the person who is in charge of giving anesthesia and checking its effects. What can I do ...

  1. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hao-Tsai Cheng

    2016-01-01

    Full Text Available Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images.

  2. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  3. Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae.

    Science.gov (United States)

    Badet, Thomas; Peyraud, Rémi; Raffaele, Sylvain

    2015-01-01

    Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.

  4. Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae

    Directory of Open Access Journals (Sweden)

    Thomas eBadet

    2015-09-01

    Full Text Available Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.

  5. Proteomic comparison between maturation drying and prematurely imposed drying of Zea mays seeds reveals a potential role of maturation drying in preparing proteins for seed germination, seedling vigor, and pathogen resistance.

    Science.gov (United States)

    Wang, Wei-Qing; Ye, Jian-Qing; Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna I; Jensen, Ole Nørregaard; Møller, Ian Max; Song, Song-Quan

    2014-02-07

    We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p may contribute to the acquisition of seed germinability. However, a relatively large number of proteins changed in the embryo (47 spots) and endosperm (76 spots) specifically during maturation drying. Among these proteins, storage proteins in the embryo and defense proteins in the endosperm may be particularly important for seedling vigor and resistance to fungal infection, respectively.

  6. Sample preparation guidelines for two-dimensional electrophoresis.

    Science.gov (United States)

    Posch, Anton

    2014-12-01

    Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.

  7. Urine sample preparation for proteomic analysis.

    Science.gov (United States)

    Olszowy, Pawel; Buszewski, Boguslaw

    2014-10-01

    Sample preparation for both environmental and more importantly biological matrices is a bottleneck of all kinds of analytical processes. In the case of proteomic analysis this element is even more important due to the amount of cross-reactions that should be taken into consideration. The incorporation of new post-translational modifications, protein hydrolysis, or even its degradation is possible as side effects of proteins sample processing. If protocols are evaluated appropriately, then identification of such proteins does not bring difficulties. However, if structural changes are provided without sufficient attention then protein sequence coverage will be reduced or even identification of such proteins could be impossible. This review summarizes obstacles and achievements in protein sample preparation of urine for proteome analysis using different tools for mass spectrometry analysis. The main aim is to present comprehensively the idea of urine application as a valuable matrix. This article is dedicated to sample preparation and application of urine mainly in novel cancer biomarkers discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers......, are reported. The aim is to depict how the elucidation of the interplay of structures requires the interplay of methods....

  9. Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core.

    Science.gov (United States)

    Gates, Zachary P; Baxa, Michael C; Yu, Wookyung; Riback, Joshua A; Li, Hui; Roux, Benoît; Kent, Stephen B H; Sosnick, Tobin R

    2017-02-28

    The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.

  10. Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study.

    Science.gov (United States)

    Zakharov, Boris; Fisyuk, Alexander; Fitch, Andy; Watier, Yves; Kostyuchenko, Anastasia; Varshney, Dushyant; Sztucki, Michael; Boldyreva, Elena; Shalaev, Evgenyi

    2016-07-01

    Ice formation and recrystallization is a key phenomenon in freezing and freeze-drying of pharmaceuticals and biopharmaceuticals. In this investigation, high-resolution synchrotron X-ray diffraction is used to quantify the extent of disorder of ice crystals in binary aqueous solutions of a cryoprotectant (sorbitol) and a protein, bovine serum albumin. Ice crystals in more dilute (10 wt%) solutions have lower level of microstrain and larger crystal domain size than these in more concentrated (40 wt%) solutions. Warming the sorbitol-water mixtures from 100 to 228 K resulted in partial ice melting, with simultaneous reduction in the microstrain and increase in crystallite size, that is, recrystallization. In contrast to sorbitol solutions, ice crystals in the BSA solutions preserved both the microstrain and smaller crystallite size on partial melting, demonstrating that BSA inhibits ice recrystallization. The results are consistent with BSA partitioning into quasi-liquid layer on ice crystals but not with a direct protein-ice interaction and protein sorption on ice surface. The study shows for the first time that a common (i.e., not-antifreeze) protein can have a major impact on ice recrystallization and also presents synchrotron X-ray diffraction as a unique tool for quantification of crystallinity and disorder in frozen aqueous systems. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  11. International perspectives on coal preparation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-12-31

    The report consists of the vugraphs from the presentations which covered the following topics: Summaries of the US Department of Energy`s coal preparation research programs; Preparation trends in Russia; South African coal preparation developments; Trends in hard coal preparation in Germany; Application of coal preparation technology to oil sands extraction; Developments in coal preparation in China; and Coal preparation in Australia.

  12. Preparation and characterization of antigenic properties of ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... liquid chromatography (HPLC). Then the activated KLH and OVA were conjugated .... detecting the hydrosulfuryl content of bio-samples with colorimetric determination, showing a maximum peak at 412 nm. .... Principles and methods for the preparation of drug protein conjugates for immunological studies.

  13. Molecular modeling and structural characterization of a high glycine-tyrosine hair keratin associated protein.

    Science.gov (United States)

    Singh, Rakesh S; Palmer, Jeremy C; Pudney, Paul D A; Paul, Prem K C; Johannessen, Christian; Debenedetti, Pablo G; Raut, Janhavi; Lee, Ken; Noro, Massimo; Tiemessen, David

    2017-03-22

    High glycine-tyrosine (HGT) proteins are an important constituent of the keratin associated proteins (KAPs) present in human hair. The glassy state physics of hair fibres are thought to be largely regulated by KAPs, which exist in an amorphous state and are readily affected by environmental conditions. However, there are no studies characterizing the individual KAPs. In this paper, we present the first step to fill this gap by computational modeling and experimental studies on a HGT protein, KAP8.1. In particular, we have modeled the three-dimensional structure of this 63-residue protein using homology information from an anti-freeze protein in snow flea. The model for KAP8.1 is characterized by four strands of poly-proline II (or PPII) type helical secondary structures, held together by two cysteine disulphide bridges. Computer simulations confirm the stability of the modelled structure and show that the protein largely samples the PPII and β-sheet conformations during the molecular dynamics simulations. Spectroscopic studies including Raman, IR and vibrational circular dichroism have also been performed on synthesized KAP8.1. The experimental studies suggest that KAP8.1 is characterised by β-sheet and PPII structures, largely consistent with the simulation studies. The model built in this work is a good starting point for further simulations to study in greater depth the glassy state physics of hair, including its water sorption isotherms, glass transition, and the effect of HGT proteins on KAP matrix plasticization. These results are a significant step towards our goal of understanding how the properties of hair can be affected and manipulated under different environmental conditions of temperature, humidity, ageing and small molecule additives.

  14. Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids

    Science.gov (United States)

    Cao, Lixue; Huang, Qiao; Wu, Zhichao; Cao, Dong-dong; Ma, Zhanling; Xu, Qianghua; Hu, Peng; Fu, Yanxia; Shen, Yu; Chan, Jiulin; Zhou, Cong-zhao; Zhai, Wanying; Chen, Liangbiao

    2016-01-01

    The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP–ice interaction mechanism distinct from the known antifreeze proteins. PMID:27698404

  15. Materials Preparation Center

    Data.gov (United States)

    Federal Laboratory Consortium — MPC is recognized throughout the worldwide research community for its unique capabilities in purification, preparation, and characterization of: rare earth metals,...

  16. Documents preparation and review

    International Nuclear Information System (INIS)

    1999-01-01

    Ignalina Safety Analysis Group takes active role in assisting regulatory body VATESI to prepare various regulatory documents and reviewing safety reports and other documentation presented by Ignalina NPP in the process of licensing of unit 1. The list of main documents prepared and reviewed is presented

  17. Proteomic Comparison between Maturation Drying and Prematurely Imposed Drying of Zea mays Seeds Reveals a Potential Role of Maturation Drying in Preparing Proteins for Seed Germination, Seedling Vigor, and Pathogen Resistance

    DEFF Research Database (Denmark)

    Wang, Wei-Qing; Ye, Jian-Qing; Rogowska-Wrzesinska, Adelina

    2014-01-01

    We have studied the role(s) of maturation drying in the acquisition of germinability, seedling vigor and pathogen resistance by comparing the proteome changes in maize embryo and endosperm during mature and prematurely imposed drying. Prematurely imposed dried seeds at 40 days after pollination...... (DAP) germinated almost as well as mature seeds (at 65 DAP), but their seedling growth was slower and they were seriously infected by fungi. A total of 80 and 114 proteins were identified to change at least two-fold (p...

  18. PREPARATIVE SKIN PREPARATION AND SURGICAL WOUND INFECTION

    Directory of Open Access Journals (Sweden)

    Anjanappa

    2015-01-01

    Full Text Available BACKGROUND AND OBJECTIVE: It is an established fact now that the normal skin of healthy human beings harbours a rich bacterial fl ora. Normally considered non - pathogenic , these organisms way be a potential source of infection of the surgical wound. Approximately 20% of the resident flora is beyond the reach of surgical scrubs and antiseptics. The goal of surgical preparation of the skin with antiseptics is to remove transient and pathogenic microorganisms on the skin surface and to reduce the resident flora to a low level. Povidone iodine (I odophors and chlorhexidine are most often used antiseptics for pre - operative skin preparation. OBJECTIVES : To evaluate the efficacy of povidone iodine alone and in combination with antiseptic agent containing alcoholic chlorhexidine in preoperative skin p reparation by taking swab culture. (2 To compare the rate of postoperative wound infection in both the groups. METHODS: One hundred patients (fifty in each group undergoing clean elective surgery with no focus of infection on the body were included in th e study. The pre - operative skin preparation in each group is done with the respective antiseptic regimen. In both the groups after application of antiseptics , sterile saline swab culture was taken immediately from site of incision. In cases which showed gr owth of organisms , the bacteria isolated were identified by their morphological and cultural characteristics. Grams staining , coagulase test and antibiotic sensitivity test were done wherever necessary and difference in colonization rates was determined as a measure of efficacy of antiseptic regimen. RESULTS: The results of the study showed that when compared to povidone iodine alone , using a combination of povidone iodine and alcoholic solution of chlorhexidine , the colonization rates of the site of incisi on were reduced significantly. As for the rate of post - operative wound infection , it is also proven that wound infections are also

  19. Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

    Science.gov (United States)

    Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

    2017-02-01

    A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh 3 )] (Where R=H (1); CH 3 (2); C 2 H 5 (3); C 6 H 5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh 3 )] (3) >[Ni(4-Msal-mtsc)(PPh 3 )] (2) >[Ni(4-Msal-tsc)(PPh 3 )] (1) >[Ni(4-Msal-ptsc)(PPh 3 )] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes

  20. Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation

    DEFF Research Database (Denmark)

    Malmström, Johan; Larsen, Kristoffer; Hansson, Lennart

    2002-01-01

    -dimensional electrophoresis was interfaced to miniaturized sample preparation techniques using microcapillary extraction. Four protein groups were identified; cytoskeletal, adhesion, scavenger and metabolic proteins. These patient's proteomes showed a high degree of heterogeneity between patients but larger homogeneity...

  1. PREPARATION OF URANIUM MONOSULFIDE

    Science.gov (United States)

    Yoshioka, K.

    1964-01-28

    A process is given for preparing uranium monosulfide from uranium tetrafluoride dissolved in molten alkali metal chloride. A hydrogen-hydrogen sulfide gas mixture passed through the solution precipitates uranium monosulfide. (AEC)

  2. Sperm preparation for fertilization

    NARCIS (Netherlands)

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen

  3. Thyroid preparation overdose

    Science.gov (United States)

    Thyroid preparations are medicines used to treat thyroid gland disorders. Overdose occurs when someone takes more than the normal or recommended amount of this medicine. This can be by accident or ...

  4. Dukovany ASSET mission preparation

    International Nuclear Information System (INIS)

    Kouklik, I.

    1996-01-01

    We are in the final stages of the Dukovany ASSET mission 1996 preparation. I would like to present some of our recent experiences. Maybe they would be helpful to other plants, that host ASSET missions in future

  5. Preparation of bromine fluoride

    International Nuclear Information System (INIS)

    Domange, Pr; Duflo, J.

    1958-05-01

    This note addresses the preparation of bromine fluoride. It indicates the implemented process for the reaction, used products (fluorine and bromine), and column characteristics. It describes the operating mode. Apparatus drawing is provided

  6. Microencapsulation III: Preparation of invertase microcapsules.

    Science.gov (United States)

    Rambourg, P; Lévy, J; Lévy, M C

    1982-07-01

    Invertase was incorporated into polyamide microcapsules. The following parameters were studied: pH of the aqueous phase during interfacial polymerization; duration of the polymerization; surfactant concentration; stirring rate; improvements in the isolation procedure; effect of lyophilization. The inactivation of the encapsulated enzyme by pepsin was shown to be related to the acidic incubation medium and prompted incorporation of protective proteins in the microcapsules. This process allowed relative protection of the enzyme. In a second set of experiments, an emulsification-reticulation method was developed, which encapsulated invertase in a cross-linked protein. Various proteins and bifunctional acylating agents were tested. Microcapsules of immobilized invertase were prepared through cross-linking of the enzyme protein itself.

  7. TEM Specimen Preparation

    OpenAIRE

    sprotocols

    2014-01-01

    Author: House Ear Institute ### Preparative Techniques for the TEM For routine transmission electron microscopy (TEM), it is generally accepted that specimens should be thin, dry and contain molecules which diffract electrons. Biological specimens, which are large and consist of large amounts of water, also do not defract electrons and are therefore difficult to see in the TEM. Preparing biological specimens for the TEM, whilst retaining the structural morphology of the material, is a...

  8. Preparing Students for Globalization

    DEFF Research Database (Denmark)

    Friesel, Anna

    2010-01-01

    A. Friesel. Preparing Students for Globalization Working with International Teams with Projects // Electronics and Electrical Engineering. - Kaunas: Technologija, 2019. - No. 6(102). - P. 111-114. This paper summarizes the activities, contents and overall outcomes of our experiences with internat......A. Friesel. Preparing Students for Globalization Working with International Teams with Projects // Electronics and Electrical Engineering. - Kaunas: Technologija, 2019. - No. 6(102). - P. 111-114. This paper summarizes the activities, contents and overall outcomes of our experiences...

  9. Polymers for Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Gianfranco Pasut

    2014-01-01

    Full Text Available Polyethylene glycol (PEG at the moment is considered the leading polymer for protein conjugation in view of its unique properties, as well as to its low toxicity in humans, qualities which have been confirmed by its extensive use in clinical practice. Other polymers that are safe, biodegradable and custom-designed have, nevertheless, also been investigated as potential candidates for protein conjugation. This review will focus on natural polymers and synthetic linear polymers that have been used for protein delivery and the results associated with their use. Genetic fusion approaches for the preparation of protein-polypeptide conjugates will be also reviewed and compared with the best known chemical conjugation ones.

  10. 21 CFR 102.22 - Protein hydrolysates.

    Science.gov (United States)

    2010-04-01

    ... “hydrolyzed milk protein” is not an acceptable name for this ingredient because it is not specific to the ingredient (hydrolysates can be prepared from other milk proteins). The names “hydrolyzed vegetable protein... derived. (a) “Hydrolyzed wheat gluten,” “hydrolyzed soy protein,” and “autolyzed yeast extract” are...

  11. Batter and method for preparing a pasta

    OpenAIRE

    Wind, P.; Linden, van der, E.

    2011-01-01

    This invention describes a batter that is suitable for preparing a pasta. The batter comprises water, a starch and a protein, whereby the weight ratio between the protein and the total amount of starch in the batter is represented by the symbol y and whereby the weight percentage of the total amount of starch, with respect to the weight of the batter, is represented by the symbol x; whereby the following applies at the same time: 0.1 = y = 2; 10 = x = 80; y = 11/150x + 290/150; y =11/150x+895...

  12. Interfacial Protein-Protein Associations

    OpenAIRE

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2013-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for ...

  13. Immunostimulatory mouse granuloma protein.

    Science.gov (United States)

    Fontan, E; Fauve, R M; Hevin, B; Jusforgues, H

    1983-10-01

    Earlier studies have shown that from subcutaneous talc-induced granuloma in mice, a fraction could be extracted that fully protected mice against Listeria monocytogenes. Using standard biochemical procedures--i.e., ammonium sulfate fractionation, preparative electrophoresis, gel filtration chromatography, isoelectric focusing, and preparative polyacrylamide gel electrophoresis--we have now purified an active factor to homogeneity. A single band was obtained in NaDodSO4/polyacrylamide gel with an apparent Mr of 55,000. It migrated with alpha 1-globulins and the isoelectric point was 5 +/- 0.1. The biological activity was destroyed with Pronase but not with trypsin and a monospecific polyclonal rabbit antiserum was obtained. The intravenous injection of 5 micrograms of this "mouse granuloma protein" fully protects mice against a lethal inoculum of L. monocytogenes. Moreover, after their incubation with 10 nM mouse granuloma protein, mouse peritoneal cells became cytostatic against Lewis carcinoma cells.

  14. A rare polyglycine type II-like helix motif in naturally occurring proteins.

    Science.gov (United States)

    Warkentin, Eberhard; Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Heider, Johann; Ermler, Ulrich

    2017-11-01

    Common structural elements in proteins such as α-helices or β-sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen-bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP II or PG II ) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self-contained hydrogen-bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine-rich PG II -like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG II -like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen-bonding network of the main-chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG II -like helix surrounded by six nearly parallel PG II -like helices in a hexagonal array, plus an additional PG II -like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG II -helix are saturated by either intra- or intermolecular hydrogen-bonds, resulting in a self-contained hydrogen-bonding network. Similar, but incomplete PG II -helix patterns were also previously identified in a GTP-binding protein and an antifreeze protein. © 2017 Wiley Periodicals, Inc.

  15. Surface preparation of niobium

    International Nuclear Information System (INIS)

    Kneisel, P.

    1980-01-01

    Any discussion of surface preparation for superconducting rf-surfaces is certainly connected with the question what is the best recipe for achieving high Q-values and high break-down fields. Since the break-down in a cavity is not understood so far and because several mechanisms play a role, it also is not possible to give one recipe which always works. Nevertheless in the past certain preparation techniques for niobium surfaces have been developed and certain rules for preparation can be applied. In the following the to-days state of the art will be described and it is attempted to give a short description of the surface in conjunction with the methods of surface treatments, which generally can be applied to niobium cavities. (orig./WTR)

  16. Preparation of alumina microspheres

    International Nuclear Information System (INIS)

    Santos, W.R. dos; Abrao, A.

    1980-01-01

    Inorganic exchangers are widely used for adsorption and column partition chromatography. The main difficulty of using commercial alumina (in powder) for column chromatography is related to its packing, and the operations through the column become diffcult and time-consuming; also it turns to be virtually impossible to use large dimension columns. In order to eliminate these problems, a process for the preparation of alumina micro-spheres was developed as an adaptation of a similar process used to prepare nuclear fuel microspheres (UO 2 , ThO 2 ). The flowsheet of this process is presented together with the analytical results of sphericity after calcination, granulometry, density and characterization by X-ray diffractometry. Solubility tests showed that the so-prepared microspheres are well resistant to strong acids and bases; retention tests showed their efficiency, mainly to copper. (C.L.B.) [pt

  17. Soluble protein isolated from low cost fish and fish wastes

    OpenAIRE

    Lekshmy Nair, A.; Gopakumar, K.

    1982-01-01

    The method of preparation, composition, amino acid content, protein efficiency ratio and areas of possible application of water soluble protein isolates from low cost fish and fish wastes are discussed in detail in this communication.

  18. Teaching Preparation Program (TPP).

    Science.gov (United States)

    California Univ., Los Angeles. Dept. of Geography.

    Because most graduate geography students will engage in professional teaching activities, the Teaching Preparation Program of UCLA's department of geography is viewed as an important part of graduate training. The program, co-directed by a graduate student and faculty member, is available to all graduate students on a voluntary basis and consists…

  19. Neoglycoconjugates: preparation and applications

    National Research Council Canada - National Science Library

    Lee, Reiko T; Lee, Y. C

    1994-01-01

    ... and ApplicationsNeoglycoconjugates: Preparation and Applications Edited by Y. C. LEE Biology The Johns Baltimore, D e p a r t m e n t Hopkins University M a r y l a n d REIKO T. LEE Biology D e...

  20. PREPARATION, CHARACTERIZATION AND OPTICAL ...

    African Journals Online (AJOL)

    The prepared host-guest composite material shows luminescence. The material has the potentiality as luminescence material. KEY WORDS: Host-guest nanocomposite material, Nanometer MCM-41 molecular sieve host, Rhodamine B guest, Characterization, Luminescence Bull. Chem. Soc. Ethiop. 2009, 23(1), 145-150.

  1. Preparation of 1-bromoheptacosane

    International Nuclear Information System (INIS)

    Hernandez, L.; Hernandez, A.; Gonzalez, J.C.

    1996-01-01

    Alkybromides are ones of the main organic precursors for fatty acids and alcohols labelling with Carbon 1-14. In this work the preparation of 1-bromoheptacosane by bromodescarboxylation of 1-octacosanoic acid is described. The synthesis yielded 80.5% of final product and more than 97% of chemical purity. Clean-up procedure modifications and spectral data bromoheptacosane are also reported

  2. Preparation of uranium nitride

    International Nuclear Information System (INIS)

    Potter, R.A.; Tennery, V.J.

    1976-01-01

    A process is described for preparing actinide-nitrides from massive actinide metal which is suitable for sintering into low density fuel shapes by partially hydriding the massive metal and simultaneously dehydriding and nitriding the dehydrided portion. The process is repeated until all of the massive metal is converted to a nitride

  3. Electrophoretic transfer protein zymography.

    Science.gov (United States)

    Pan, Daniel; Hill, Adam P; Kashou, Anthony; Wilson, Karl A; Tan-Wilson, Anna

    2011-04-15

    Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  5. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  6. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register ...

  7. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place in the afternoons of 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance via Ind...

  8. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance ...

  9. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2009-01-01

    The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 11, 13, 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned and the limited capacity of the main auditorium, you are requested to register in advance via ...

  10. Overview of Site Preparation

    International Nuclear Information System (INIS)

    Garin, P.

    2006-01-01

    The preparation of Cadarache as the host of ITER is organised at a double level: Europe, since the beginning of the candidature in 2001, is coordinating the so-called European ITER Site Studies; France, as the host country, has put in place a dedicated structure at a decisional level (close to the government), and operational level in the PACA region with two entities: The Agency Iter France (AIF), inside the CEA, interlocutor of international and European entities, in charge of site preparation and fund recollection; An accompanying prefectoral mission, in charge mainly of road adaptation and the international school. The paper will cover all the aspects related to the preparation of the implementation of ITER: Technical aspects: the progress of site preparation itself, its servicing (water supply, electrical supply, Internet...), the road adaptation between the large harbour of Fos-sur-mer and Cadarache, etc. will be detailed. Regulatory procedures: in the framework of the delegation that the ITER partners gave to the CEA/AIF on 14 th September 2005, two main large files are in progress: The public debate, organised by an independent authority, informs the population of the challenges and impacts of ITER in Provence; The safety documents: the writing of the preliminary safety report, which will be submitted to the Nuclear Safety Authority and the files submitted to the public during the public enquiries are ongoing. Socioeconomic aspects: the welcome of ITER staff and their families is operational, via a dedicated Welcome Office; the location of an international school in Manosque leads now to its pre-figuration. The overall organisation will be described, as well as all planning forecast for the coming years, leading to the start of construction. (author)

  11. PREPARING A MARKETING STRATEGY

    OpenAIRE

    Grönholm, Eija

    2010-01-01

    The objective of this thesis was to prepare a marketing strategy for Living City Centre of Kotka Association. The work was implemented with the members of the association and the executive director Reijo Saksa. Living City Centre of Kotka Association was founded in spring 2006 for promoting living, enjoyable and safe centers in the City of Kotka. The association has two permanent employees. The main duties are managing the Kotka market places and promoting the stakeholder connections betw...

  12. PREPARATION OF URANIUM HEXAFLUORIDE

    Science.gov (United States)

    Lawroski, S.; Jonke, A.A.; Steunenberg, R.K.

    1959-10-01

    A process is described for preparing uranium hexafluoride from carbonate- leach uranium ore concentrate. The briquetted, crushed, and screened concentrate is reacted with hydrogen fluoride in a fluidized bed, and the uranium tetrafluoride formed is mixed with a solid diluent, such as calcium fluoride. This mixture is fluorinated with fluorine and an inert diluent gas, also in a fluidized bed, and the uranium hexafluoride obtained is finally purified by fractional distillation.

  13. Preparative radiation chemistry

    International Nuclear Information System (INIS)

    Drawe, H.

    1978-01-01

    Preparative synthesis of compounds with the aid of radiation chemistry is increasingly used in laboratories as well as on a technical scale. A large number of new compounds has been produced with the methods of radiation chemistry. With the increasing number of available radiation sources, also the number of synthesis metods in radiation chemistry has increased. This paper can only briefly mention the many possible ways of synthesis in radiation chemistry. (orig./HK) [de

  14. Sperm preparation for fertilization

    OpenAIRE

    Gadella, B.M.

    2014-01-01

    Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen cryopreservation; evaluation of semen in the andrology laboratory; genetic aspects of male reproduction; emerging techniques and future development of semen evaluation and handling and applied androlo...

  15. TORIS Data Preparation Guidelines

    Energy Technology Data Exchange (ETDEWEB)

    Guinn, H.; Remson, D.

    1999-03-11

    The objective of this manual is to present guidelines and procedures for the preparation of new data for the Tertiary Oil Recovery Information System (TORIS) data base. TORIS is an analytical system currently maintained by the Department of Energy's (DOE) Bartlesville Project Office. It uses an extensive field- and reservoir-level data base to evaluate the technical and economic recovery potential of specific crude oil reservoirs.

  16. Esterase, Total Protein and Seed Storage Protein Diversity in Okra ...

    African Journals Online (AJOL)

    USER

    The protein content of okra seeds varies between 15% and 26%, and edible oil content of more than 14% has been reported (NARP, 1993). A mucilaginous preparation from the pod can be used as a plasma replacement or blood volume expander. Also, mucilage from the stem and roots is used for clarifying sugar juice in.

  17. Adhesives from modified soy protein

    Science.gov (United States)

    Sun, Susan [Manhattan, KS; Wang, Donghai [Manhattan, KS; Zhong, Zhikai [Manhattan, KS; Yang, Guang [Shanghai, CN

    2008-08-26

    The present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

  18. Radio decontamination of the biological preparation iron-protein

    International Nuclear Information System (INIS)

    Otero, I.; Chavez, A.; Aznar, E.; Gonzalez, R.; Prieto, E.

    1996-01-01

    Is showed the gamma radiation application for to diminish the bio burden of the experimental lots possessing an elevated bio burden. The gamma radiation no change the quality parameter of product. Is no possible to apply others sterilization methods why changed composition of this medicine Instituto Nacional de Endocrinologia

  19. Simulating and Optimizing Preparative Protein Chromatography with ChromX

    Science.gov (United States)

    Hahn, Tobias; Huuk, Thiemo; Heuveline, Vincent; Hubbuch, Ju¨rgen

    2015-01-01

    Industrial purification of biomolecules is commonly based on a sequence of chromatographic processes, which are adapted slightly to new target components, as the time to market is crucial. To improve time and material efficiency, modeling is increasingly used to determine optimal operating conditions, thus providing new challenges for current and…

  20. Methods of preparation and the energy, protein and mineral values ...

    African Journals Online (AJOL)

    La teneur en eau, cendre, protéine, lipide, fibre et glucide est déterminée par les méthodes standards. Les minéraux (magnésium, cuivre, fer et zinc) par spectrophotométrie d'absorption atomique. L'enquête auprès des vendeuses a donné les quantités d'aliments vendues actuellement sur le marché pour chacun des plats ...

  1. [Preparation of monolithic materials and their applications in proteomic analysis].

    Science.gov (United States)

    Liang, Yu; Zhang, Lihua; Zhang, Yukui

    2011-09-01

    Proteomics is one of the core contents of life science in the post-genomic era, among which it is very important to develop the analytical techniques with high resolution, high sensitivity, high accuracy and high throughput. With the advantages of facile preparation, fast mass transfer, low backpressure and easy modification, monolithic materials have been widely used in proteomic analysis. This review summarizes the preparation methods of different kinds of monolithic materials (including organic polymer monoliths, silica-based monoliths, organic-inorganic hybrid silica monoliths) and their applications in proteomic study such as the digestion of proteins, the separation of proteins or peptides, high throughput analysis integrating online digestion, separation and identification.

  2. Preparates hardened by radiation

    International Nuclear Information System (INIS)

    Carder, C.H.; Osborn, C.L.; Hodakowski, L.E.

    1979-01-01

    Ink and coating masses hardened by radiation have proved themsleves as suitable preparates which have originated from the reaction of a poly (alcylene oxy) polyol or polyester polyol, on organic di-isocyanate and hydroxy alkyl acrylate. At temperatures between 45 and 60 0 C, 2.5 to 8.0 equivalents of the organic di-isocyanate and 1.5 to 6.0 equivalents of the hydroxy alkyl acrylate are converted for every hydroxy equivalent in polyol. 9 examples supplement the very extensive description of the fabrication possibilities. (UWI) [de

  3. Potassium fluorotitanate preparation

    International Nuclear Information System (INIS)

    Perillo, Patricia; Ares, Osvaldo; Botbol, Jose.

    1989-01-01

    In order to determine the best conditions for potassium fluotitanate preparation as intermediate step in the electrolytic production of metalic titanium, the effects of a number of experimental variables have been studied. This method is a process of sintering titanium dioxide with potassium fluosilicate and potassium chloride, followed by leaching with boiling water and further crystallization by cooling the solution. An overall yield of 90% has been attained under the following conditions: working temperature: 750 deg C; heating time for sintering: 3 hours; molar ratio: titanium dioxide: potassium fluosilicate: potassium chloride: 1 : 2 : 0.4; number of leachings: 6. (Author) [es

  4. A Standardized and Reproducible Urine Preparation Protocol for Cancer Biomarkers Discovery

    Directory of Open Access Journals (Sweden)

    Julia Beretov

    2014-01-01

    Full Text Available A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA alongside high speed centrifugation (HSC provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis.

  5. Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

    NARCIS (Netherlands)

    Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

    1995-01-01

    Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

  6. Protein Extractability

    African Journals Online (AJOL)

    Results showed that protein extractability was dependent on pH, type of salt, salt concentrations and extraction time. Salts extracted more proteins from the moringa seed flour than water. Maximum extraction of protein was. 85.06% and 84.72% with 0.5 M CaCl and 0.75 M NaCl respectively. On varying the pH, maximum ...

  7. Physiologic effects of bowel preparation

    DEFF Research Database (Denmark)

    Holte, Kathrine; Nielsen, Kristine Grubbe; Madsen, Jan Lysgård

    2004-01-01

    healthy volunteers (median age, 63 years) underwent bowel preparation with bisacodyl and sodium phosphate. Fluid and food intake were standardized according to weight, providing adequate calorie and oral fluid intake. Before and after bowel preparation, weight, exercise capacity, orthostatic tolerance...... preparation has significant adverse physiologic effects, which may be attributed to dehydration. The majority of these findings is small and may not be of clinical relevance in otherwise healthy patients undergoing bowel preparation and following recommendations for oral fluid intake....

  8. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2007-01-01

    The Department of Human Resources is organising a preparation for retirement seminar which will take place on the four successive afternoons of 2 to 5 October 2007. Similar seminars in the past have always proved highly successful. Retirement marks the end of one’s working life and the start of a new period of life. This period of transition and change is experienced differently from one individual to another. In any case, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above as well as those who have retired during the year have been sent a personal invitation to attend. Spouses are welcome. Staff members below 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned and the limited capacity of th...

  9. Integrated coal preparation

    International Nuclear Information System (INIS)

    Buchanan, D.J.; Jones, T.F.

    1992-01-01

    Perceptions of quality have changed over the years. The attributes of a certain coal (its rank, slagging propensity, ash content etc) are traditionally referred to as its quality. However, the subject of this paper is quality in a much wider sense: quality as fitness for purpose: and all that such a wide definition entails. British Standard BS 5750 (ISO 9000) Quality Systems defines a systems approach to quality, and includes both the supplier of raw materials and the final customer within this boundary. Coal preparation starts at the production face. The greater the proportion of dirt in run-of-mine product the greater the challenge in satisfying the customer's needs. Significant advances have been made in minimizing mined dirt. For example, the sue of vertical steering on longwall faces improves productivity and quality. Unfortunately modern mining methods produce large quantities of fines, despite efforts to reduce them at the point of production and during transportation to the surface. Coal preparation also produces further fines. It has been estimated that fine coal costs 2.5 times as much to clean as large coal, and the costs of handing wet fine coal product will inflate this estimate. Handling considerations rightly concern our customers and are part of the wider meaning of quality. In this paper the authors address some novel solutions to the challenge posed by fines

  10. Data Breach Preparation

    Energy Technology Data Exchange (ETDEWEB)

    Belangia, David Warren [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-03-13

    The Home Depot Data Breach is the second largest data breach on record. It has or will affect up to 56 million debit or credit cards. A trusted vendor account, coupled with the use of a previously unknown variant of malware that allowed the establishment of a foothold, was the entry point into the Home Depot network. Once inside the perimeter, privilege escalation provided an avenue to obtain the desired information. Home Depot did, however, learn some lessons from Target. Home Depot certainly communicated better than Target, procured insurance, and instituted as secure an environment as possible. There are specific measures an institution should undertake to prepare for a data breach, and everyone can learn from this breach. Publicly available information about the Home Depot Data Breach provides insight into the attack, an old malware variant with a new twist.While the malware was modified as to be unrecognizable with tools, it probably should have been detected. There are also concerns with Home Depot’s insurance and the insurance provider’s apparent lack of fully reimbursing Home Depot for their losses. The effect on shareholders and Home Depot’s stock price was short lived. This story is still evolving but provides interesting lessons learned concerning how an organization should prepare for it inevitable breach.

  11. prepare_taxa_charts

    Science.gov (United States)

    Lakhujani, Vijay; Badapanda, Chandan

    2017-06-01

    QIIME (Quantitative Insights Into Microbial Ecology) is one of the most popular open-source bioinformatics suite for performing metagenome, 16S rRNA amplicon and Internal Transcribed Spacer (ITS) data analysis. Although, it is very comprehensive and powerful tool, it lacks a method to provide publication ready taxonomic pie charts. The script plot_taxa_summary . py bundled with QIIME generate a html file and a folder containing taxonomic pie chart and legend as separate images. The images have randomly generated alphanumeric names. Therefore, it is difficult to associate the pie chart with the legend and the corresponding sample identifier. Even if the option to have the legend within the html file is selected while executing plot_taxa_summary . py , it is very tedious to crop a complete image (having both the pie chart and the legend) due to unequal image sizes. It requires a lot of time to manually prepare the pie charts for multiple samples for publication purpose. Moreover, there are chances of error while identifying the pie chart and legend pair due to random alphanumeric names of the images. To bypass all these bottlenecks and make this process efficient, we have developed a python based program, prepare_taxa_charts . py , to automate the renaming, cropping and merging of taxonomic pie chart and corresponding legend image into a single, good quality publication ready image. This program not only augments the functionality of plot_taxa_summary . py but is also very fast in terms of CPU time and user friendly.

  12. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2011-01-01

    The Human Resources Department is organizing a Preparation for Retirement Seminar, which will take place on 18 and 21 October 2011 in the afternoon in the Main Auditorium and on 19 October and 15 and 16 November 2011 in the afternoon in the Council Chamber. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned, you are r...

  13. Preparation for Retirement Seminar

    CERN Multimedia

    HR Department

    2011-01-01

    The Human Resources Department is organizing a Preparation for Retirement Seminar, which will take place on 18 and 21 October 2011 in the afternoon in the Main Auditorium and on 19 October and 15 and 16 November 2011 in the afternoon in the Council Chamber. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to availability of places. Registration: In view of the number of people concerned, you are ...

  14. Preparation for retirement seminar

    CERN Multimedia

    HR Department

      The Human Resources Department is organizing a preparation for retirement seminar, which will take place on the afternoons of the 25 and 27 November 2009. Similar seminars in the past have always proved highly successful. Retirement marks the end of a person’s working life and the start of a new chapter. This period of transition is experienced differently from one individual to another. In all cases, being well-informed and prepared greatly facilitates the change in lifestyle. We would like to draw your attention to the following information: Staff concerned: All staff members aged 58 and above have been sent a personal invitation to attend. Spouses are welcome. Staff members under the age of 58 who are interested in attending the seminar may also apply. Their applications will be accepted subject to the availability of places. Registration: In view of the number of people concerned and the limited capacity of the Main Auditorium, you are requested to register in advance via Indico. &a...

  15. PREPARATION FOR RETIREMENT PROGRAMME

    CERN Multimedia

    Human Resources Division

    2001-01-01

    27 March 2001 from 2.00 p.m. to 5.30 p.m. 28 March 2001 from 2.00 p.m. to 5.30 p.m. 29 March 2001 from 2.00 p.m. to 5.30 p.m. 30 March 2001 from 2.00 p.m. to 4.45 p.m. Auditorium (Main Building) After the success of the preparation seminars held in recent years, it has been decided that the programme should continue. The forthcoming seminar has been prepared in close collaboration with the CERN Pensioners' Association. The programme will be organised over several half-day sessions. Once again this year, a special session will be devoted to the 10th revision of the Swiss state pension scheme, the 'AVS' (Assurance-Vieillesse et Survivants), and the consequences for international civil servants. A talk will be given by Mrs Danièle Siebold, Director of the Caisse Cantonale Genevoise de Compensation, aimed mainly at those residing in or intending to move to Switzerland, or who worked in Switzerland before joining CERN. To enable Mrs Siebold to respond to your concerns as effectively as possible, please ...

  16. Design and functionality of dense protein particles

    NARCIS (Netherlands)

    Saglam, D.

    2012-01-01

    Food products that contain high levels of protein can help to control food intake and to maintain a healthy body weight due to their strong satiating properties. They are also beneficial in the nutrition of elderly and commonly used in medical nutrition. Preparation of food products at high protein

  17. Full replacement of menhaden fish meal protein by low-gossypol cottonseed flour protein in the diet of juvenile black sea bass Centropristis striata

    Science.gov (United States)

    Eight iso-nitrogeneous (46% crude protein) and iso-lipidic (14% crude lipid) diets were formulated and prepared to replace menhaden fish meal (FM) protein (59.5% CP) by low-gossypol glandless meal (GCSM) protein (50.4% CP), solvent-extracted cottonseed meal (SCSM) protein (53.8% protein) and high go...

  18. Prepare Healthy Foods with Toddlers

    Science.gov (United States)

    Izumi-Taylor, Satomi; Rike, Cheryl

    2011-01-01

    Toddlers--from about 16 to 36 months--can learn a variety of skills as they prepare food and follow recipes in developmentally appropriate ways. Early childhood teachers are encouraged to support young children's healthy eating habits by offering simple food preparation experiences. When toddlers--and preschoolers--safely prepare healthy snacks,…

  19. Microspheres for protein delivery prepared from amphiphilic multiblock copolymers. 1. influence of preparation techniques on particle characteristics and protein delivery

    NARCIS (Netherlands)

    Bezemer, J.M.; Radersma, R.; Grijpma, Dirk W.; Dijkstra, Pieter J.; van Blitterswijk, Clemens; Feijen, Jan

    2000-01-01

    The entrapment of lysozyme in amphiphilic multiblock copolymer microspheres by emulsification and subsequent solvent removal processes was studied. The copolymers are composed of hydrophilic poly(ethylene glycol) (PEG) blocks and hydrophobic poly(butylene terephthalate) (PBT) blocks. Direct solvent

  20. Thermoplastic starch materials prepared from rice starch

    International Nuclear Information System (INIS)

    Pontes, Barbara R.B.; Curvelo, Antonio A.S.

    2009-01-01

    Rice starch is a source still little studied for the preparation of thermoplastic materials. However, its characteristics, such as the presence of proteins, fats and fibers may turn into thermoplastics with a better performance. The present study intends the evaluation of the viability of making starch thermoplastic from rice starch and glycerol as plasticizer. The results of X-ray diffraction and scanning electronic microscopy demonstrate the thermoplastic acquisition. The increase of plasticizer content brings on more hydrophilic thermoplastics with less resistance to tension and elongation at break. (author)

  1. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  2. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat de Protéine de Petit-Lait, Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas ...

  3. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  4. Preparing for public policy

    Science.gov (United States)

    Plapp, Brendan

    2002-03-01

    In the early 1990s, the tight job market for Ph.D. recipients in physics led to a reexamination of graduate programs by some departments. The speaker participated in this reanalysis at his graduate institution and arranged presentations of alternative careers to the physics graduate student body. What became clear was that diverse options were open; job seekers just needed flexible expectations. However, there are a number of additions or modifications to graduate programs which could further help to prepare Ph.D. recipients as they move into non-traditional roles, such as additional and more formal experience in communicating science to a wide range of audiences. In particular, it would be advantageous to learn how to explain the role that basic scientific research projects play in the larger public policy arena. Examples from the speaker's experience of working as a staff member in the U.S. Congress will be presented to illustrate the skills needed in that environment.

  5. Preparing for faster filling

    CERN Multimedia

    CERN Bulletin

    2010-01-01

    Following the programmed technical stop last week, operators focussed on preparing the machine for faster filling, which includes multibunch injection and a faster pre-cycle phase.   The LHC1 screen shot during the first multibunch injection operation. The LHC operational schedule incorporates a technical stop for preventive maintenance roughly every six weeks of stable operation, during which several interventions on the various machines are carried out. Last week these included the replacement of a faulty magnet in the SPS pre-accelerator, which required the subsequent re-setting of the system of particle extraction and transfer to the LHC. At the end of last week, all the machines were handed back for operation and work could start on accommodating all the changes made into the complex systems in order for normal operation to be resumed. These ‘recovery’ operations continued through the weekend and into this week. At the beginning of this week, operators succeeded in pro...

  6. Decommissioning. Success with preparation

    International Nuclear Information System (INIS)

    Klasen, Joerg; Schulz, Rolf; Wilhelm, Oliver

    2017-01-01

    The decommissioning of a nuclear power plant poses a significant challenge for the operating company. The business model is turned upside down and a working culture developed for power operation has to be adapted while necessary know- how for the upcoming tasks has to be built up. The trauma for the employees induced by the final plant shut-down has to be considered and respected. The change of working culture in the enterprise has to be managed and the organization has to be prepared for the future. Here the methods of Change-Management offer a systematic and effective approach. Confidence in the employee's competencies is one of the key success factors for the change into the future.

  7. Uranium dioxide preparation

    International Nuclear Information System (INIS)

    Watt, G.W.; Baugh, D.W. Jr.

    1977-01-01

    An actinide dioxide, e.g., uranium dioxide, is prepared by reacting an actinide nitrate or hydrate or tetrahydrofuranate thereof, e.g., uranyl nitrate, a hydrate of uranyl nitrate, or a tetrahydrofuranate of uranyl nitrate with an alkali or alkaline earth metal adduct of a monocyclic or polycyclic hydrocarbon in the presence of an inert organic solvent. Typically, the starting material may be uranyl nitrate dihydrate or uranyl nitrate ditetrahydrofuranate (the latter material is a novel composition of matter) with a reactant such as the sodium adduct of naphthalene in the presence of a solvent such as tetrahydrofuran. The resultant uranium dioxide may be further purified by heating it in the presence of hydrogen. 15 claims

  8. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  9. Protein-stabilized magnetic fluids

    Energy Technology Data Exchange (ETDEWEB)

    Soenen, S.J.H. [Interdisciplinary Research Center, Katholieke Universiteit Leuven-Campus Kortrijk, University Campus, B-8500 Kortrijk (Belgium); Hodenius, M.; Schmitz-Rode, T. [Helmholtz Institute, Applied Medical Engineering, RWTH Aachen University, Aachen (Germany); De Cuyper, M. [Interdisciplinary Research Center, Katholieke Universiteit Leuven-Campus Kortrijk, University Campus, B-8500 Kortrijk (Belgium)], E-mail: Marcel.DeCuyper@KULeuven-Kortrijk.be

    2008-03-15

    The adsorption of bovine serum albumin (BSA) and egg yolk phosvitin on magnetic fluid particles was investigated. Incubation mixtures were prepared by mixing an alkaline suspension of tetramethylammonium-coated magnetite cores with protein solutions at various protein/Fe{sub 3}O{sub 4} ratios, followed by dialysis against a 5 mM TES buffer (pH 7.0), after which separation of bound and non-bound protein by high-gradient magnetophoresis was executed. Both the kinetic profiles as well as the isotherms of adsorption strongly differed for both proteins. In case of the spherical BSA, initially, abundant adsorption occurred, then it decreased and-at high protein concentrations-it slowly raised again. In contrast, with the highly phosphorylated phosvitin, binding slowly started and the extent of protein adsorption remained unchanged both as a function of time and phosvitin concentration. Competition binding studies, using binary protein mixtures composed of equal weight amounts of BSA and phosvitin, showed that binding of the latter protein is 'unrealistically' high. Based on the geometry of the two proteins, putative pictures on their orientation on the particle's surface in the various experimental conditions were deduced.

  10. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  11. Effect of soya bean diet preparations on some haematological and ...

    African Journals Online (AJOL)

    Effects of Soya bean diet preparations on the hematocrit, hemoglobin concentration, total plasma protein, plasma albumin, sodium, potassium and chloride concentrations were studied in male albino rats. The animals were fed diets containing 75%, 50% and 25% Soya bean in groups II, III and IV respectively. Group I rats ...

  12. Preparation and characterization of the polyclonal antibody against ...

    African Journals Online (AJOL)

    To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR ...

  13. Preparation, Characterization and In vitro Evaluation of Theophylline ...

    African Journals Online (AJOL)

    Methods: SPI-based nanoparticles were prepared with soy protein-dextran conjugates obtained by titanium dioxide (TiO2) photocatalysis using a simple ionic gelation method. Formation of the conjugates was monitored spectrophotometrically for free amino group content (A340nm) and by Fourier transform infrared ...

  14. Formation of spherical protein nanoparticles without impacting protein integrity

    International Nuclear Information System (INIS)

    Montalvo, Brenda L; Pacheco, Yamaris; Sosa, Brian A; Velez, Denisse; Sanchez, Giselle; Griebenow, Kai

    2008-01-01

    Protein formulation at the nanoscale is challenging because of protein susceptibility to chemical and physical degradation during processing. Herein, we present a straightforward method to prepare spherical protein nanoparticles by co-lyophilizing five structurally different enzymes (horseradish peroxidase, carbonic anhydrase, lysozyme, subtilisin Carlsberg and α-chymotrypsin) with methyl-β-cyclodextrin followed by suspension of the powders in ethyl acetate. The size distribution was narrow and varied from 88 ± 14 to 148 ± 16 nm as determined by dynamic light scattering. Scanning and transmission electron micrographs confirmed the size and spherical morphology of the protein nanoparticles. Residual activities for all enzymes tested were 100% upon dissolving the nanoparticles in buffer and no insoluble aggregates were formed.

  15. Prepared for internship?

    LENUS (Irish Health Repository)

    Abuhusain, H

    2009-03-01

    Preparedness of medical school graduates for the intern year is one of the emphasised objectives of undergraduate medical training. We have evaluated the perceived preparedness of graduates undertaking the intern year in the Republic of Ireland. A 9-page questionnaire was mailed to all 497 interns in Ireland following commencement of the intern year in July 2005. Data obtained included demographics, perceived preparedness and assessment of perceived clinical skills (four sub-domains: core competencies, communication, emergencies, and educational environment). Information on intern induction was also collected. 99 questionnaires were returned (19.9%). Most of the cohort were Irish and worked in large medical school teaching hospitals. The majority of interns felt \\'unprepared\\' for the intern year. Interns perceived themselves \\'poor\\' in all areas of clinical skills assessed. Intern induction was attended by the majority and most stated it was too short. Medical schools are actively seeking innovative methods, through early patient contact and sub-internships, to better prepare undergraduates for the intern year. The deficiencies identified in this study are significant and emphasise the need for continued reform in the undergraduate curriculum.

  16. Preparing for the future

    CERN Multimedia

    Panos Charitos

    2016-01-01

    The second annual meeting of the Future Circular Collider (FCC) design study took place from 11 to 15 April in Rome.   The participants in the second annual meeting of the FCC design study. (Photo: Vinicio Tullio/INFN) More than 450 scientists, researchers and leaders of high-tech industry gathered in Rome to review the progress of the Future Circular Collider (FCC) design study. The study was kicked off in 2014 as a response to a statement in the European Strategy for Particle Physics, and today embraces 74 institutes from 26 countries. With the LHC programme well under way, particle physicists are at an exciting juncture. New results from the 13 TeV run could show that we are on the threshold of an eye-opening era that presents new challenges and calls for developments. “To prepare for its future, CERN should continue to develop a vibrant R&D programme that should take advantage of its strengths and uniqueness, pursue design studies for...

  17. Preparation for retirement seminar

    CERN Multimedia

    HR Department

    2010-01-01

    (Health insurance and wealth and succession planning) During the preparation for retirement seminar in November 2009, the sessions on health insurance in Switzerland and in France unfortunately had to be postponed. Participants in the seminar also expressed interest in an information session on “How to manage your wealth and organize your succession”. The sessions on health insurance will be held on 16 March 2010 and those on managing wealth and succession on 18 March 2010. Programme for Tuesday 16 March 2010 (TH Theory Conference Room, Building 4/3-006): 09:00 Health insurance in Switzerland by Mr. Sandro Breitenstein, Service de l'Assurance Maladie du Canton de Genève 10:00 Coffee break 10:20 Health insurance in France by Mr. Dominique Curtiaud, Caisse Primaire d'Assurance Maladie de l'Ain Programme for Thursday 18 March 2010 (TH Theory Conference Room, Building 4/3-006): 09:00 How to manage your wealth and organize your succession in Switzerland by Mr. Jean-Marc W...

  18. Protein deamidation

    OpenAIRE

    Robinson, Noah E.

    2002-01-01

    A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically ...

  19. Preradiation preparation of cancer patients

    International Nuclear Information System (INIS)

    Il'in, V.I.

    1991-01-01

    The main stages in preradiation preparation of cancer patients were defined. Immediate preparation must include both clinical and psychological aspects. It was shown that in preradiation preparation of cancer patients it was necessary to make a plan of radiotherapy. The plan of radiotherapy was made of three processes: determination of tumor nature and localization, choice of the corresponding irradiation conditions, definition of degree of radiation complications rick

  20. Micro-algae as a source of protein.

    Science.gov (United States)

    Becker, E W

    2007-01-01

    About five decades ago, the mass production of certain protein-rich micro-algae was considered as a possibility to close the predicted so called "protein gap". Comprehensive analyses and nutritional studies have demonstrated that these algal proteins are of high quality and comparable to conventional vegetable proteins. However, due to high production costs as well as technical difficulties to incorporate the algal material into palatable food preparations, the propagation of algal protein is still in its infancy. To date, the majority of micro-algal preparations are marketed as health food, as cosmetics or as animal feed.

  1. Fundamentals of Protein NMR Spectroscopy

    CERN Document Server

    Rule, Gordon S

    2006-01-01

    NMR spectroscopy has proven to be a powerful technique to study the structure and dynamics of biological macromolecules. Fundamentals of Protein NMR Spectroscopy is a comprehensive textbook that guides the reader from a basic understanding of the phenomenological properties of magnetic resonance to the application and interpretation of modern multi-dimensional NMR experiments on 15N/13C-labeled proteins. Beginning with elementary quantum mechanics, a set of practical rules is presented and used to describe many commonly employed multi-dimensional, multi-nuclear NMR pulse sequences. A modular analysis of NMR pulse sequence building blocks also provides a basis for understanding and developing novel pulse programs. This text not only covers topics from chemical shift assignment to protein structure refinement, as well as the analysis of protein dynamics and chemical kinetics, but also provides a practical guide to many aspects of modern spectrometer hardware, sample preparation, experimental set-up, and data pr...

  2. Applications of reversible covalent chemistry in analytical sample preparation.

    Science.gov (United States)

    Siegel, David

    2012-12-07

    Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

  3. A comparative evaluation of the ultrastructure and protein yields of ...

    African Journals Online (AJOL)

    ... efficient as the detergent solubilized technique. Additionally some axial filaments were also released. The protein yield of L. hardjo from the two techniques of leptospire protein preparation was significantly lower than the other test serovars (P<0.01). Overall, the protein yield of antigens produced by the SDS solubilization ...

  4. Limited hydrolysis of soybean protein concentrate and isolate with ...

    African Journals Online (AJOL)

    Soy protein concentrate (SPC) and soy protein isolate (SPI) were limited hydrolyzed with trypsin or neutrase under controlled hydrolysis conditions. Eight soybean protein hydrolysates, 4 SPC hydrolysates and 4 SPI hydrolysates were prepared with degree of hydrolysis (DH) of 1 or 2%. SDS-PAGE was used to determine ...

  5. Modulating surface rheology by electrostatic protein/polysaccharide interactions

    NARCIS (Netherlands)

    Ganzevles, R.A.; Zinoviadou, K.; Vliet, T. van; Stuart, M.A.C.; Jongh, H.H.J. de

    2006-01-01

    There is a large interest in mixed protein/polysaccharide layers at air-water and oil-water interfaces because of their ability to stabilize foams and emulsions. Mixed protein/polysaccharide adsorbed layers at air-water interfaces can be prepared either by adsorption of soluble protein/

  6. LHC preparations change gear

    International Nuclear Information System (INIS)

    Anon.

    1995-01-01

    After the formal approval by CERN Council in December (January, page 1) of the LHC protonproton collider for CERN's 27- kilometre LEP tunnel, preparations for the new machine change gear. Lyndon Evans becomes LHC Project Leader, and CERN's internal structure will soon be reorganized to take account of the project becoming a definite commitment. On the experimental side, the full Technical Proposals for the big general purpose ATLAS and CMS detectors were aired at a major meeting of the LHC Committee at CERN in January. These Technical Proposals are impressive documents each of some several hundred pages. (Summaries of the detector designs will appear in forthcoming issues of the CERN Courier.) The ALICE heavy ion experiment is not far behind, and plans for other LHC experiments are being developed. Playing an important role in this groundwork has been the Detector Research and Development Committee (DRDC), founded in 1990 to foster detector development for the LHC experimental programme and structured along the lines of a traditional CERN Experiments Committee. Established under the Director Generalship of Carlo Rubbia and initially steered by Research Director Walter Hoogland, the DRDC has done sterling work in blazing a trail for LHC experiments. Acknowledging that the challenge of LHC experimentation needs technological breakthroughs as well as specific detector subsystems, DRDC proposals have covered a wide front, covering readout electronics and computing as well as detector technology. Its first Chairman was Enzo larocci, succeeded in 1993 by Michal Turala. DRDC's role was to evaluate proposals, and make recommendations to CERN's Research Board for approval and resource allocation, not an easy task when the LHC project itself had yet to be formally approved. Over the years, a comprehensive portfolio of detector development has been built up, much of which has either led to specific LHC detector subsystems for traditional detector tasks

  7. Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

    DEFF Research Database (Denmark)

    Ingversen, J.

    1975-01-01

    Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS...... with a granular component. Particles with the same structure were present in the protein body preparation from the mutant, where, however, the granular component was the most prominent. Amino-acid composition and SDS-polyacrylamide gel electrophoresis of the proteins from the protein body preparation revealed...

  8. Protein composition in tofu of corrected quality

    Directory of Open Access Journals (Sweden)

    Stanojević Slađana P.

    2010-01-01

    Full Text Available Soybeans are an inexpensive, high-quality protein source. Soybeans have long been a staple of the human diet in Asia, especially as tofu, which is prepared from soymilk. In this study, tofu was made using a new production method which includes hydrothermal cooking (HTC and rennin-pepsin coagulant. The effects of the addition of gallic acid to the slurry during tofu processing were studied. Tofu was made from two soybean genotypes: Lana and Balkan. The observed genotypes are characterized by relatively high content of total proteins in flour, from 45.88% to 48.83%. The prepared tofu samples are characterized by extremely high content of total proteins (52.17% - Lana tofu and 56.08% - Balkan tofu. The presence of gallic acid significantly affects the solubility of tofu protein. The applied modifications of traditional procedure of tofu production significantly improved sensory properties of soybean protein products.

  9. Cavity preparation machine for the standardization of in vitro preparations

    Directory of Open Access Journals (Sweden)

    Carlos José Soares

    2008-09-01

    Full Text Available Several in vitro studies employ the confection of cavity preparations that are difficult to standardize by means of manual high speed handpieces. This study presents the development of a cavity preparation machine designed to standardize in vitro cavity preparations. A metal base of 25 mm x 25 mm x 4 mm (length x width x height was coupled to a small mobile table which was designed to be able to move by means of two precision micrometers (0.01-mm accuracy in the horizontal directions (right-left, and back-front. A high speed handpiece was coupled to a metallic connecting rod which had an accurate dial indicator enabling control of the vertical movement. The high speed handpiece is also able to move 180° around its longitudinal axis and 360° around its transversal axis. The suggested cavity preparation machine precisely helps in the standardization of cavity preparations for in vitro studies.

  10. Purification of thyrotropin from human hypophysis: preliminary preparation

    International Nuclear Information System (INIS)

    Borghi, V.C.; Lin, L.H.; Bartolini, P.

    1988-07-01

    The adequacy of stored crude preparations for isolation of human tyrotropin (TSH) was evaluated according to Ross et al from a side fraction obtained during the purification of growth hormone from frozen pituitaries (SOMATORMON). Six crude TSH preparations were stored at - 20 0 C during several years for further purification. One of these preparations was purified by sucessive chromatographies on Sephadex G-100, hydroxylapatite and SP-Sephadex C50. The TSH content present in the chromatographic fractions and in the pools was assayed by specific radioimmunoassay developed at our laboratory. The protein determination of the fractions and pools was performed by absorbance at 280 nm and by the method of Lowry at al, respectively. The TSH activity increased eight times during the purification and the TSH purified had a radioimmunological potency around half that de scribed by Roos at al. The results suggest the fitness of long time stored preparations in the attainment of pure TSH. (author) [pt

  11. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    Science.gov (United States)

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  12. Preparation and properties of a neurotoxin purified from the venom of black widow spider (Latrodectus mactans tredecimguttatus).

    Science.gov (United States)

    Grasso, A

    1976-08-09

    A neurotoxin of the venom of the spider Latrodectus mactans tredecimguttatus, has been prepared in a homogeneous form and examined by a variety of techniques. The protein has a molecular weight of 130 000 and its toxicity in mice is about 49 000 LD50 mg pure protein/g body weight. The toxin releases norepinephrine from synaptosomes prepared from rat brain and shows most of the toxic effects of the crude venom preparation.

  13. TFTR DT preparation project status

    Energy Technology Data Exchange (ETDEWEB)

    Perry, E.D.; Dudek, L.E.

    1993-11-01

    The Tokamak Fusion Test Reactor (TFTR) research program is preparing to commence the first high power Deuterium-Tritium (DT) experiments of the US Fusion Program. Hardware upgrades to TFTR required for DT operations have been completed. This paper discusses these hardware preparations.

  14. A Simple Preparation of Hexadeuteriocholesterol

    DEFF Research Database (Denmark)

    Holm, Torkil; Crossland, Ingolf

    1996-01-01

    26,26,26,27,27,27 Hxadeuterio cholesterol has been prepared in 9 stages from pregnenolone and hexadeuterio acetone with an overall yield of 7 %.......26,26,26,27,27,27 Hxadeuterio cholesterol has been prepared in 9 stages from pregnenolone and hexadeuterio acetone with an overall yield of 7 %....

  15. Preparing Students for Romantic Relationships

    Science.gov (United States)

    Weissbourd, Richard; Peterson, Amelia; Weinstein, Emily

    2014-01-01

    One of the most important aspects in our lives is learning how to have mutual, caring romantic relationships. Yet while schools and many other industries in this country devote tremendous attention and resources to preparing the young for work, they do remarkably little to prepare them for generous, self-respecting sex and love. Educators and…

  16. Communication Games Reveal Preparation Contextuality

    Science.gov (United States)

    Hameedi, Alley; Tavakoli, Armin; Marques, Breno; Bourennane, Mohamed

    2017-12-01

    A communication game consists of distributed parties attempting to jointly complete a task with restricted communication. Such games are useful tools for studying limitations of physical theories. A theory exhibits preparation contextuality whenever its predictions cannot be explained by a preparation noncontextual model. Here, we show that communication games performed in operational theories reveal the preparation contextuality of that theory. For statistics obtained in a particular family of communication games, we show a direct correspondence with correlations in spacelike separated events obeying the no-signaling principle. Using this, we prove that all mixed quantum states of any finite dimension are preparation contextual. We report on an experimental realization of a communication game involving three-level quantum systems from which we observe a strong violation of the constraints of preparation noncontextuality.

  17. Preparation of Radix Arnebiae ointment

    Science.gov (United States)

    Ji, Y. B.; Zhao, H.; Xin, G. S.; Wei, C.; Zhu, H. J.

    2017-12-01

    Aim of this study is to prepare Arnebiae ointment research, screening, get the best preparation technology. The preparation process of different frying temperature, different frying time, different oil quantity and dosage ratio were prepared by frying method. The orthogonal method was used in this study. The content of L-shikonin in Arnebiae ointment was used as the research factor, and the preparation process of Arnebiae ointment t was optimized by this method. The experimental results show that the optimum extraction process is the extraction temperature of 110°C, the holding time is 1.5min, and when the ratio of oil to oil is 1:8, the quality of Arnebiae ointment the best. The extraction process can effectively improve the L-shikonin in the Arnebiae Ointment, and the extraction process is reasonable, efficient and economical.

  18. Physiologic effects of bowel preparation

    DEFF Research Database (Denmark)

    Holte, Kathrine; Nielsen, Kristine Grubbe; Madsen, Jan Lysgård

    2004-01-01

    , plasma and extracellular volume, balance function, and biochemical parameters were measured. RESULTS: Bowel preparation led to a significant decrease in exercise capacity (median, 9 percent) and weight (median, 1.2 kg). Plasma osmolality was significantly increased from 287 to 290 mmol kg(-1), as well......PURPOSE: Despite the universal use of bowel preparation before colonoscopy and colorectal surgery, the physiologic effects have not been described in a standardized setting. This study was designed to investigate the physiologic effects of bowel preparation. METHODS: In a prospective study, 12...... healthy volunteers (median age, 63 years) underwent bowel preparation with bisacodyl and sodium phosphate. Fluid and food intake were standardized according to weight, providing adequate calorie and oral fluid intake. Before and after bowel preparation, weight, exercise capacity, orthostatic tolerance...

  19. Communication Games Reveal Preparation Contextuality.

    Science.gov (United States)

    Hameedi, Alley; Tavakoli, Armin; Marques, Breno; Bourennane, Mohamed

    2017-12-01

    A communication game consists of distributed parties attempting to jointly complete a task with restricted communication. Such games are useful tools for studying limitations of physical theories. A theory exhibits preparation contextuality whenever its predictions cannot be explained by a preparation noncontextual model. Here, we show that communication games performed in operational theories reveal the preparation contextuality of that theory. For statistics obtained in a particular family of communication games, we show a direct correspondence with correlations in spacelike separated events obeying the no-signaling principle. Using this, we prove that all mixed quantum states of any finite dimension are preparation contextual. We report on an experimental realization of a communication game involving three-level quantum systems from which we observe a strong violation of the constraints of preparation noncontextuality.

  20. Protein Crystallizability.

    Science.gov (United States)

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.