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Sample records for protein sbpa obtained

  1. 7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

    PubMed Central

    Norville, Julie E.; Kelly, Deborah F.; Knight, Thomas F.; Belcher, Angela M.; Walz, Thomas

    2007-01-01

    Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date. PMID:17638580

  2. Molecular characterization of the S-layer gene, sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen.

    PubMed

    Ilk, Nicola; Völlenkle, Christine; Egelseer, Eva M; Breitwieser, Andreas; Sleytr, Uwe B; Sára, Margit

    2002-07-01

    The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices. PMID:12089001

  3. Obtaining a protein concentrate from integral defatted sunflower flour.

    PubMed

    Ordóñez, C; Asenjo, M G; Benitez, C; González, J L

    2001-06-01

    Proteins which are found in integral defatted sunflower flour (27% of protein in dry weight) allow us to produce a protein concentrate by means of extraction of proteins with a basic pH solution, followed by their precipitation with an acid pH solution. Once the suitable conditions for pH and temperature were fixed in order to carry out these processes, a solid proteic concentrate (71% of protein in dry weight) was obtained which was rich in glutamic and aspartic acids, with a liquid supernatant very rich in phosphorus and potassium, which might be used as an agricultural fertilizer. PMID:11333039

  4. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  5. Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films

    NASA Astrophysics Data System (ADS)

    Lejardi, Ainhoa; López, Aitziber Eleta; Sarasua, José R.; Sleytr, U. B.; Toca-Herrera, José L.

    2013-09-01

    Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (Tg) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below Tg. The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 μm2), followed by PDLLA (0.034 μm2), and PLGA (0.039 μm2), and the largest size for PLCL (0.09 μm2). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (˜1800 ng cm-2) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest Tg). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

  6. Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

    PubMed Central

    Fogen, Dawson; Wu, Sau-Ching; Ng, Kenneth Kai-Sing; Wong, Sui-Lam

    2015-01-01

    To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation–a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability. PMID:26406477

  7. Methods to obtain protein concentrates from jumbo squid (Dosidicus gigas) and evaluation of their functionality.

    PubMed

    Galvez-Rongel, A; Ezquerra-Brauer, J M; Ocano-Higuera, V M; Ramirez-Wong, B; Torres-Arreola, W; Rouzaud-Sandez, O; Marquez-Rios, E

    2014-03-01

    Jumbo squid is an important fishery resource in Mexico, and its muscle is lean and white and it has a very low price in the market. It is abundant, but with little or nothing added value, therefore is necessary to search alternatives of processing. Due to muscle characteristics, the aim of this study was to obtain protein concentrates using different methods. They were obtained by means of acidic (acid protein concentrates) and alkaline (alkaline protein concentrates) dissolution. Moreover, a protein concentrate was obtained by direct isoelectric precipitation and by the traditional method (neutral protein concentrates). The yield with better results was alkaline protein concentrates (63.58 ± 1.8%). The gel hardness was significantly different (p < 0.05), especially for the alkaline protein concentrates. The acid protein concentrates, isoelectric precipitation and alkaline protein concentrates were better with regard to the neutral protein concentrates, concerning the emulsifying and foaming properties. The protein concentrates by means of alkaline dissolution gave a better gelling property, but all the processes had the potential to obtain protein with emulsifying and foaming properties. PMID:23733825

  8. Characterization of rice starch and protein obtained by a fast alkaline extraction method.

    PubMed

    Souza, Daiana de; Sbardelotto, Arthur Francisco; Ziegler, Denize Righetto; Marczak, Ligia Damasceno Ferreira; Tessaro, Isabel Cristina

    2016-01-15

    This study evaluated the characteristics of rice starch and protein obtained by a fast alkaline extraction method on rice flour (RF) derived from broken rice. The extraction was conducted using 0.18% NaOH at 30°C for 30min followed by centrifugation to separate the starch rich and the protein rich fractions. This fast extraction method allowed to obtain an isoelectric precipitation protein concentrate (IPPC) with 79% protein and a starchy product with low protein content. The amino acid content of IPPC was practically unchanged compared to the protein in RF. The proteins of the IPPC underwent denaturation during extraction and some of the starch suffered the cold gelatinization phenomenon, due to the alkaline treatment. With some modifications, the fast method can be interesting in a technological point of view as it enables process cost reduction and useful ingredients obtention to the food and chemical industries. PMID:26258699

  9. Bioactive properties of peptides obtained from Argentinian defatted soy flour protein by Corolase PP hydrolysis.

    PubMed

    Coscueta, Ezequiel R; Amorim, Maria M; Voss, Glenise B; Nerli, Bibiana B; Picó, Guillermo A; Pintado, Manuela E

    2016-05-01

    Enzymatic hydrolysis of soybean meal protein isolate (SPI) obtained under two temperature conditions with Corolase PP was studied, assessing the impact of hydrolysis on potential antioxidant and antihypertensive activities. The protein was isolated from soybean meal under controlled conditions of time and temperature (70 °C, 1h; 90 °C, 30 min). Degree of hydrolysis assessed the progress of hydrolysis at different sampling times. For hydrolysates the antioxidant and angiotensin-converting-enzyme (ACE) inhibitory activities were measured. As observed, the DH was increasing until reaching 20% at 10h with disappearance of globular proteins and generation of low molecular weight peptides (less than 3kDa). A significant increase in antioxidant and ACE inhibitory capacities was observed. Five main peptides were identified, which may explain through their sequences the bioactive properties analyzed. Through this study was possible to obtain for the first time with Corolase PP soy hydrolysates with potential antioxidant and ACE inhibitory activities, which can be used to obtain new added value functional ingredients from soy meal. PMID:26769502

  10. Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

    PubMed

    Rego, J P A; Moura, A A; Nouwens, A S; McGowan, M R; Boe-Hansen, G B

    2015-09-01

    The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively. PMID:26282524

  11. Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity

    PubMed Central

    Karamać, Magdalena; Kosińska-Cagnazzo, Agnieszka; Kulczyk, Anna

    2016-01-01

    The antioxidant activity of flaxseed protein hydrolysates obtained using five different enzymes was evaluated. Proteins were isolated from flaxseed cake and were separately treated with papain, trypsin, pancreatin, Alcalase and Flavourzyme. The degree of hydrolysis (DH) was determined as the percentage of cleaved peptide bonds using a spectrophotometric method with o-phthaldialdehyde. The distribution of the molecular weights (MW) of the hydrolysis products was profiled using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC) separations. The antioxidant activities of the protein isolate and hydrolysates were probed for their radical scavenging activity using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+) and photochemiluminescence (PCL-ACL) assays, and for their ferric reducing antioxidant power (FRAP) and ability to bind Fe2+. The hydrolysates were more effective as antioxidants than the protein isolate in all systems. The PCL-ACL values of the hydrolysates ranged from 7.2 to 35.7 μmol Trolox/g. Both the FRAP and ABTS•+ scavenging activity differed among the hydrolysates to a lower extent, with the ranges of 0.20–0.24 mmol Fe2+/g and 0.17–0.22 mmol Trolox/g, respectively. The highest chelating activity (71.5%) was noted for the pancreatin hydrolysate. In general, the hydrolysates obtained using Alcalase and pancreatin had the highest antioxidant activity, even though their DH (15.4% and 29.3%, respectively) and the MW profiles of the peptides varied substantially. The O2•− scavenging activity and the ability to chelate Fe2+ of the Flavourzyme hydrolysate were lower than those of the Alcalase and pancreatin hydrolysates. Papain was the least effective in releasing the peptides with antioxidant activity. The study showed that the type of enzyme used for flaxseed protein hydrolysis determines the antioxidant activity of

  12. Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity.

    PubMed

    Karamać, Magdalena; Kosińska-Cagnazzo, Agnieszka; Kulczyk, Anna

    2016-01-01

    The antioxidant activity of flaxseed protein hydrolysates obtained using five different enzymes was evaluated. Proteins were isolated from flaxseed cake and were separately treated with papain, trypsin, pancreatin, Alcalase and Flavourzyme. The degree of hydrolysis (DH) was determined as the percentage of cleaved peptide bonds using a spectrophotometric method with o-phthaldialdehyde. The distribution of the molecular weights (MW) of the hydrolysis products was profiled using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and size exclusion-high performance liquid chromatography (SE-HPLC) separations. The antioxidant activities of the protein isolate and hydrolysates were probed for their radical scavenging activity using 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(•+)) and photochemiluminescence (PCL-ACL) assays, and for their ferric reducing antioxidant power (FRAP) and ability to bind Fe(2+). The hydrolysates were more effective as antioxidants than the protein isolate in all systems. The PCL-ACL values of the hydrolysates ranged from 7.2 to 35.7 μmol Trolox/g. Both the FRAP and ABTS(•+) scavenging activity differed among the hydrolysates to a lower extent, with the ranges of 0.20-0.24 mmol Fe(2+)/g and 0.17-0.22 mmol Trolox/g, respectively. The highest chelating activity (71.5%) was noted for the pancreatin hydrolysate. In general, the hydrolysates obtained using Alcalase and pancreatin had the highest antioxidant activity, even though their DH (15.4% and 29.3%, respectively) and the MW profiles of the peptides varied substantially. The O₂(•-) scavenging activity and the ability to chelate Fe(2+) of the Flavourzyme hydrolysate were lower than those of the Alcalase and pancreatin hydrolysates. Papain was the least effective in releasing the peptides with antioxidant activity. The study showed that the type of enzyme used for flaxseed protein hydrolysis determines the antioxidant activity

  13. Can a pairwise contact potential stabilize native protein folds against decoys obtained by threading?

    PubMed

    Vendruscolo, M; Najmanovich, R; Domany, E

    2000-02-01

    We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading. PMID:10656261

  14. The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers

    NASA Astrophysics Data System (ADS)

    Hu, Yufeng; Chen, Zhenhang; Fu, Yanjun; He, Qingzhong; Jiang, Lun; Zheng, Jiangge; Gao, Yina; Mei, Pinchao; Chen, Zhongzhou; Ren, Xueqin

    2015-03-01

    Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

  15. Structural requirements to obtain highly potent and selective 18 kDa Translocator Protein (TSPO) Ligands.

    PubMed

    Taliani, Sabrina; Pugliesi, Isabella; Da Settimo, Federico

    2011-01-01

    The (18 kDa) Translocator Protein (TSPO), was initially identified in 1977 as peripheral binding site for the benzodiazepine diazepam and named "Peripheral-type benzodiazepine receptor (PBR)". It is an evolutionarily well-conserved protein particularly located at the outer/inner mitochondrial membrane contact sites, in closely association with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT), thus forming the mitochondrial permeability transition pore (MPTP). TSPO is ubiquitary expressed in peripheral tissues (steroid producing tissues, liver, heart, kidney, lung, immune system) and in lower levels in the central nervous system, where it is mainly located in glial cells, and in neurons. TSPO is involved in a variety of biological processes such as cholesterol transport, steroidogenesis, calcium homeostasis, lipid metabolism, mitochondrial oxidation, cell growth and differentiation, apoptosis induction, and regulation of immune functions. In the last decade, many studies have reported that TSPO basal expression is altered in a number of human pathologies, such as cancer and neurodegenerative disorders (Huntington's and Alzheimer's diseases), as well as in various forms of brain injury and inflammation and anxiety. Consequently, TSPO has not only been suggested as a promising drug target for a number of therapeutic applications (anticonvulsant, anxiolytic, immunomodulating, etc.), but also as valid diagnostic marker for related-disease state and progression, prompting the development of specific labelled ligands as powerful tools for imaging techniques. A number of structurally different classes of ligands have been reported, showing high affinity and selectivity towards TSPO. Indeed, most of these ligands have been designed starting from selective CBR ligands which were structurally modified in order to shift their affinity towards TSPO. Extensive structure-activity relationship studies were performed allowing to

  16. Food Protein-polysaccharide Conjugates Obtained via the Maillard Reaction: A Review.

    PubMed

    de Oliveira, Fabíola Cristina; Coimbra, Jane Sélia Dos Reis; de Oliveira, Eduardo Basílio; Zuñiga, Abraham Damian Giraldo; Rojas, Edwin E Garcia

    2016-05-18

    The products formed by glycosylation of food proteins with carbohydrates via the Maillard reaction, also known as conjugates, are agents capable of changing and improving techno-functional characteristics of proteins. The Maillard reaction uses the covalent bond between a group of a reducing carbohydrates and an amino group of a protein. This reaction does not require additional chemicals as it occurs naturally under controlled conditions of temperature, time, pH, and moisture. Moreover, there is growing interest in modifying proteins for industrial food applications. This review analyses the current state of art of the Maillard reaction on food protein functionalities. It also discusses the influence of the Maillard reaction on the conditions and formulation of reagents that improve desirable techno-functional characteristics of food protein. PMID:24824044

  17. Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

    NASA Astrophysics Data System (ADS)

    Rusen, L.; Mustaciosu, C.; Mitu, B.; Filipescu, M.; Dinescu, M.; Dinca, V.

    2013-08-01

    Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm-2. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

  18. A protein map of the yeast activated spliceosome as obtained by electron microscopy.

    PubMed

    Sun, Chengfu; Rigo, Norbert; Fabrizio, Patrizia; Kastner, Berthold; Lührmann, Reinhard

    2016-09-01

    We have elucidated the spatial arrangement of proteins and snRNP subunits within the purified spliceosomal B(act) complex from Saccharomyces cerevisiae, using negative-stain immunoelectron microscopy. The B(act) spliceosome exhibits a mushroom-like shape with a main body connected to a foot and a steep and a shallow slope. The U5 core components, including proteins Snu114 and Prp8, are located in the main body and foot, while Brr2 is on the shallow slope. U2 snRNP components and the RNA helicase Prp2 were predominantly located in the upper regions of both slopes. While several proteins of the "nineteen complex" are located on the steep slope, Prp19, Cef1, and the U6 snRNA-binding protein Cwc2 are on the main body. Our results also indicate that the catalytic core RNP of the spliceosome resides in its main body. We thus assign distinct domains of the B(act) complex to its snRNP and protein components, and we provide first structural insights into the remodeling events at the spliceosome during its transformation from the B to the B(act) complex. PMID:27368340

  19. Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology.

    PubMed

    Wiśniewski, Jacek R; Nagaraj, Nagarjuna; Zougman, Alexandre; Gnad, Florian; Mann, Matthias

    2010-06-01

    Taking advantage of the recently developed Filter Assisted Sample Preparation (FASP) method for sample preparation, we performed an in-depth analysis of phosphorylation sites in mouse brain. To maximize the number of detected phosphorylation sites, we fractionated proteins by size exclusion chromatography (SEC) or separated tryptic peptides on an anion exchanger (SAX) prior or after the TiO(2)-based phosphopeptide enrichment, respectively. SEC allowed analysis of minute tissue samples (1 mg total protein), and resulted in identification of more than 4000 sites in a single experiment, comprising eight fractions. SAX in a pipet tip format offered a convenient and rapid way to fractionate phosphopeptides and mapped more than 5000 sites in a single six fraction experiment. To enrich peptides containing phosphotyrosine residues, we describe a filter aided antibody capturing and elution (FACE) method that requires only the uncoupled instead of resin-immobilized capture reagent. In total, we identified 12,035 phosphorylation sites on 4579 brain proteins of which 8446 are novel. Gene Ontology annotation reveals that 23% of identified sites are located on plasma membrane proteins, including a large number of ion channels and transporters. Together with the glycosylation sites from a recent large-scale study, they can confirm or correct predicted membrane topologies of these proteins, as we show for the examples calcium channels and glutamate receptors. PMID:20415495

  20. Direct structural evidence of protein redox regulation obtained by in-cell NMR.

    PubMed

    Mercatelli, Eleonora; Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2016-02-01

    The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately. PMID:26589182

  1. Obtaining structural information of small proteins using solid-state nanopores and high-bandwidth measurements

    NASA Astrophysics Data System (ADS)

    Niedzwiecki, David; Lanci, Christopher; Saven, Jeffery; Drndic, Marija

    2015-03-01

    The use of biological nanopores sensors to characterize proteins has proved a fruitful field of study. Solid-state nanopores hold several advantages over their biological counterparts, including the ability to tune pore diameter and their robustness to external conditions. Despite these advantages, the use of solid-state nanopores for protein analysis has proved difficult due to rapid translocation times of proteins and poor signal-to-noise of small peptides. Recently, improvements in high-bandwidth acquisition and in signal-to-noise have made the study of small peptides using solid-state nanopores feasible. Here we report on the detection and characterization of peptides as small as 33 amino-acids in length using sub-10 nm thin silicon nitride nanopores, giving high signal levels, combined with high-bandwidth electronics. In addition we show differentiation between monomers and dimer forms of the GCN-4 p1 leucine zipper, a coil-coil structure, and compare this with the unstructured 33-mer. The differentiation between these two forms demonstrates the possibility of extracting useful structural information from short peptide structures using modern solid-state nanopore systems.

  2. INSIGHTS ON SCRAPIE PRION PROTEIN (PrPSc) STRUCTURE OBTAINED BY LIMITED PROTEOLYSIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elucidation of the structure of PrPSc, essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research, and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on...

  3. Scale-up of the process to obtain functional ingredients based in plasma protein concentrates from porcine blood.

    PubMed

    Parés, Dolors; Toldrà, Mònica; Saguer, Elena; Carretero, Carmen

    2014-01-01

    The feasibility of a scaled-up process to obtain two protein concentrates from porcine blood plasma, i.e. serum and albumin, for use as functional food ingredients was assessed. The process consisted of fractionating plasma proteins by salting out, concentrating and purifying fractions by means of membrane technology, and subsequently dehydrating through spray-drying. The fractionation process allowed a good isolation of the desired proteins, which were then concentrated and desalted in a tangential flow filtration (TFF) process combining ultra and diafiltration. Purification, pre-concentration and dehydration were successfully achieved. The functional properties of dehydrated serum and albumin were determined. As compared to the same hemoderivatives obtained by a lab-scale production system, serum maintained the gelling properties; albumin exhibited similar foaming properties; and both serum and albumin concentrates showed slightly improved emulsifying properties. PMID:23927918

  4. Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins.

    PubMed

    Orsini Delgado, María C; Nardo, Agustina; Pavlovic, Marija; Rogniaux, Hélène; Añón, María C; Tironi, Valeria A

    2016-04-15

    The objective of the present work was to separate and identify antioxidant peptides from a simulated gastrointestinal digest (Id) from Amaranthus mantegazzianus proteins (I), which has previously been demonstrated to have this activity. I and Id were separated by preparative RP-HPLC. Fractions were evaluated by the ORAC method and the more active ones were analyzed by LC-MS/MS. Each fraction presented diverse peptides from different proteins, most of them from the 11S globulin. After grouping the peptides from 11S globulin according to their overlapping sequences, and based on previous information about structure-activity relationships, ten sequences were synthesized, in order to evaluate their antioxidant activity. Four peptides presented interesting activity: AWEEREQGSR>YLAGKPQQEH∼IYIEQGNGITGM∼TEVWDSNEQ. They exhibited some of the structural characteristics already known to demonstrate this activity, all of them containing at least one bulky aromatic residue. All belonged to little structured, internal or exposed regions of the acid subunit of the 11S globulin. PMID:26675853

  5. Ultrastructural studies on scrapie prion protein crystals obtained from reverse micellar solutions.

    PubMed Central

    Wille, H; Prusiner, S B

    1999-01-01

    The structural transition from the cellular prion protein (PrPC) that is rich in alpha-helices to the pathological form (PrPSc) that has a high beta-sheet content seems to be the fundamental event underlying the prion diseases. Determination of the structure of PrPSc and the N-terminally truncated PrP 27-30 has been complicated by their insolubility. Here we report the solubilization of PrP 27-30 through a system of reverse micelles that yields monomeric and dimeric PrP. Although solubilization of PrP 27-30 was not accompanied by any recognizable change in secondary structure as measured by FTIR spectroscopy, it did result in a loss of prion infectivity. The formation of small two- and three-dimensional crystals upon exposure to uranyl salts argues that soluble PrP 27-30 possesses considerable tertiary structure. The crystals of PrP 27-30 grown from reverse micellar solutions suggest a novel crystallization mechanism that might be applicable for other membrane proteins. A variety of different crystal lattices diffracted up to 1.85 nm by electron microscopy. Despite the lack of measurable biological activity, the structure of PrP 27-30 in these crystals may provide insight into the structural transition that occurs during PrPSc formation. PMID:9916037

  6. Production of Hyaluronic Acid by Streptococcus zooepidemicus on Protein Substrates Obtained from Scyliorhinus canicula Discards.

    PubMed

    Vázquez, José A; Pastrana, Lorenzo; Piñeiro, Carmen; Teixeira, José A; Pérez-Martín, Ricardo I; Amado, Isabel R

    2015-10-01

    This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs. PMID:26512678

  7. Production of Hyaluronic Acid by Streptococcus zooepidemicus on Protein Substrates Obtained from Scyliorhinus canicula Discards

    PubMed Central

    Vázquez, José A.; Pastrana, Lorenzo; Piñeiro, Carmen; Teixeira, José A.; Pérez-Martín, Ricardo I.; Amado, Isabel R.

    2015-01-01

    This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs. PMID:26512678

  8. Ion-Specific Control of the Self-Assembly Dynamics of a Nanostructured Protein Lattice

    PubMed Central

    2015-01-01

    Self-assembling proteins offer a potential means of creating nanostructures with complex structure and function. However, using self-assembly to create nanostructures with long-range order whose size is tunable is challenging, because the kinetics and thermodynamics of protein interactions depend sensitively on solution conditions. Here we systematically investigate the impact of varying solution conditions on the self-assembly of SbpA, a surface-layer protein from Lysinibacillus sphaericus that forms two-dimensional nanosheets. Using high-throughput light scattering measurements, we mapped out diagrams that reveal the relative yield of self-assembly of nanosheets over a wide range of concentrations of SbpA and Ca2+. These diagrams revealed a localized region of optimum yield of nanosheets at intermediate Ca2+ concentration. Replacement of Mg2+ or Ba2+ for Ca2+ indicates that Ca2+ acts both as a specific ion that is required to induce self-assembly and as a general divalent cation. In addition, we use competitive titration experiments to find that 5 Ca2+ bind to SbpA with an affinity of 67.1 ± 0.3 μM. Finally, we show via modeling that nanosheet assembly occurs by growth from a negligibly small critical nucleus. We also chart the dynamics of nanosheet size over a variety of conditions. Our results demonstrate control of the dynamics and size of the self-assembly of a nanostructured lattice, the constituents of which are one of a class of building blocks able to form novel hybrid nanomaterials. PMID:25494454

  9. Ion-specific control of the self-assembly dynamics of a nanostructured protein lattice.

    PubMed

    Rad, Behzad; Haxton, Thomas K; Shon, Albert; Shin, Seong-Ho; Whitelam, Stephen; Ajo-Franklin, Caroline M

    2015-01-27

    Self-assembling proteins offer a potential means of creating nanostructures with complex structure and function. However, using self-assembly to create nanostructures with long-range order whose size is tunable is challenging, because the kinetics and thermodynamics of protein interactions depend sensitively on solution conditions. Here we systematically investigate the impact of varying solution conditions on the self-assembly of SbpA, a surface-layer protein from Lysinibacillus sphaericus that forms two-dimensional nanosheets. Using high-throughput light scattering measurements, we mapped out diagrams that reveal the relative yield of self-assembly of nanosheets over a wide range of concentrations of SbpA and Ca(2+). These diagrams revealed a localized region of optimum yield of nanosheets at intermediate Ca(2+) concentration. Replacement of Mg(2+) or Ba(2+) for Ca(2+) indicates that Ca(2+) acts both as a specific ion that is required to induce self-assembly and as a general divalent cation. In addition, we use competitive titration experiments to find that 5 Ca(2+) bind to SbpA with an affinity of 67.1 ± 0.3 μM. Finally, we show via modeling that nanosheet assembly occurs by growth from a negligibly small critical nucleus. We also chart the dynamics of nanosheet size over a variety of conditions. Our results demonstrate control of the dynamics and size of the self-assembly of a nanostructured lattice, the constituents of which are one of a class of building blocks able to form novel hybrid nanomaterials. PMID:25494454

  10. Sequentially Integrated Optimization of the Conditions to Obtain a High-Protein and Low-Antinutritional Factors Protein Isolate from Edible Jatropha curcas Seed Cake.

    PubMed

    León-López, Liliana; Dávila-Ortiz, Gloria; Jiménez-Martínez, Cristian; Hernández-Sánchez, Humberto

    2013-01-01

    Jatropha curcas seed cake is a protein-rich byproduct of oil extraction which could be used to produce protein isolates. The purpose of this study was the optimization of the protein isolation process from the seed cake of an edible provenance of J. curcas by an alkaline extraction followed by isoelectric precipitation method via a sequentially integrated optimization approach. The influence of four different factors (solubilization pH, extraction temperature, NaCl addition, and precipitation pH) on the protein and antinutritional compounds content of the isolate was evaluated. The estimated optimal conditions were an extraction temperature of 20°C, a precipitation pH of 4, and an amount of NaCl in the extraction solution of 0.6 M for a predicted protein content of 93.3%. Under these conditions, it was possible to obtain experimentally a protein isolate with 93.21% of proteins, 316.5 mg 100 g(-1) of total phenolics, 2891.84 mg 100 g(-1) of phytates and 168 mg 100 g(-1) of saponins. The protein content of the this isolate was higher than the content reported by other authors. PMID:25937971

  11. Sequentially Integrated Optimization of the Conditions to Obtain a High-Protein and Low-Antinutritional Factors Protein Isolate from Edible Jatropha curcas Seed Cake

    PubMed Central

    León-López, Liliana; Dávila-Ortiz, Gloria; Jiménez-Martínez, Cristian; Hernández-Sánchez, Humberto

    2013-01-01

    Jatropha curcas seed cake is a protein-rich byproduct of oil extraction which could be used to produce protein isolates. The purpose of this study was the optimization of the protein isolation process from the seed cake of an edible provenance of J. curcas by an alkaline extraction followed by isoelectric precipitation method via a sequentially integrated optimization approach. The influence of four different factors (solubilization pH, extraction temperature, NaCl addition, and precipitation pH) on the protein and antinutritional compounds content of the isolate was evaluated. The estimated optimal conditions were an extraction temperature of 20°C, a precipitation pH of 4, and an amount of NaCl in the extraction solution of 0.6 M for a predicted protein content of 93.3%. Under these conditions, it was possible to obtain experimentally a protein isolate with 93.21% of proteins, 316.5 mg 100 g−1 of total phenolics, 2891.84 mg 100 g−1 of phytates and 168 mg 100 g−1 of saponins. The protein content of the this isolate was higher than the content reported by other authors. PMID:25937971

  12. Statistical Estimation of the Protein-Ligand Binding Free Energy Based On Direct Protein-Ligand Interaction Obtained by Molecular Dynamics Simulation

    PubMed Central

    Fukunishi, Yoshifumi; Nakamura, Haruki

    2012-01-01

    We have developed a method for estimating protein-ligand binding free energy (ΔG) based on the direct protein-ligand interaction obtained by a molecular dynamics simulation. Using this method, we estimated the ΔG value statistically by the average values of the van der Waals and electrostatic interactions between each amino acid of the target protein and the ligand molecule. In addition, we introduced fluctuations in the accessible surface area (ASA) and dihedral angles of the protein-ligand complex system as the entropy terms of the ΔG estimation. The present method included the fluctuation term of structural change of the protein and the effective dielectric constant. We applied this method to 34 protein-ligand complex structures. As a result, the correlation coefficient between the experimental and calculated ΔG values was 0.81, and the average error of ΔG was 1.2 kcal/mol with the use of the fixed parameters. These results were obtained from a 2 nsec molecular dynamics simulation. PMID:24281257

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

    PubMed

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

    2013-01-01

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

  14. INSECTICIDAL ACTIVITY OF PROTEIN EXTRACTS OBTAINED FROM BULBS OF CHILEAN AMARYLLIDACEAE AGAINST TRIALEURODES VAPORARIORUM WESTWOOD AND PSEUDOCOCCUS VIBURNI SIGNORET.

    PubMed

    Zapata, N; Vargas, M; Coronado, A; Van Damme, E J M; Smagghe, G

    2015-01-01

    Entomotoxic proteins are produced by plants in defence against insect herbivory. Some carbohydrate-binding proteins exhibit strong insecticidal activity affecting the survival, growth, development and feeding behavior of phytophagous insects. The occurrence of entomotoxic lectins is well documented in the Amaryllidaceae, a plant family spread world-wide. In Chile, this family is represented by numerous species, many of which are also of high ornamental value. Protein extracts were obtained from bulbs of five different species of Chilean Amaryllidaceae. A dose-response assay was carried out with two important pests: the greenhouse whitefly Trialeurodes vaporariorum Westwood and the mealybug Pseudococcus viburni Signoret. The extracts were offered to insects in a liquid artificial diet for three days and the mortality was scored. The Phycella australis Ravenna extract caused the highest insecticidal activity (T. vaporariorum LC₅₀: 7200 µg/mL; P. viburni LC₅₀: 9500 µg/mL). Applied at 1000 µg/mL in the diet the P. australis extract did not repel feeding of these pests. A mannose-binding lectin isolated from the bulbs of P. australis proved to be moderately toxic for these pests (T. vaporariorum LC₅₀: 1127 µg/mL; P. viburni LC₅₀: 2320 µg/mL). PMID:27145586

  15. Comparative Composition and Antioxidant Activity of Peptide Fractions Obtained by Ultrafiltration of Egg Yolk Protein Enzymatic Hydrolysates

    PubMed Central

    Chay Pak Ting, Bertrand P.; Mine, Yoshinori; Juneja, Lekh R.; Okubo, Tsutomu; Gauthier, Sylvie F.; Pouliot, Yves

    2011-01-01

    The objective of the study was to compare the antioxidant activity of two distinct hydrolysates and their peptide fractions prepared by ultrafiltration (UF) using membranes with molecular weight cut-off of 5 and 1 kDa. The hydrolysates were a delipidated egg yolk protein concentrate (EYP) intensively hydrolyzed with a combination of two bacterial proteases, and a phosphoproteins (PPP) extract partially hydrolyzed with trypsin. Antioxidant activity, as determined by the oxygen radical absorbance capacity (ORAC) assay, was low for EYP and PPP hydrolysates with values of 613.1 and 489.2 μM TE·g−1 protein, respectively. UF-fractionation of EYP hydrolysate increased slightly the antioxidant activity in permeate fractions (720.5–867.8 μM TE·g−1 protein). However, ORAC values were increased by more than 3-fold in UF-fractions prepared from PPP hydrolysate, which were enriched in peptides with molecular weight lower than 5 kDa. These UF-fractions were characterized by their lower N/P atomic ratio and higher phosphorus content compared to the same UF-fractions obtained from EYP-TH. They also contained high amounts of His, Met, Leu, and Phe, which are recognized as antioxidant amino acids, but also high content in Lys and Arg which both represent target amino acids of trypsin used for the hydrolysis of PPP. PMID:24957729

  16. A new hyaluronic acid pH sensitive derivative obtained by ATRP for potential oral administration of proteins.

    PubMed

    Fiorica, Calogero; Pitarresi, Giovanna; Palumbo, Fabio Salvatore; Di Stefano, Mauro; Calascibetta, Filippo; Giammona, Gaetano

    2013-11-30

    Atom transfer radical polymerization (ATRP) has been successfully employed to obtain a new derivative of hyaluronic acid (HA) able to change its solubility as a function of external pH and then to be potentially useful for intestinal release of bioactive molecules, included enzymes and proteins. In particular, a macroinitiator has been prepared by linking 2-bromo-2-methypropionic acid (BMP) to the amino groups of ethylenediamino derivative of tetrabutyl ammonium salt of HA (HA-TBA-EDA). This macroinititor, named HA-TBA-EDA-BMP has been used for the ATRP of sodium methacrylate (MANa) using a complex of Cu(I) and 2,2'-bipyridyl (Byp) as a catalyst. The resulting copolymer, named HA-EDA-BMP-MANa, has been characterized by (1)H NMR and size exclusion chromatography (SEC) analyses. A turbidimetric analysis has showed its pH sensitive behavior, being insoluble in simulated gastric fluid but soluble when pH increases more than 2.5. To confirm the ability of HA-EDA-BMP-MANa in protecting peptides or proteins from denaturation in acidic medium, α-chymotrypsin has been chosen as a model of protein molecule and its activity has been evaluated after entrapment into HA-EDA-BMP-MANa chains and treatment under simulated gastric conditions. Finally, cell compatibility has been evaluated by performing a MTS assay on murine dermal fibroblasts cultured with HA-EDA-BMP-MANa solutions. PMID:24060369

  17. Structure and Antioxidant Activity of Soy Protein Isolate-Dextran Conjugates Obtained by TiO2 Photocatalysis

    PubMed Central

    Jin, Bei; Zhou, Xiaosong; Li, Bing; Chen, Caiyan; Zhang, Xiaosa; Chen, Siqiao

    2015-01-01

    The aim of this study was to investigate the structural characteristics and antioxidant activities of soy protein isolate- (SPI-) dextran conjugates obtained by TiO2 photocatalysis treatment. Results revealed that the UV-vis absorption and the fluorescence intensity increased as the photocatalytic power increased (P < 0.05). Higher photocatalytic power could promote the extent of glycation and the formation of high molecular weight SPI-dextran conjugates, which were evidenced by free amino group content and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The Fourier transform infrared (FT-IR) spectra suggested that the amide I, II, and III bands of SPI were altered by the glycation induced by TiO2 photocatalysis. Moreover, significant changes of secondary structure occurred in SPI-dextran conjugates. The α-helix, β-sheet, β-turns, and random coil were changed from approximately 10.6%, 37.9%, 12.9%, and 38.6% to 3.8%, 10.4%, 17.7%, and 68.8%, respectively, after treatment at photocatalytic power of 1000 W. In addition, SPI-dextran conjugates obtained by TiO2 photocatalysis treatment exhibited high hydroxyl radical scavenging activity and possessed increased reducing power. All data indicated that TiO2 photocatalysis was an efficient method for promoting protein-polysaccharide copolymerisation. PMID:26495283

  18. Heterotetramers formed by an S-layer-streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development.

    PubMed

    Huber, Carina; Liu, Jing; Egelseer, Eva M; Moll, Dieter; Knoll, Wolfgang; Sleytr, Uwe B; Sára, Margit

    2006-01-01

    Based on the S-layer protein SbpA of Bacillus sphaericus CCM 2177, an S-layer-streptavidin fusion protein was constructed. After heterologous expression, isolation of the fusion protein, and refolding, functional heterotetramers were obtained that had retained the ability to recrystallize into the square-lattice structure on plain gold chips and on gold chips precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Monolayers generated by recrystallization of heterotetramers on plain gold chips or on gold chips precoated with thiolated SCWP were exploited for the binding of biotinylated oligonucleotides (30-mers). Hybridization experiments with complementary fluorescently labeled oligonucleotides carrying one mismatch or no mismatch (both 15-mers) were performed and evaluated with surface-plasmon-field-enhanced fluorescence spectroscopy. For surfaces generated by the recrystallization of heterotetramers on SCWP-coated gold chips, a detection limit of 1.57 pM could be determined, whereas for surfaces obtained by direct recrystallization of heterotetramers on plain gold chips, a detection limit of 8.2 pM was found. Measuring the association and dissociation processes of oligonucleotides carrying no mismatch led to a dissociation constant of K(D)=6.3 x 10(-10) m, whereas for oligonucleotides carrying one mismatch a dissociation constant of K(D)=7.9 x 10(-9) m was determined. This finding was confirmed by measuring the whole Langmuir isotherm, which resulted in a dissociation constant of K(D)=2.6 x 10(-8) m. PMID:17193570

  19. Cloning and expression of a gene encoding a protein obtained from earthworm secretion that is a chemoattractant for garter snakes.

    PubMed

    Liu, W; Wang, D; Chen, P; Halpern, M

    1997-10-24

    The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736-8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF' host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-beta-D-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes. PMID:9341189

  20. Differential Gene Expression of Heat Shock Protein 90 (Hsp90) of Candida albicans obtained from Malaysian and Iranian Patients

    PubMed Central

    Khalili, Vajihe; Shokri, Hojjatollah; Md Akim, Abdah; Khosravi, Ali Reza

    2016-01-01

    Background Candida albicans (C. albicans) has several virulence factors, in particular heat shock protein 90 (Hsp90), which is expressed by Hsp90 gene. The purposes of this study were to assess the expression of Hsp90 gene in clinical and control isolates of C. albicans obtained from different geographical regions (Malaysia and Iran), different temperatures (25°C, 37°C and 42°C) and mice with candidiasis. Methods C. albicans isolates were cultured onto sabouraud dextrose agar (SDA). The assessment of the expression of Hsp90 gene was performed using real time-polymerase chain reaction (RT-PCR). Results The results showed a significant increase in the expression of C. albicans Hsp90 gene under high thermal shock (42°C) when compared to other temperatures tested (P-value = 0.001). The mean differences in the expression of Hsp90 gene at 37°C were 0.20 (95% confidence interval (CI) 0.13–0.29) between Malaysian and Iranian controls (P-value = 0.040) and 0.47 (95% CI 0.27–0.60) between Malaysian and Iranian patients (P-value = 0.040). Conclusion The results demonstrated that the expression of C. albicans Hsp90 gene varied between Malaysian and Iranian subjects, representing the efficacy of geographical and thermal conditions on virulence gene expression.

  1. Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

    PubMed Central

    Dean, D A; Fikes, J D; Gehring, K; Bassford, P J; Nikaido, H

    1989-01-01

    Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process. Images PMID:2644203

  2. Emulsifying and Foaming Properties of Different Protein Fractions Obtained from a Novel Lupin Variety AluProt-CGNA(®) (Lupinus luteus).

    PubMed

    Burgos-Díaz, César; Piornos, José A; Wandersleben, Traudy; Ogura, Takahiro; Hernández, Xaviera; Rubilar, Mónica

    2016-07-01

    The use of vegetable proteins as food ingredient is becoming increasingly important due to their high versatility and environmental acceptability. This work describes a chemical characterization and techno-functional properties (emulsifying and foaming properties) of 3 protein fractions obtained from a protein-rich novel lupin variety, AluProt-CGNA(®) . This nongenetically modified variety have a great protein content in dehulled seeds (60.6 g protein/100 g, dry matter), which is higher than soybean and other lupin varieties. A simple procedure was utilized to obtain 3 different fractions by using alkali solubilization and isoelectric precipitation. Fractions 1 and 3 were mainly composed of protein and polysaccharides (NNE), whereas fraction 2 was mainly composed by protein (97%, w/w). Fraction 3 presented interesting and potential foaming properties in comparison to the other fractions evaluated in the study. Besides, its solubility, foaming and emulsifying capacity were practically not affected by pH variations. The 3 fractions also presented good emulsion stability, reaching values above a 95%. SDS-PAGE showed that fractions 1 and 2 contained mainly conglutin α, β, and δ, but in different ratios, whereas fraction 3 contained mainly conglutin γ and albumins. The results of this work will provide better understanding for the utilization of each protein fractions as potential ingredients in food industry. PMID:27232549

  3. Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition.

    PubMed

    Blommel, Paul G; Becker, Katie J; Duvnjak, Petar; Fox, Brian G

    2007-01-01

    The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 mug/mL of expression culture were identified. PMID:17506520

  4. Larger red-shift in optical emissions obtained from the thin films of globular proteins (BSA, lysozyme) - polyelectrolyte (PAA) complexes

    NASA Astrophysics Data System (ADS)

    Talukdar, Hrishikesh; Kundu, Sarathi; Basu, Saibal

    2016-09-01

    Globular proteins (lysozyme and BSA) and polyelectrolyte (sodium polyacrylic acid) are used to form protein-polyelectrolyte complexes (PPC). Out-of-plane structures of ≈30-60 nm thick PPC films and their surface morphologies have been studied by using X-ray reflectivity and atomic force microscopy, whereas optical behaviors of PPC and protein conformations have been studied by using UV-vis, photoluminescence and FTIR spectroscopy respectively. Our study reveals that thin films of PPC show a larger red-shift of 23 and 16 nm in the optical emissions in comparison to that of pure protein whereas bulk PPC show a small blue-shift of ≈3 nm. A small amount of peak-shift is found to occur due to the heat treatment or concentration variation of the polyelectrolyte/protein in bulk solution but cannot produce such film thickness independent larger red-shift. Position of the emission peak remains nearly unchanged with the film thickness. Mechanism for such larger red-shift has been proposed.

  5. Recombinant bovine alpha-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli.

    PubMed

    Wang, M; Scott, W A; Rao, K R; Udey, J; Conner, G E; Brew, K

    1989-12-15

    A cDNA clone containing the entire coding region for bovine pre-alpha-lactalbumin (LA) together with 27 base pairs of 5'-noncoding and 268 base pairs of 3'-noncoding sequences was isolated from a bovine mammary cDNA plasmid library in the Okayama-Berg vector system using a synthetic oligonucleotide probe and sequenced. The coding segment for mature LA was subcloned into the T7 expression system of Studier and co-workers (Studier, F.W., and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113-130; Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W. (1987) Gene (Amst.) 56, 125-135) and expressed as a 21-kDa fusion protein that consisted of the mature bovine LA sequence connected to the NH2-terminal 50 residues of human cathepsin D by a linker sequence containing protease cleavage sites. This fusion protein was expressed in an insoluble form and accumulated to about 50% of the total bacterial protein within 3 h after induction of T7 RNA polymerase synthesis. The protein was solubilized, purified by gel filtration, and converted to an active form by treatment with mixtures of reduced and oxidized glutathione in the presence of Ca2+. The maximum specific activity of the fusion protein was about 25% of that of native LA, suggesting that the attachment of an NH2-terminal extension sterically hinders but does not prevent the interaction with galactosyltransferase. The extension also does not block the binding of the regulatory Ca2+ ion that is required for folding from the reduced denatured state. Trypsin cleaved the folded fusion protein specifically at a Lys-Glu bond at the junction with the mature LA sequence to give a product indistinguishable in structure and activity from native LA. PMID:2687274

  6. Protein synthesis underlies post-retrieval memory consolidation to a restricted degree only when updated information is obtained

    PubMed Central

    Rodriguez-Ortiz, Carlos J.; De la Cruz, Vanesa; Gutiérrez, Ranier; Bermudez-Rattoni, Federico

    2005-01-01

    Consolidation theory proposes that through the synthesis of new proteins recently acquired memories are strengthened over time into a stable long-term memory trace. However, evidence has accumulated suggesting that retrieved memory is susceptible to disruption, seeming to consolidate again (reconsolidate) to be retained in long-term storage. Here we show that intracortical blockade of protein synthesis in the gustatory cortex after retrieval of taste-recognition memory disrupts previously consolidated memory to a restricted degree only if the experience is updated. Our results suggest that retrieved memory can be modified as part of a mechanism for incorporating updated information into previously consolidated memory. PMID:16166395

  7. Modeling data from titration, amide H/D exchange, and mass spectrometry to obtain protein-ligand binding constants.

    PubMed

    Zhu, Mei M; Rempel, Don L; Gross, Michael L

    2004-03-01

    We recently reported a new method for quantification of protein-ligand interaction by mass spectrometry, titration and H/D exchange (PLIMSTEX) for determining the binding stoichiometry and affinity of a wide range of protein-ligand interactions. Here we describe the method for analyzing the PLIMSTEX titration curves and evaluate the effect of various models on the precision and accuracy for determining binding constants using H/D exchange and a titration. The titration data were fitted using a 1:n protein:ligand sequential binding model, where n is the number of binding sites for the same ligand. An ordinary differential equation was used for the first time in calculating the free ligand concentration from the total ligand concentration. A nonlinear least squares regression method was applied to minimize the error between the calculated and the experimentally measured deuterium shift by varying the unknown parameters. A resampling method and second-order statistics were used to evaluate the uncertainties of the fitting parameters. The interaction of intestinal fatty-acid-binding protein (IFABP) with a fatty-acid carboxylate and that of calmodulin with Ca(2+) are used as two tests. The modeling process described here not only is a new tool for analyzing H/D exchange data acquired by ESI-MS, but also possesses novel aspects in modeling experimental titration data to determine the affinity of ligand binding. PMID:14998541

  8. Protein Synthesis Underlies Post-Retrieval Memory Consolidation to a Restricted Degree Only when Updated Information Is Obtained

    ERIC Educational Resources Information Center

    Rodriguez-Ortiz, Carlos J.; De la Cruz, Vanesa; Gutierrez, Ranier; Bermudez-Rattoni, Federico

    2005-01-01

    Consolidation theory proposes that through the synthesis of new proteins recently acquired memories are strengthened over time into a stable long-term memory trace. However, evidence has accumulated suggesting that retrieved memory is susceptible to disruption, seeming to consolidate again (reconsolidate) to be retained in long-term storage. Here…

  9. Data on endogenous chicken sperm peptides and small proteins obtained through Top-Down High Resolution Mass Spectrometry.

    PubMed

    Soler, L; Labas, V; Thélie, A; Teixeira-Gomes, A P; Grasseau, I; Bouguereau, L; Blesbois, E

    2016-09-01

    The endogenous peptides and small proteins present in chicken sperm were identified in the context of the characterization of a fertility-diagnostic method based on the use of ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The interpretation and description of these data can be found in a research article, "Intact cell MALDI-TOF MS on sperm: a molecular test for male fertility diagnosis" (Soler et al., 2016) [1], and raw data derived from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768. Here, we describe the inventory of all the molecular species identified, along with their biochemical features and functional analysis. This peptide/protein catalogue can be further employed as reference for other studies and reveal that the use of proteomics allows for a global evaluation of sperm cells functions. PMID:27617276

  10. A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

    PubMed

    Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

    2013-11-29

    The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. PMID:24139506

  11. Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria sp.).

    PubMed

    Laohakunjit, Natta; Selamassakul, Orrapun; Kerdchoechuen, Orapin

    2014-09-01

    An enzymatic bromelain seaweed protein hydrolysate (eb-SWPH) was characterised as the precursor for thermally processed seafood flavour. Seaweed (Gracilaria fisheri) protein after agar extraction was hydrolysed using bromelain (enzyme activity=119,325 U/g) at 0-20% (w/w) for 0.5-24 h. Optimal hydrolysis conditions were determined using response surface methodology. The proposed model took into account the interaction effect of the enzyme concentration and hydrolysis time on the physicochemical properties and volatile components of eb-SWPH. The optimal hydrolysis conditions for the production of eb-SWPH were 10% bromelain for 3h, which resulted in a 38.15% yield and a 62.91% degree of hydrolysis value. Three free amino acids, arginine, lysine, and leucine, were abundant in the best hydrolysate. Ten volatile flavours of the best eb-SWPH were identified using gas chromatography/mass spectrometry. The predominant odourants were hexanal, hexanoic acid, nonanoic acid, and dihydroactinidiolide. The thermally processed seafood flavour produced from eb-SWPH exhibited a roasted seafood-like flavouring. PMID:24731327

  12. New user-friendly approach to obtain an Eisenberg plot and its use as a practical tool in protein sequence analysis.

    PubMed

    Keller, Rob C A

    2011-01-01

    The Eisenberg plot or hydrophobic moment plot methodology is one of the most frequently used methods of bioinformatics. Bioinformatics is more and more recognized as a helpful tool in Life Sciences in general, and recent developments in approaches recognizing lipid binding regions in proteins are promising in this respect. In this study a bioinformatics approach specialized in identifying lipid binding helical regions in proteins was used to obtain an Eisenberg plot. The validity of the Heliquest generated hydrophobic moment plot was checked and exemplified. This study indicates that the Eisenberg plot methodology can be transferred to another hydrophobicity scale and renders a user-friendly approach which can be utilized in routine checks in protein-lipid interaction and in protein and peptide lipid binding characterization studies. A combined approach seems to be advantageous and results in a powerful tool in the search of helical lipid-binding regions in proteins and peptides. The strength and limitations of the Eisenberg plot approach itself are discussed as well. The presented approach not only leads to a better understanding of the nature of the protein-lipid interactions but also provides a user-friendly tool for the search of lipid-binding regions in proteins and peptides. PMID:22016610

  13. Thermodynamic features characterizing good and bad folding sequences obtained using a simplified off-lattice protein model

    NASA Astrophysics Data System (ADS)

    Amatori, A.; Ferkinghoff-Borg, J.; Tiana, G.; Broglia, R. A.

    2006-06-01

    The thermodynamics of the small SH3 protein domain is studied by means of a simplified model where each beadlike amino acid interacts with the others through a contact potential controlled by a 20×20 random matrix. Good folding sequences, characterized by a low native energy, display three main thermodynamical ensembles, namely, a coil-like ensemble, an unfolded globule, and a folded ensemble (plus two other states, frozen and random coils, populated only at extreme temperatures). Interestingly, the unfolded globule has some regions already structured. Poorly designed sequences, on the other hand, display a wide transition from the random coil to a frozen state. The comparison with the analytic theory of heteropolymers is discussed.

  14. Standard free energies of binding of solute to proteins in aqueous medium. Part 2. Analysis of data obtained from equilibrium dialysis and isopiestic experiments.

    PubMed

    Chattoraj, D K; Biswas, S C; Mahapatra, P K; Chatterjee, S

    1999-03-01

    In an earlier publication by Chattoraj et al. [Biophysical Chemistry 63 (1996) 37], a generalized equation for standard free energy of (delta G0) interaction of surfactant, inorganic salts and aqueous solvent with protein, forming a single phase has been deduced on strict thermodynamic grounds. In the present paper, this equation has been utilized to calculate delta G0 in kilojoules per kilogram of different proteins for the change of bulk surfactant activity from zero to unity in the mole fraction scale. Values of binding interactions of CTAB, MTAB, DTAB and SDS to BSA, beta-lactoglobulin, gelatin, casein, myosin, lysozyme and their binary and ternary mixtures had already been determined in this laboratory at different surfactant concentrations, pH, ionic strength and temperature using an equilibrium dialysis technique. Values of delta G0 for saturated protein-surfactant complexes as well as unsaturated complexes are found to be equal. delta G0 is also found to vary linearly with maximum moles of surfactants bound to a kilogram of protein or protein mixture and the slope of this linear plot represents standard free energy delta G0B for the transfer of 1 mol of surfactant from the bulk for binding reaction with protein; -delta G0 values for different systems vary widely and the order of their magnitudes represents relative affinities of surfactants to proteins. Magnitude of -delta G0B on the other hand varies within a narrow range of 32-37 kJ/mol of surfactant. For interaction of SDS with BSA, close to the CMC, values of delta G0 are very high due to the formation of micelles of protein-bound surfactants. Values of delta G0 for negative binding of inorganic salts to proteins and protein mixtures have been evaluated using our generalized equation in which excess binding values of water and salts have been calculated from the data obtained from our previous isopiestic experiments. delta G0 values in these cases are positive due to the excess hydration of proteins

  15. Immune reactivity of sera obtained from brucellosis patients and vaccinated-rabbits to a fusion protein from Brucella melitensis

    PubMed Central

    Amani, Jafar; Ghasemi, Amir; Ranjbar, Reza; Shabani, Mahdi; Zandemami, Mahdi; Golmohammadi, Reza

    2015-01-01

    Objective(s): Brucella spp. are facultative intracellular pathogens which can stay alive and multiply in professional and nonprofessional phagocytes. Immunity against Brucella melitensis involves antigen-specific CD4+ and CD8+ T-cells activation and humoral immune responses. Due to negative aspects of live attenuated vaccines, much attention has been focused on finding Brucella-protective antigens to introduce them as potential subunit vaccine candidates. Materials and Methods: A chimeric gene encoding trigger factor (TF), Omp3148-74 and BP2687-111 fragments (TOB) from B. melitensis was successfully cloned, expressed in Escherichia coli BL21-DE3 and purified by Ni-NTA agarose column. Antibodies to recombinant TOB (rTOB) have been investigated in Brucella-infected human sera and a pool serum prepared from B. melitensis-vaccinated rabbits. Results: Our results showed that the immunized rabbit pool serum strongly reacted with rTOB. In addition, antibodies against rTOB were detectable in 76.5% of sera obtained from infected patients. Conclusion: These findings suggest that rTOB may provide a potential immunogenic candidate which could be considered in future vaccine studies. PMID:26019797

  16. Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar) Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

    PubMed Central

    Darewicz, Małgorzata; Borawska, Justyna; Vegarud, Gerd E.; Minkiewicz, Piotr; Iwaniak, Anna

    2014-01-01

    The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE) inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes) and ex vivo digestion (with human gastrointestinal enzymes). Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50%) of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes. PMID:25123137

  17. New User-Friendly Approach to Obtain an Eisenberg Plot and Its Use as a Practical Tool in Protein Sequence Analysis

    PubMed Central

    Keller, Rob C.A.

    2011-01-01

    The Eisenberg plot or hydrophobic moment plot methodology is one of the most frequently used methods of bioinformatics. Bioinformatics is more and more recognized as a helpful tool in Life Sciences in general, and recent developments in approaches recognizing lipid binding regions in proteins are promising in this respect. In this study a bioinformatics approach specialized in identifying lipid binding helical regions in proteins was used to obtain an Eisenberg plot. The validity of the Heliquest generated hydrophobic moment plot was checked and exemplified. This study indicates that the Eisenberg plot methodology can be transferred to another hydrophobicity scale and renders a user-friendly approach which can be utilized in routine checks in protein–lipid interaction and in protein and peptide lipid binding characterization studies. A combined approach seems to be advantageous and results in a powerful tool in the search of helical lipid-binding regions in proteins and peptides. The strength and limitations of the Eisenberg plot approach itself are discussed as well. The presented approach not only leads to a better understanding of the nature of the protein–lipid interactions but also provides a user-friendly tool for the search of lipid-binding regions in proteins and peptides. PMID:22016610

  18. Multiemission wavelength picosecond time-resolved fluorescence decay data obtained on the millisecond time scale: application to protein:DNA interactions and protein-folding reactions

    NASA Astrophysics Data System (ADS)

    Beechem, Joseph M.

    1992-04-01

    One of the major aspects of fluorescence spectroscopy which differentiates this technique from many other spectroscopic approaches is the inherent multidimensional nature of the data. For instance, the basic pulsed-laser fluorescence data set is characterized by fluorescence versus: emission wavelength, polarization state (parallel and perpendicular intensities), time of emission (picoseconds to nanoseconds), and time of biological reaction (milliseconds to minutes). Usually, this six-dimensional data set is obtained piecemeal, single dimension at a time; often complete data sets are not even collected. This is especially true of the biological time scale axis. Data acquisition times for picosecond decay data are typically seconds to minutes, and, therefore, it has not been generally possible to perform this experiment in a kinetic mode. What is described in this report is the construction of a parallel multichannel time-correlated single-photon counting (TCSPC) fluorometer which is capable of simultaneous collection of: fluorescence vs. picosecond to nanosecond time vs. emission wavelength vs. polarization state vs. millisecond to second time. Use is made of two multi-anode microchannel plate detectors, each obtaining data at two different polarization states, six different emission wavelengths, along 12 independent TCSPC channels. This instrument is interfaced to a three-syringe stepper motor controlled stop-flow apparatus, and picosecond decay data along all of these channels is stored and collected by two 33 MHz 80486 computers at rates approaching 1200 - 12000 data sets per second.

  19. Comparison of measurements of canine plasma creatinine, glucose, proteins, urea, alanine aminotransferase, and alkaline phosphatase obtained with Spotchem SP 4430 and Vitros 250 analyzers.

    PubMed

    Trumel, C; Diquélou, A; Germain, C; Palanché, F; Braun, J P

    2005-12-01

    The suitability of the Spotchem 4430 benchtop biochemistry analyzer for canine blood samples was tested for creatinine, glucose, proteins, urea, alkaline phosphatases and alanine aminotransferase. Results obtained from whole blood and corresponding heparin plasma were identical except for proteins which were higher in plasma (n=10). Between series imprecision (n=10) was <5% for substrates and <10% for enzymes. Comparison of results from 100 Li-heparin samples with those measured with a Vitros 250 analyzer showed good correlation (r>0.93). The slopes of the Passing-Bablock's regression ranged from 0.90 to 1.20 and intercepts were low. The mean biases were low, except for creatinine for which the results obtained by Spotchem (Jaffe reaction) were about 20 micromol/L higher than with the Vitros (enzymatic reaction). The results of this study show that the Spotchem analyzer is suitable for use in canine whole blood or plasma when small numbers of tests are to be performed and large analyzers are not available. PMID:16054888

  20. Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases.

    PubMed

    Bougatef, Ali; Nedjar-Arroume, Naima; Ravallec-Plé, Rozenn; Leroy, Yves; Guillochon, Didier; Barkia, Ahmed; Nasri, Moncef

    2008-11-15

    The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase(®), chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2±1.5% at 2mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P1-P8). Biological functions of all fractions were assayed, and P4 was found to display a high ACE inhibitory activity. The IC50 values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P4 were 1.2±0.09 and 0.81±0.013mg/ml, respectively. Further, P4 showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P4 was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine. PMID:26047434

  1. Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and CE-ICP-MS.

    PubMed

    Kannamkumarath, Sasi S; Wrobel, Katarzyna; Wuilloud, Rodolfo G

    2005-03-31

    In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05molL(-1) sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05molL(-1), pH 8) as compared to 0.05molL(-1) HCl, 0.05molL(-1) Tris/HCl or hot water (60 degrees C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50mmolL(-1) Tris buffer (pH 8), 150mmolL(-1) ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine. PMID:18969975

  2. High-shear, Jet-cooking, and Alkali Treatment of Corn Distillers' Dried Grains to Obtain Products with Enhanced Protein, Oil, and Phenolic Antioxidants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Distillers dried grains (DDG) have potential to be a nutritionally important source of protein, oil, and phenolic antioxidants. DDG was subjected to high-shear and jet-cooking, with or without alkaline pH adjustment and autoclaving. Soluble and insoluble fractions were analyzed for protein, oil, a...

  3. [Position statement. Protein/creatinine in a randomly obtained urine sample in the diagnosis of proteinuria in pregnant patients with arterial hypertension].

    PubMed

    2012-01-01

    Leaños Miranda and collaborators published that the measurement of protein/creatinine ratio in a single random urine sample is a reliable indicator of significant proteinuria and may be reasonably used as alternative to the 24-hours urine collection method as a diagnostic criteria for urinary protein, and it is also a criterion for identifying the disease severity. This leads us to present this successful result of the investigation as a position statement in the care of pregnant women with hypertension. PMID:23282273

  4. A comparison of the estimates of whole-body protein turnover in parenterally fed neonates obtained using three different end products.

    PubMed

    Pencharz, P; Beesley, J; Sauer, P; Van Aerde, J; Canagarayar, U; Renner, J; McVey, M; Wesson, D; Swyer, P

    1989-06-01

    Protein turnover rates in neonates have been calculated largely by measuring urinary [15N]urea enrichment following administration of [15N]glycine. Although ammonia has been increasingly recognized as an end product of nitrogen metabolism, in neonates it yields a different estimate of protein turnover than does urea. Comparisons of ammonia and urea end products in parenterally fed neonates have not previously been reported. A third and independent way of estimating protein turnover, developed for adults, is to use breath 13CO2 as an end product following administration of [1-13C]leucine. We therefore carried out simultaneous measurements of protein turnover in 10 parenterally fed neonates, using the three end products. The infants were clinically stable, weighed 2.6 +/- 0.2 kg, and received 3.1 +/- 0.2 g.kg-1.d-1 of amino acid, 2.2 +/- 0.1 g.kg-1.d-1 of lipids, and an energy intake of 90 +/- 4 kcal.kg-1.d-1 (1 kcal = 4.186 kJ). The turnover estimates derived from the 13CO2 and [15N]urea end products were very similar. The [15N]ammonia end product produced values approximately 66% (p less than 0.01) of the other two. We conclude that the ammonia and urea end products probably originate in different precursor pools. The similarity of the urea and breath carbon dioxide results helps validate the use of the urea end product in studying the nitrogen metabolism of parenterally fed neonates. Ideally in future studies two or more end products should be used, since they provide information about different aspects of the neonates' protein metabolism. PMID:2505915

  5. Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures.

    PubMed

    Moskovets, Eugene V; Ivanov, Alexander R

    2016-06-01

    This work demonstrates that the chromatographic separation performed at highly stabilized elevated temperature results in significant improvements in sensitivity, quantitative accuracy, chromatographic resolution, and run-to-run reproducibility of nanoLC-MS analysis of complex peptides mixtures. A newly developed platform was shown to provide conditions for accurate temperature stabilization and temperature homogeneity when performing nanoLC-ESI MS analysis. We quantitatively assessed and compared the recovery of peptides and small proteins from nanoLC columns at room and elevated temperatures. We found that analyses performed at highly stabilized elevated temperatures led to improved detection sensitivity, reproducibility, and chromatographic resolution in reversed-phase LC separation of unmodified peptides (both hydrophilic and hydrophobic), post-translationally modified peptides (O-phosphorylated), and small intact proteins. The analytical benefits of elevated temperatures for qualitative and quantitative proteomic LC-MS profiling were demonstrated using mixtures of synthetic peptides, tryptic digests of mixtures of model proteins, and digested total lysates of isolated rat kidney mitochondria. The effect of elevated temperature on the ion suppression was also demonstrated. Graphical Abstract A fragment of overlaid LC retention time-m/z planar views demonstrates the improved separation performance in the analysis of a complex peptide mixture at elevated temperature. Retention time-m/z 2D peptide features detected at 60 °C (magenta) were matched and aligned with features detected at room temperature (green). PMID:26898204

  6. New vectors for co-expression of proteins : structure of bacillus subtilis ScoAB obtained by high-throughput protocols.

    SciTech Connect

    Stols, L.; Zhou, M.; Eschenfeldt, W. H.; Millard, C. S.; Abdullah, J.; Collart, F. R.; Kim, Y.; Donnelly, M. I.; Biosciences Division

    2007-06-01

    The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.

  7. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

    PubMed Central

    Bhatia, Prateek A.; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodopetra frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp c-DNA, using a baculovirus expression system. The resulting CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCPR) column The binding affinities (Ki values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [3H]-etoposide on the CMAC(Sf9MRP1) column to a greater extent than (R)-verapamil and the relative IC50 values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC50 values were consistent with previously reported data. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system. PMID:20441926

  8. Angiotensin I-Converting Enzyme inhibitory and antioxidant activities and surfactant properties of protein hydrolysates as obtained of Amaranthus hypochondriacus L. grain.

    PubMed

    Soriano-Santos, J; Escalona-Buendía, H

    2015-04-01

    Even though some research has been carried out on surfactant properties of amaranth protein hydrolysates, their bio-functionality has not been studied yet. In this work amaranth grain Alb 1 and Glob were hydrolyzed (Alb 1H, Glob H) and foams and emulsions at optimal conditions (t, E/S, pH5) were prepared in order to assess techno-functional properties such as foaming (F) and emulsifying (E) (capacity (C) and stability (S)). FC and EC were much better for Glob H than for Alb H. Angiotensin I-converting enzyme-inhibitory activity was higher for Alb 1H (roughly 50 %) than that of Glob H (roughly 30 %). Scavenging of radicals activity (DPPH· or ABTS· (+) ) of Alb 1H and Glob H, at 2 mg/mL, was similar (approx. 40 %), but lower than Alb 1 (approx. 70 %), which was the best antioxidant. The low reducing power showed that hydrolysates barely donate an electron or hydrogen. Chelating activity on Cu(2+) was lower than that exhibited by Fe(2+,) which was remarkable, approx. 80 % as long as DH% > 10 %, where hydrolysates displayed high solubility (Alb 1H = 85 %, Glob H = 70 %) because of occurrence of 1-10 kDa peptides. Amaranth foams and emulsions prepared with protein hydrolysates have a potential as a nutraceutical food. PMID:25829587

  9. Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam

    PubMed Central

    Sawaya, Michael R.; Cascio, Duilio; Gingery, Mari; Rodriguez, Jose; Goldschmidt, Lukasz; Colletier, Jacques-Philippe; Messerschmidt, Marc M.; Boutet, Sébastien; Koglin, Jason E.; Williams, Garth J.; Brewster, Aaron S.; Nass, Karol; Hattne, Johan; Botha, Sabine; Doak, R. Bruce; Shoeman, Robert L.; DePonte, Daniel P.; Park, Hyun-Woo; Federici, Brian A.; Sauter, Nicholas K.; Schlichting, Ilme; Eisenberg, David S.

    2014-01-01

    It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information. PMID:25136092

  10. Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

    PubMed

    Pleschberger, Magdalena; Neubauer, Angela; Egelseer, Eva M; Weigert, Stefan; Lindner, Brigitte; Sleytr, Uwe B; Muyldermans, Serge; Sára, Margit

    2003-01-01

    Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology. PMID:12643755

  11. Influence of plasma total antioxidant ability on lipid and protein oxidation products in plasma and erythrocyte ghost obtained from developing and adult rats pretreated with two vitamin K formulations.

    PubMed

    Hadi, Ansari Hadipour; Abdolamir, Allameh; Mahtab, Hajhaidari; Abolfazl, Dadkhah; Yusef, Rasmi

    2004-12-01

    Ferric reducing ability of plasma (FRAP), as an index of total antioxidant capacity of plasma was found to be enhanced significantly (p < 0.05) in suckling rats pretreated either with vitamin K1 (28, 56 or 84 mg/kg/3 days) or menadione (vitamin K3) at a dose of 15 mg/kg b.w./3 days. The effect of vitamin K1 on FRAP was dose-dependent and it was inversely related to the formation of lipid peroxidation products in plasma as judged by thiobarbituric acid reacting substances (TBARS). Lack of influence of the drugs on FRAP in adults was corroborated with elevation in the levels of plasma TBARS. Possible role of FRAP on the rate of lipid peroxidation and protein oxidation (protein carbonyls) on erythrocyte membrane was also investigated following isolation of erythrocyte ghost from control and treated rats. Vitamin K1 as well as menadione failed to change the levels of protein carbonyls in erythrocyte ghost obtained from both the age groups. Analysis of major erythrocyte membrane proteins, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) substantiated these results. In spite of higher antioxidant capacity of plasma and erythrocytes obtained from young rats, the rate of lipid peroxidation in erythrocyte ghost preparation was also high in this age group (p < 0.05). These results suggest that the drug-related induction in FRAP occurs only in immature animals as a part of protective mechanism against lipid peroxidation products generated in plasma. PMID:15663201

  12. Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence

    PubMed Central

    Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B.; Puckett, Latisha M.; Phan, N. H.; Starch, Joel A.; Okimoto, Mark R.; Gidden, Jennifer; Stites, Wesley E.; Lay, Jackson O.

    2012-01-01

    Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a “snapshot” of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔGH2O. In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured

  13. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

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

  14. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Obtaining and maintaining funding

    SciTech Connect

    Beverly Hartline

    1996-04-01

    Obtaining and maintaining funding is important for individuals, groups, institutions, and fields. This challenge is easier during times of abundant and growing resources than it is now, when funding is tight and shrinking. Thus, to obtain and maintain funding will require: maintaining healthy funding levels for all of science; maintaining healthy funding levels for the field(s) you work in; and competing successfully for the available funds. Everyone should pay attention to the overall prospects for science funding and dedicate some effort to working with others to grow the constituency for science. Public support is likely an important prerequisite for keeping future science budgets high. In this context, researchers should share with society at large the benefits of their research, so that taxpayers can see and appreciate some return from the federal investment in science. Assuming this effort is successful, and there continue to be government and private organizations with substantial resources to invest in research, what can the individual investigator do to improve her chances? She can be clear about her goal(s) and carefully plan her effort to make maximum progress for minimum resources, especially early in her career while she is establishing a solid professional reputation. Specific useful strategies include: brainstorm funding options and select the most promising one(s); be persistent but flexible, responsive to new information and changing circumstances; provide value and assistance to prospective funding sources both before and after receiving funding; know the funding agents and what their goals are, they are the customers; promise a lot and always deliver more; build partnerships and collaboration to leverage interest and resources; and develop capabilities and ideas with a promising, irresistible future. There is no guarantee of success. For the best chances, consistently contribute positively and productively in all your efforts, and continue to

  16. A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

    PubMed

    Ilk, Nicola; Küpcü, Seta; Moncayo, Gerald; Klimt, Sigrid; Ecker, Rupert C; Hofer-Warbinek, Renate; Egelseer, Eva M; Sleytr, Uwe B; Sára, Margit

    2004-04-15

    The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis. PMID:14725506

  17. A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.

    PubMed Central

    Ilk, Nicola; Küpcü, Seta; Moncayo, Gerald; Klimt, Sigrid; Ecker, Rupert C; Hofer-Warbinek, Renate; Egelseer, Eva M; Sleytr, Uwe B; Sára, Margit

    2004-01-01

    The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis. PMID:14725506

  18. Employment Obtaining and Business Starting

    ERIC Educational Resources Information Center

    Lan, Jian

    2009-01-01

    The implementation of business starting education in higher vocational colleges is of important and realistic meanings for cultivating advanced technology application-type talents and for releasing the employment obtaining pressure of higher vocational students. Based on the analysis on the employment situation of higher vocational graduates, this…

  19. Obtaining Public Records: Reporter Guide

    ERIC Educational Resources Information Center

    Hammond, Betsy

    2013-01-01

    Obtaining public records is essential to covering public education. Fortunately, the law is on the side of reporters: Public agencies generally must disclose their records to the public and to the media--with important exceptions. Public agencies are often reluctant to hand over records, however, even when the law clearly says they should.…

  20. Truncated States Obtained by Iteration

    NASA Astrophysics Data System (ADS)

    Cardoso B., W.; Almeida G. de, N.

    2008-02-01

    We introduce the concept of truncated states obtained via iterative processes (TSI) and study its statistical features, making an analogy with dynamical systems theory (DST). As a specific example, we have studied TSI for the doubling and the logistic functions, which are standard functions in studying chaos. TSI for both the doubling and logistic functions exhibit certain similar patterns when their statistical features are compared from the point of view of DST.

  1. Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification.

    PubMed

    Völlenkle, Christine; Weigert, Stefan; Ilk, Nicola; Egelseer, Eva; Weber, Viktoria; Loth, Fritz; Falkenhagen, Dieter; Sleytr, Uwe B; Sára, Margit

    2004-03-01

    The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA(31-1068)/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-microm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm(2), whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm(2) was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system. PMID:15006773

  2. 48 CFR 1353.107 - Obtaining forms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Obtaining forms. 1353.107 Section 1353.107 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CLAUSES AND FORMS FORMS General 1353.107 Obtaining forms. The DOC forms may be obtained from any DOC contracting office....

  3. 48 CFR 1353.107 - Obtaining forms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Obtaining forms. 1353.107 Section 1353.107 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CLAUSES AND FORMS FORMS General 1353.107 Obtaining forms. The DOC forms may be obtained from any DOC contracting office....

  4. 48 CFR 1353.107 - Obtaining forms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Obtaining forms. 1353.107 Section 1353.107 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CLAUSES AND FORMS FORMS General 1353.107 Obtaining forms. The DOC forms may be obtained from any DOC contracting office....

  5. 48 CFR 1353.107 - Obtaining forms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Obtaining forms. 1353.107 Section 1353.107 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CLAUSES AND FORMS FORMS General 1353.107 Obtaining forms. The DOC forms may be obtained from any DOC contracting office....

  6. 48 CFR 1353.107 - Obtaining forms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Obtaining forms. 1353.107 Section 1353.107 Federal Acquisition Regulations System DEPARTMENT OF COMMERCE CLAUSES AND FORMS FORMS General 1353.107 Obtaining forms. The DOC forms may be obtained from any DOC contracting office....

  7. SOFIA Observatory Obtains 'First Light' Images

    NASA Video Gallery

    NASA's Stratospheric Observatory for Infrared Astronomy, or SOFIA, successfully obtained its "First Light"" images during an overnight flight May 26. Scientists are now processing the data gathered...

  8. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    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.

  9. 47 CFR 54.615 - Obtaining services.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 3 2011-10-01 2011-10-01 false Obtaining services. 54.615 Section 54.615 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES (CONTINUED) UNIVERSAL SERVICE Universal Service Support for Health Care Providers § 54.615 Obtaining services. (a) Selecting a provider. In selecting...

  10. 42 CFR 442.101 - Obtaining certification.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... section states the requirements for obtaining notice of an ICF/MR's certification before a Medicaid agency... Secretary for an ICF/MR located on an Indian reservation. (c) The agency must obtain notice of certification... provisions pertains to the ICF/MR: (1) An ICF/MR meets the conditions of participation set forth in subpart...

  11. Astrophysical Reaction Rates Obtained By Indirect Techniques

    SciTech Connect

    Tribble, R. E.; Al-Abdullah, T.; Alharbi, A.; Banu, A.; Chen, X.; Clark, H. L.; Fu, C.; Gagliardi, C. A.; Hardy, J. C.; Iacob, V. E.; Lui, Y.-W.; McCleskey, M.; Mukhamedzhanov, A.; Nica, N.; Park, H. I.; Roeder, B.; Simmons, E.; Tabacaru, G.; Tokimoto, Y.; Trache, L.

    2010-08-12

    Indirect techniques have been used to obtain information about reaction rates for several proton capture reactions that occur on short-lived nuclei. The techniques used to carry out the measurements are reviewed and the results obtained are presented. Also future prospects for further measurements with a new facility, T-REX are discussed.

  12. 33 CFR 118.20 - Obtaining information.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Obtaining information. 118.20 Section 118.20 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES BRIDGE LIGHTING AND OTHER SIGNALS § 118.20 Obtaining information. Persons desiring information concerning...

  13. 38 CFR 21.5725 - Obtaining benefits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Obtaining benefits. 21... benefits. (a) Actions required of the individual. In order to obtain benefits under the educational assistance and subsistence allowance program, an individual must— (1) File a claim for benefits with VA,...

  14. 33 CFR 118.20 - Obtaining information.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Obtaining information. 118.20 Section 118.20 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES BRIDGE LIGHTING AND OTHER SIGNALS § 118.20 Obtaining information. Persons desiring information concerning...

  15. 38 CFR 21.5725 - Obtaining benefits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2014-07-01 2014-07-01 false Obtaining benefits. 21... benefits. (a) Actions required of the individual. In order to obtain benefits under the educational assistance and subsistence allowance program, an individual must— (1) File a claim for benefits with VA,...

  16. 38 CFR 21.5725 - Obtaining benefits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2013-07-01 2013-07-01 false Obtaining benefits. 21... benefits. (a) Actions required of the individual. In order to obtain benefits under the educational assistance and subsistence allowance program, an individual must— (1) File a claim for benefits with VA,...

  17. 38 CFR 21.5725 - Obtaining benefits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2012-07-01 2012-07-01 false Obtaining benefits. 21... benefits. (a) Actions required of the individual. In order to obtain benefits under the educational assistance and subsistence allowance program, an individual must— (1) File a claim for benefits with VA,...

  18. 38 CFR 21.5725 - Obtaining benefits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Obtaining benefits. 21... benefits. (a) Actions required of the individual. In order to obtain benefits under the educational assistance and subsistence allowance program, an individual must— (1) File a claim for benefits with VA,...

  19. Ready Reference. How To Obtain an ISBN; How To Obtain an ISSN; How To Obtain an SAN.

    ERIC Educational Resources Information Center

    Koltay, Emery

    2003-01-01

    These three articles describe ISBNs (International Standard Book Numbers); ISSNs (International Standard Serial Numbers); and SANs (Standard Address Numbers), for organizations served by the book industry; and explains how to apply to obtain the appropriate numbers. (LRW)

  20. 25 CFR 162.539 - Must I obtain a WEEL before obtaining a WSR lease?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... AND PERMITS Wind and Solar Resource Leases Wsr Leases § 162.539 Must I obtain a WEEL before obtaining... direct result of energy resource information gathered from a WEEL activity, obtaining a WEEL is not...

  1. 25 CFR 162.539 - Must I obtain a WEEL before obtaining a WSR lease?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... AND PERMITS Wind and Solar Resource Leases Wsr Leases § 162.539 Must I obtain a WEEL before obtaining... direct result of energy resource information gathered from a WEEL activity, obtaining a WEEL is not...

  2. 40 CFR 35.6305 - Obtaining supplies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ....6325 through 35.6340, and 35.6350. Supplies obtained with Core Program funds must be for non-site-specific purposes. All purchases of supplies under the Core Program must comply with the requirements...

  3. 47 CFR 54.615 - Obtaining services.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... provided under § 54.621, that the requester cannot obtain toll-free access to an Internet service provider... thing of value; (6) If the service or services are being purchased as part of an aggregated...

  4. 47 CFR 54.615 - Obtaining services.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... provided under § 54.621, that the requester cannot obtain toll-free access to an Internet service provider... thing of value; (6) If the service or services are being purchased as part of an aggregated...

  5. Mass culture of photobacteria to obtain luciferase

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Rich, E., Jr.

    1969-01-01

    Inoculating preheated trays containing nutrient agar with photobacteria provides a means for mass culture of aerobic microorganisms in order to obtain large quantities of luciferase. To determine optimum harvest time, growth can be monitored by automated light-detection instrumentation.

  6. 48 CFR 53.107 - Obtaining forms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... (see 41 CFR 101-26.302). Standard forms adapted for computer preparation (see 53.105) or with special... Government Printing Office (GPO). (b) Contractors and other parties may obtain standard and optional...

  7. Treatment of biomass to obtain ethanol

    DOEpatents

    Dunson, Jr., James B.; Elander, Richard T.; Tucker, III, Melvin P.; Hennessey, Susan Marie

    2011-08-16

    Ethanol was produced using biocatalysts that are able to ferment sugars derived from treated biomass. Sugars were obtained by pretreating biomass under conditions of high solids and low ammonia concentration, followed by saccharification.

  8. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  9. SOY PROTEIN NANOPARTICLES AND NANOCOMPOSITES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein isolate (SPI) is obtained from soybean by removing soybean oil and soy carbohydrates. SPI contains more than 90% protein. Structurally, SPI is a globular protein and its aggregates in water consist of sphere-like protein particles. The number average aggregate size of SPI at pH=5.2 is...

  10. A monomeric L-aspartase obtained by in vitro selection.

    PubMed

    Kong, Xiangduo; Li, Zhengqiang; Gou, Xiaojun; Zhu, Shizhen; Zhang, Hongying; Wang, Xiaoping; Zhang, Jin

    2002-07-01

    By mimicking the partial spatial structure of the dimer of the l-aspartase subunit, the central ten-helix bundle, and an "active site" between the cleft of domain 1 (D1) and domain 3 (D3) from different subunits, we designed l-aspartase variants, in which D1D2 and D2D3 were ligated with a random hexapeptide loop. As expected, we obtained the variant with the highest activity (relative activity is 21.3% of the native enzyme, named as drAsp017) by in vitro selection. The molecular weight of this variant, obtained from size-exclusion column chromatography, is about 81 kDa, which indicates that it is indeed a monomer, whereas native l-aspartase is a tetramer. The activity-reversibility of drAsp017 (10(-7) m) was 80% after incubation for 30 min at 50 degrees C, while native enzyme only retained about 17% under the same conditions. Reactivation of drAsp017 denatured in 4 m guanidine HCl was independent of protein concentration at up to 20 x 10(-8) m at 25 degrees C, whereas the protein concentration of native enzyme strongly affected its reactivation under the above conditions. The sensitivity of drAsp017 (10(-7) m) to effective factors in the fumarate-amination reaction compared with native enzyme was also determined. Half-saturating concentrations of the activator l-aspartate and Mg2+ for drAsp017 (0.8 and 0.5 mm, respectively) are much higher than that of the native enzyme (0.10 and 0.15 mm, respectively). The data show that a monomeric l-aspartase is obtained by in vitro selection. Thus, the conversion of oligomeric proteins into their functional monomers could have important applications. PMID:11983692

  11. 15 CFR 285.15 - Obtaining documents.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...; phone: 301-975-4016; fax: 301-926-2884; e-mail: nvlap@nist.gov. (b) Copies of all ISO/IEC documents are..., Gaithersburg, MD. For access to the NIST campus, please contact NVLAP by phone at 301-975-4016 or by e-mail at NVLAP@nist.gov to obtain instructions for visitor registration....

  12. Obtaining Your License: Careers in Real Estate.

    ERIC Educational Resources Information Center

    Lyon, Robert

    Two steps are required to obtain a real estate salesperson's license in Texas: (1) selecting a broker to serve as an advisor, and (2) meeting personal requirements (at least 18 years old, a Texas resident, completion of a minimum of 12 semester hours of real estate and related courses, application, acceptable score on state exam, and payment of…

  13. 15 CFR 285.15 - Obtaining documents.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Obtaining documents. 285.15 Section 285.15 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, DEPARTMENT OF COMMERCE ACCREDITATION AND ASSESSMENT PROGRAMS...

  14. Obtaining Funding and Support for Undergraduate Research

    ERIC Educational Resources Information Center

    Dorff, Michael; Narayan, Darren A.

    2013-01-01

    Over the past decade there has been a dramatic increase in undergraduate research activities at colleges and universities nationwide. However, this comes at a time when budgets are being tightened and some institutions do not have the resources to pursue new initiatives. In this article we present some ideas for obtaining funding and support for…

  15. 40 CFR 761.208 - Obtaining manifests.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... print the manifest under 40 CFR 262.21 (c) and (e). A registered source may be a: (i) State agency; (ii) Commercial printer; (iii) PCB waste generator, transporter or, designated facility; or (iv) PCB waste broker... PROHIBITIONS PCB Waste Disposal Records and Reports § 761.208 Obtaining manifests. (a)(1) A generator may...

  16. 40 CFR 761.208 - Obtaining manifests.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... print the manifest under 40 CFR 262.21 (c) and (e). A registered source may be a: (i) State agency; (ii) Commercial printer; (iii) PCB waste generator, transporter or, designated facility; or (iv) PCB waste broker... PROHIBITIONS PCB Waste Disposal Records and Reports § 761.208 Obtaining manifests. (a)(1) A generator may...

  17. 42 CFR 442.101 - Obtaining certification.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 4 2012-10-01 2012-10-01 false Obtaining certification. 442.101 Section 442.101 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS STANDARDS FOR PAYMENT TO NURSING FACILITIES AND INTERMEDIATE...

  18. 42 CFR 442.101 - Obtaining certification.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 4 2013-10-01 2013-10-01 false Obtaining certification. 442.101 Section 442.101 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS STANDARDS FOR PAYMENT TO NURSING FACILITIES AND INTERMEDIATE...

  19. 42 CFR 442.101 - Obtaining certification.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Obtaining certification. 442.101 Section 442.101 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS STANDARDS FOR PAYMENT TO NURSING FACILITIES AND INTERMEDIATE...

  20. Method of Obtaining Uniform Coatings on Graphite

    DOEpatents

    Campbell, I. E.

    1961-04-01

    A method is given for obtaining uniform carbide coatings on graphite bodies. According to the invention a metallic halide in vapor form is passed over the graphite body under such conditions of temperature and pressure that the halide reacts with the graphite to form a coating of the metal carbide on the surface of the graphite.

  1. METHOD OF OBTAINING UNIFORM COATINGS ON GRAPHITE

    DOEpatents

    Campbell, I.E.

    1961-04-01

    A method is given for obtaining uniform carbide coatings on graphite bodies. According to the invention a metallic halide in vapor form is passed over the graphite body under such conditions of temperature and pressure that the halide reacts with the graphite to form a coating of the metal carbide on the surface of the graphite.

  2. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  3. How to obtain confirmation for revalidation.

    PubMed

    Middleton, Lyn; Llewellyn, Denise

    2016-07-27

    Rationale and key points This is the seventh in a series of eight articles providing information about the Nursing and Midwifery Council revalidation process. This article focuses on obtaining confirmation for revalidation. » Revalidation is a mandatory process for nurses and midwives, enabling them to demonstrate their ability to practise safely and effectively. » Confirmation provides assurance that nurses and midwives have met the requirements of revalidation. » Confirmation does not involve making judgements about whether a nurse or midwife is fit to practise. Reflective activity 'How to' revalidate articles can help to update your practice and provide information about the revalidation process, including how you can obtain confirmation for revalidation. Reflect on and write a short account of: 1. The information that nurses and midwives are required to collect to meet the revalidation requirements. 2. How you could use this article to educate your colleagues. Subscribers can upload their reflective accounts at: rcni.com/portfolio. PMID:27461327

  4. Processing to obtain high-purity gallium

    NASA Astrophysics Data System (ADS)

    Bautista, Renato G.

    2003-03-01

    Gallium has become increasingly popular as a substrate material for electronic devices. Aside from ore, gallium can be obtained from such industrial sources as the Bayer process caustic liquor that is a byproduct of bauxite processing, flue dust removed from the fume-collection system in plants that produce aluminum by the electrolytic process, zinc refinery residues, gallium scrap materials, and coal fly ash. The purification process for gallium can start with solvent-extraction processes where the concentrations of impurities, especially metals, are reduced to the ppm range. This article describes how ultra-purification techniques can be employed to reduce the undesirable impurities to the low ppb range. The various procedures described give an idea as to the extent of work needed to obtain and prepare high-purity gallium for electronic application.

  5. Obtaining growth hormone from calf blood

    NASA Technical Reports Server (NTRS)

    Kalchev, L. A.; Ralchev, K. K.; Nikolov, I. T.

    1979-01-01

    The preparation of a growth hormone from human serum was used for the isolation of the hormone from calf serum. The preparation was biologically active - it increased the quantity of the free fatty acids released in rat plasma by 36.4 percent. Electrophoresis in Veronal buffer, ph 8.6, showed the presence of a single fraction having mobility intermediate between that of alpha and beta globulins. Gel filtration through Sephadex G 100 showed an elutriation curve identical to that obtained by the growth hormone prepared from pituitary glands.

  6. Obtaining information by dynamic (effortful) touching.

    PubMed

    Turvey, M T; Carello, Claudia

    2011-11-12

    Dynamic touching is effortful touching. It entails deformation of muscles and fascia and activation of the embedded mechanoreceptors, as when an object is supported and moved by the body. It is realized as exploratory activities that can vary widely in spatial and temporal extents (a momentary heft, an extended walk). Research has revealed the potential of dynamic touching for obtaining non-visual information about the body (e.g. limb orientation), attachments to the body (e.g. an object's height and width) and the relation of the body both to attachments (e.g. hand's location on a grasped object) and surrounding surfaces (e.g. places and their distances). Invariants over the exploratory activity (e.g. moments of a wielded object's mass distribution) seem to ground this 'information about'. The conception of a haptic medium as a nested tensegrity structure has been proposed to express the obtained information realized by myofascia deformation, by its invariants and transformations. The tensegrity proposal rationalizes the relative indifference of dynamic touch to the site of mechanical contact (hand, foot, torso or probe) and the overtness of exploratory activity. It also provides a framework for dynamic touching's fractal nature, and the finding that its degree of fractality may matter to its accomplishments. PMID:21969694

  7. Obtaining useful information from expert based sources.

    PubMed Central

    Slawson, D. C.; Shaughnessy, A. F.

    1997-01-01

    Clinicians rely heavily on expert based systems-consultation with colleagues, journal reviews and textbooks, and continuing education activities-to obtain new information. The usefulness of sources such as these depends on the relevance and validity of the information and the work it takes to obtain it. Useful information can be distinguished from the useless by asking three questions: Does the information focus on an outcome that my patients care about? Is the issue common to my practice, and is the intervention feasible? If the information is true, will it require me to change my practice? If the answer to all three questions is yes, then the information is a common POEM (patient oriented evidence that matters), capable of improving the lives of your patients and must be evaluated for validity. Conclusions based on results of well designed clinical trials are more likely to be valid than those drawn from observations based on experience in clinical practice. Both members of the team, clinicians and experts, must take responsibility for their respective roles. PMID:9099121

  8. Obtaining information by dynamic (effortful) touching

    PubMed Central

    Turvey, M. T.; Carello, Claudia

    2011-01-01

    Dynamic touching is effortful touching. It entails deformation of muscles and fascia and activation of the embedded mechanoreceptors, as when an object is supported and moved by the body. It is realized as exploratory activities that can vary widely in spatial and temporal extents (a momentary heft, an extended walk). Research has revealed the potential of dynamic touching for obtaining non-visual information about the body (e.g. limb orientation), attachments to the body (e.g. an object's height and width) and the relation of the body both to attachments (e.g. hand's location on a grasped object) and surrounding surfaces (e.g. places and their distances). Invariants over the exploratory activity (e.g. moments of a wielded object's mass distribution) seem to ground this ‘information about’. The conception of a haptic medium as a nested tensegrity structure has been proposed to express the obtained information realized by myofascia deformation, by its invariants and transformations. The tensegrity proposal rationalizes the relative indifference of dynamic touch to the site of mechanical contact (hand, foot, torso or probe) and the overtness of exploratory activity. It also provides a framework for dynamic touching's fractal nature, and the finding that its degree of fractality may matter to its accomplishments. PMID:21969694

  9. Acoustic barriers obtained from industrial wastes.

    PubMed

    Garcia-Valles, M; Avila, G; Martinez, S; Terradas, R; Nogués, J M

    2008-07-01

    Acoustic pollution is an environmental problem that is becoming increasingly more important in our society. Likewise, the accumulation of generated waste and the need for waste management are also becoming more and more pressing. In this study we describe a new material--called PROUSO--obtained from industrial wastes. PROUSO has a variety of commercial and engineering, as well as building, applications. The main raw materials used for this environmentally friendly material come from slag from the aluminium recycling process, dust from the marble industry, foundry sands, and recycled expanded polystyrene from recycled packaging. Some natural materials, such as plastic clays, are also used. To obtain PROUSO we used a conventional ceramic process, forming new mineral phases and incorporating polluted elements into the structure. Its physical properties make PROUSO an excellent acoustic and thermal insulation material. It absorbs 95% of the sound in the frequency band of the 500 Hz. Its compressive strength makes it ideal for use in ceramic wall building. PMID:18514765

  10. Obtaining railpad properties via dynamic mechanical analysis

    NASA Astrophysics Data System (ADS)

    Oregui, M.; de Man, A.; Woldekidan, M. F.; Li, Z.; Dollevoet, R.

    2016-02-01

    In this paper, we propose combining dynamic mechanical analysis (DMA) and the time-temperature superposition principle to determine various railpad dynamic properties. Having accurate information regarding the dynamic properties of a railpad is a fundamental requirement for designing tracks and understanding track deterioration. By testing three different railpad types, we demonstrate that the dynamic behavior of railpads over a wide frequency range can be successfully obtained under different preloads and temperatures if time-temperature superposition can be applied. To investigate railpad aging, worn railpads taken from a mainline in the Netherlands are tested. In this case, worn railpads are softer and possess a lower damping capacity than new railpads. In addition to performing these measurements, a Prony series material model is proposed to reproduce the dynamic behavior of railpads. The Prony series model is in good agreement with the measurements. Measured railpad dynamic properties and the corresponding Prony series numerical model provide valuable information for track design and modeling.

  11. First results obtained by RUNJOB campaign

    NASA Astrophysics Data System (ADS)

    Kamioka, E.; Apanasenko, A. V.; Berezovskaya, V. A.; Fujii, M.; Fukuda, T.; Hareyama, M.; Hashimoto, G.; Ichimura, M.; Kobayashi, T.; Kopenkin, V.; Kuramata, S.; Lapshin, V. I.; Managadze, A. K.; Matsutani, H.; Misnikova, N. P.; Misu, T.; Mukhamedshin, R. A.; Nakamura, A.; Namiki, M.; Nanjo, H.; Nikolsky, S. I.; Ogura, K.; Ohta, S.; Oshuev, D. S.; Publichencko, P. A.; Rakobolskaya, I. V.; Roganova, T. M.; Sazhina, G. P.; Semba, H.; Shibata, T.; Shiota, T.; Sugimoto, H.; Sveshnikova, L. G.; Taran, V. M.; Watanabe, Z.; Yajima, N.; Yamagami, T.; Yashin, I. V.; Zamchalova, E. A.; Zatsepin, G. T.; Zayarnaya, I. S.; Runjob Collaboration ( Russia-Nippon Joint Balloon-Program)

    We report experimental results obtained by using a wide-gap type emulsion chamber flown in the first Japanese-Russo joint balloon project, called RUNJOB ( RUssia- Nippon JOint Balloon-program). Two balloons were launched from Kamchatka in July 1995, and both were recovered successfully near the Volga River. The exposure time was 130 hours for the first flight and 168 hours for the second. The mean ceiling altitude, in both flights, was 32 km corresponding to 10 g/cm 2. Total area of the emulsion chamber was 0.8 m 2, and the thickness 0.385 and 2.28 collision m.f.p.'s for vertically incident proton- and iron-primaries, respectively. We detected 381 showers using Fuji-#200-type X-ray film; of these 174 showers were due to atmospheric secondary γ-rays, and the rest 207 came from nuclear components. The energy range covers 20˜200 TeV for proton-primary, 3˜30 TeV/nucleon for helium-primary, and 0.7˜5 TeV/nucleon for iron-primary. We give the energy spectra for various elements (proton, helium, …, iron) as well as the all-particle spectrum and the average mass of the cosmic-ray primaries.

  12. Plasticity characteristics obtained through hardness measurement

    SciTech Connect

    Milman, Y.V.; Galanov, B.A.; Chugunova, S.I. )

    1993-09-01

    A characteristic of material plasticity [delta][sub H] is proposed. [delta][sub H] is determined as a part of plastic deformation in the total deformation during indentation. The following analytic expressions for the elastic deformation [epsilon][sub e] and for the total deformation [epsilon] on the contact area indenter-specimen in the direction of loading force are obtained, [epsilon][sub e] = 1.08(1 [minus] v [minus] 2v[sup 2])H[sub V/E], [epsilon] [approx] 0.076, where H[sub V] is the Vickers hardness, E is Young's modulus, v is the Poisson ratio, and [delta][sub H] = 1 [minus] ([epsilon][sub e]/[epsilon]). The [delta][sub H] value is calculated for various crystalline materials at different temperatures and in different structural states. [delta][sub H] is consistent with the concept of plasticity established before, and to characterize the influence of temperature, alloying and strain hardening on plasticity. The necessary condition for revealing ductility at tension and bending is [delta][sub H] [>=] 0.9. [delta][sub H] can be used as a plasticity characteristic of brittle materials.

  13. Utilizing therapists to obtain death penalty verdicts.

    PubMed

    Weinstock, R

    1994-01-01

    As a result of recent decisions by the United States and California Supreme Courts, therapists now have been placed in a position in which they can be forced to testify in death penalty cases for the only purposes of achieving a conviction and a death penalty verdict. Zeal for the death penalty seems to have overcome any concern for the ethics of psychiatrists or even for the welfare of society. In California, therapists can now be forced to testify against their own patients in capital cases even if the patient does not tender his mental state as an issue, despite the presence of a psychotherapist-patient privilege in the state for criminal matters. In California, the only option for therapists who wish to treat potentially dangerous patients may be to conduct the therapy under the umbrella of attorney-client privilege. Otherwise they may not be able to avoid serious ethical problems and personal danger if the patient actually does kill someone during or after therapy. They may be unable honestly and ethically to treat such patients without obtaining truly informed consent to therapy under these potentially "undercover policeman" circumstances. Hopefully, professional organizations will take a more activist position, and courts will appreciate the folly of these decisions and reverse them. Otherwise, they may spread to other states, for which California frequently sets precedents. PMID:8193387

  14. Bias annealing: A method for obtaining transition paths de novo

    NASA Astrophysics Data System (ADS)

    Hu, Jie; Ma, Ao; Dinner, Aaron R.

    2006-09-01

    Computational studies of dynamics in complex systems require means for generating reactive trajectories with minimum knowledge about the processes of interest. Here, we introduce a method for generating transition paths when an existing one is not already available. Starting from biased paths obtained from steered molecular dynamics, we use a Monte Carlo procedure in the space of whole trajectories to shift gradually to sampling an ensemble of unbiased paths. Application to basin-to-basin hopping in a two-dimensional model system and nucleotide-flipping by a DNA repair protein demonstrates that the method can efficiently yield unbiased reactive trajectories even when the initial steered dynamics differ significantly. The relation of the method to others and the physical basis for its success are discussed.

  15. Ligand Release Pathways Obtained with WExplore: Residence Times and Mechanisms.

    PubMed

    Dickson, Alex; Lotz, Samuel D

    2016-06-23

    The binding of ligands with their molecular receptors is of tremendous importance in biology. Although much emphasis has been placed on characterizing binding sites and bound poses that determine the binding thermodynamics, the pathway by which a ligand binds importantly determines the binding kinetics. The computational study of entire unbiased ligand binding and release pathways is still an emerging field, made possible only recently by advances in computational hardware and sampling methodologies. We have developed one such method (WExplore) that is based on a weighted ensemble of trajectories, which we apply to ligand release for the first time, using a set of three previously characterized interactions between low-affinity ligands and the protein FKBP-12 (FK-506 binding protein). WExplore is found to be more efficient that conventional sampling, even for the nanosecond-scale unbinding events observed here. From a nonequilibrium ensemble of unbinding trajectories, we obtain ligand residence times and release pathways without using biasing forces or a Markovian assumption of transitions between regions. We introduce a set of analysis tools for unbinding transition pathways, including using von Mises-Fisher distributions to model clouds of ligand exit points, which provide a quantitative proxy for ligand surface diffusion. Differences between the transition pathway ensembles of the three ligands are identified and discussed. PMID:27231969

  16. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  17. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  18. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  19. Component Repair Times Obtained from MSPI Data

    SciTech Connect

    Eide, Steven A.

    2015-05-01

    Information concerning times to repair or restore equipment to service given a failure is valuable to probabilistic risk assessments (PRAs). Examples of such uses in modern PRAs include estimation of the probability of failing to restore a failed component within a specified time period (typically tied to recovering a mitigating system before core damage occurs at nuclear power plants) and the determination of mission times for support system initiating event (SSIE) fault tree models. Information on equipment repair or restoration times applicable to PRA modeling is limited and dated for U.S. commercial nuclear power plants. However, the Mitigating Systems Performance Index (MSPI) program covering all U.S. commercial nuclear power plants provides up-to-date information on restoration times for a limited set of component types. This paper describes the MSPI program data available and analyzes the data to obtain median and mean component restoration times as well as non-restoration cumulative probability curves. The MSPI program provides guidance for monitoring both planned and unplanned outages of trains of selected mitigating systems deemed important to safety. For systems included within the MSPI program, plants monitor both train UA and component unreliability (UR) against baseline values. If the combined system UA and UR increases sufficiently above established baseline results (converted to an estimated change in core damage frequency or CDF), a “white” (or worse) indicator is generated for that system. That in turn results in increased oversight by the US Nuclear Regulatory Commission (NRC) and can impact a plant’s insurance rating. Therefore, there is pressure to return MSPI program components to service as soon as possible after a failure occurs. Three sets of unplanned outages might be used to determine the component repair durations desired in this article: all unplanned outages for the train type that includes the component of interest, only

  20. Total protein

    MedlinePlus

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  1. Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.

    PubMed

    Wilson, Kate E; Marouga, Rita; Prime, John E; Pashby, D Paul; Orange, Paul R; Crosier, Steven; Keith, Alexander B; Lathe, Richard; Mullins, John; Estibeiro, Peter; Bergling, Helene; Hawkins, Edward; Morris, Christopher M

    2005-10-01

    Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD. PMID:16145713

  2. Storage Proteins

    PubMed Central

    Fujiwara, Toru; Nambara, Eiji; Yamagishi, Kazutoshi; Goto, Derek B.; Naito, Satoshi

    2002-01-01

    Plants accumulate storage substances such as starch, lipids and proteins in certain phases of development. Storage proteins accumulate in both vegetative and reproductive tissues and serve as a reservoir to be used in later stages of plant development. The accumulation of storage protein is thus beneficial for the survival of plants. Storage proteins are also an important source of dietary plant proteins. Here, we summarize the genome organization and regulation of gene expression of storage protein genes in Arabidopsis. PMID:22303197

  3. Dietary Proteins

    MedlinePlus

    ... grains and beans. Proteins from meat and other animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Most plant proteins are incomplete. You should eat different types of plant proteins every day to get ...

  4. Protein Analysis

    NASA Astrophysics Data System (ADS)

    Chang, Sam K. C.

    Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure. Food proteins are very complex. Many have been purified and characterized. Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Daltons. They are composed of elements including hydrogen, carbon, nitrogen, oxygen, and sulfur. Twenty α-amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide bonds. Nitrogen is the most distinguishing element present in proteins. However, nitrogen content in various food proteins ranges from 13.4 to 19.1% (1) due to the variation in the specific amino acid composition of proteins. Generally, proteins rich in basic amino acids contain more nitrogen.

  5. Allergenic extracts from Metarhizium canisopliae: obtainment and characterization.

    PubMed

    Barbieri, R T; Croce, J; Gandra, R F; Gagete, E; Paula, C R; Gambale, W

    2005-01-01

    Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16th day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16th day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16th day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%); 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16th day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests. PMID:16047714

  6. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe” refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  7. How many protein-protein interactions types exist in nature?

    PubMed

    Garma, Leonardo; Mukherjee, Srayanta; Mitra, Pralay; Zhang, Yang

    2012-01-01

    "Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  8. Ultrahigh-resolution study of protein atomic displacement parameters at cryotemperatures obtained with a helium cryostat.

    PubMed

    Petrova, Tatiana; Ginell, Stephan; Mitschler, Andre; Hazemann, Isabelle; Schneider, Thomas; Cousido, Alexandra; Lunin, Vladimir Y; Joachimiak, Andrzej; Podjarny, Alberto

    2006-12-01

    Two X-ray data sets for a complex of human aldose reductase (h-AR) with the inhibitor IDD 594 and the cofactor NADP(+) were collected from two different parts of the same crystal to a resolution of 0.81 A at 15 and 60 K using cold helium gas as cryogen. The contribution of temperature to the atomic B values was estimated by comparison of the independently refined models. It was found that although being slightly different for different kinds of atoms, the differences (deltaB) in the isotropic equivalents B of atomic displacement parameters (ADPs) were approximately constant (about 1.7 A(2)) for well ordered atoms as the temperature was increased from 15 to 60 K. The mean value of this difference varied according to the number of non-H atoms covalently bound to the parent atom. Atoms having a B value of higher than 8 A(2) at 15 K showed much larger deviations of deltaB from the average value, which might reflect partial occupancy of atomic sites. An analysis of the anisotropy of ADPs for individual atoms revealed an increase in the isotropy of ADPs with the increase of the temperature from 15 to 60 K. In a separate experiment, a 0.93 A resolution data set was collected from a different crystal of the same complex at 100 K using cold nitrogen as a cryogen. The effects of various errors on the atomic B values were estimated by comparison of the refined models and the temperature-dependent component was inferred. It was found that both decreasing the data redundancy and increasing the resolution cutoff led to an approximately constant increase in atomic B values for well ordered atoms. PMID:17139089

  9. A COMPARISON OF CYTOKINE MESSAGE AND PROTEIN PROFILES OBTAINED FOLLOWING DERMAL EXPOSURE TO DIISOCYANATES.

    EPA Science Inventory

    Exposure to certain low molecular weight chemicals is associated with asthma. A simple method to identify this hazard is needed. Increased expression of Th2 cytokine mRNA in draining lymph nodes following dermal exposure and increased production of Th2 cytokines by cultured cell...

  10. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  11. Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus

    PubMed Central

    Tschiggerl, Helga; Breitwieser, Andreas; de Roo, Guy; Verwoerd, Theo; Scḧaffer, Christina; Sleytr, Uwe B.

    2015-01-01

    A fusion protein based on the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and the enzyme laminarinase (LamA) from Pyrococcus furiosus was designed and overexpressed in Escherichia coli. Due to the construction principle, the S-layer fusion protein fully retained the self-assembly capability of the S-layer moiety, while the catalytic domain of LamA remained exposed at the outer surface of the formed protein lattice. The enzyme activity of the S-layer fusion protein monolayer obtained upon recrystallization on silicon wafers, glass slides and different types of polymer membranes was determined colorimetrically and related to the activity of sole LamA that has been immobilized with conventional techniques. LamA aligned within the S-layer fusion protein lattice in a periodic and orientated fashion catalyzed twice the glucose release from the laminarin polysaccharide substrate in comparison to the randomly immobilized enzyme. In combination with the good shelf-life and the high resistance towards temperature and diverse chemicals, these novel composites are regarded a promising approach for site-directed enzyme immobilization. PMID:18035441

  12. Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen

    NASA Astrophysics Data System (ADS)

    Park, Jae Hyeon; Seo, Jeongeun; Jackman, Joshua A.; Cho, Nam-Joon

    2016-06-01

    Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species.

  13. Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen

    PubMed Central

    Park, Jae Hyeon; Seo, Jeongeun; Jackman, Joshua A.; Cho, Nam-Joon

    2016-01-01

    Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species. PMID:27302853

  14. Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen.

    PubMed

    Park, Jae Hyeon; Seo, Jeongeun; Jackman, Joshua A; Cho, Nam-Joon

    2016-01-01

    Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species. PMID:27302853

  15. MAMMOTH (matching molecular models obtained from theory): an automated method for model comparison.

    PubMed

    Ortiz, Angel R; Strauss, Charlie E M; Olmea, Osvaldo

    2002-11-01

    Advances in structural genomics and protein structure prediction require the design of automatic, fast, objective, and well benchmarked methods capable of comparing and assessing the similarity of low-resolution three-dimensional structures, via experimental or theoretical approaches. Here, a new method for sequence-independent structural alignment is presented that allows comparison of an experimental protein structure with an arbitrary low-resolution protein tertiary model. The heuristic algorithm is given and then used to show that it can describe random structural alignments of proteins with different folds with good accuracy by an extreme value distribution. From this observation, a structural similarity score between two proteins or two different conformations of the same protein is derived from the likelihood of obtaining a given structural alignment by chance. The performance of the derived score is then compared with well established, consensus manual-based scores and data sets. We found that the new approach correlates better than other tools with the gold standard provided by a human evaluator. Timings indicate that the algorithm is fast enough for routine use with large databases of protein models. Overall, our results indicate that the new program (MAMMOTH) will be a good tool for protein structure comparisons in structural genomics applications. MAMMOTH is available from our web site at http://physbio.mssm.edu/~ortizg/. PMID:12381844

  16. Total protein

    MedlinePlus

    ... 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 ...

  17. Whey Protein

    MedlinePlus

    ... shows that taking whey protein in combination with strength training increases lean body mass, strength, and muscle size. ... grams/kg of whey protein in combination with strength training for 6-10 weeks. For HIV/AIDS-related ...

  18. Method for obtaining high-resolution proteomic analysis from pericarps of guarana.

    PubMed

    Souza, A L; Angelo, P C S; Nogueira, P P O; Gonçalves, J F C; Franco, A M; Astolfi-Filho, S; López-Lozano, J L; Andrade, E V

    2014-01-01

    Guarana has great agricultural potential and is largely used therapeutically and in the production of non-alcoholic energy drinks. Genomic and proteomic studies are crucial to identify proteins that play central roles in the maintenance and viability of fruits, as well as to identify proteins related to the main metabolic pathways. However, the success of any protein analysis starts with the protein extract preparation, which needs to offer an extract that is free of contaminants. This study aimed to evaluate different extraction methods to obtain high-quantity and high-quality extracts that are compatible with analysis by 2-dimensional electrophoresis and tandem mass spectrometry protein identification. Three different methods were tested: trichloroacetic acid (TCA)/acetone, sodium dodecyl sulfate (SDS)/phenol, and polyvinylpolypyrrolidone (PVPP)/SDS/phenol. The extract obtained from the TCA/acetone precipitation presented low solubility and contamination with lipids and carbohydrates. On the other hand, the quality of the extract gradually improved after using phenol and PVPP/phenol, enabling a yield up to 2 mg/g macerated tissues and the detection of 457 spots by 2-dimensional electrophoresis. The effectiveness of the procedure used was validated by identification of 10 randomly selected proteins by mass spectrometry. The procedure described here can be a starting point for applications using tissues of other organs of guarana or tissues of species that are similar to guarana. PMID:25299116

  19. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

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

  20. Research on the Distant Hybrids of Wheat Obtained via Low-Energy Ion-Beam Implantation

    NASA Astrophysics Data System (ADS)

    Chang, Shouwei; Cheng, Yuhong; Qin, Guangyong; Su, Mingjie

    2003-06-01

    The whole DNA of soybean was implanted into four varieties of wheat of Zhongyu 5, Huaiyin 9628, Wenyou 1, Jimai 5 respectively via ion-beam mediation. There were 5 plants obtained whose protein content was higher than 18.5%, the highest one was 21.44%. There were 3 plants obtained whose protein content was lower than 11.5%, the lowest one was 10.96%. We can see that the whole DNA of soybean transformed into wheat via ion beam implantation can induce the increase in wheat protein content dramatically. The result also shows that the transformation efficiency of different gene types of wheat receptor varies greatly that the implanting time has a certain effect on the efficiency of transformation.

  1. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  2. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  3. Protein folds and protein folding

    PubMed Central

    Schaeffer, R. Dustin; Daggett, Valerie

    2011-01-01

    The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. PMID:21051320

  4. The Protein Data Bank

    PubMed Central

    Berman, Helen M.; Westbrook, John; Feng, Zukang; Gilliland, Gary; Bhat, T. N.; Weissig, Helge; Shindyalov, Ilya N.; Bourne, Philip E.

    2000-01-01

    The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource. PMID:10592235

  5. MYCOPLASMA GENITALIUM PROTEIN RESEMBLING THE MYCOPLASMA PNEUMONIAE ATTACHMENT PROTEIN

    EPA Science Inventory

    In previous studies with hyperimmune rabbit sera and monoclonal antibodies against P1 protein of M. pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of M. genitalium. ecause of biological and morphological similarities between these two hum...

  6. Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds.

    PubMed

    Durak, Agata; Baraniak, Barbara; Jakubczyk, Anna; Świeca, Michał

    2013-12-01

    This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC50=0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions. PMID:23870945

  7. Protein Dynamics

    NASA Astrophysics Data System (ADS)

    Frauenfelder, Hans

    2011-03-01

    Proteins combine properties of solids, liquids, and glasses. Schrödinger anticipated the main features of biomolecules long ago by stating that they had to be solid-like, but able to assume many different conformations. Indeed proteins can assume a gigantic number of conformational substates with the same primary sequence but different conformations. The different substates are described as craters in a very-high-dimensional energy landscape. The energy landscape is organized in a hierarchy of tiers, craters within craters within craters. Protein motions are pictured as transition between substates - jumps from crater to crater. Initially we assumed that these jumps were controlled by internal barriers between substates, but experiments have shown that nature selected a different approach. Proteins are surrounded by one to two layers of water and are embedded in a bulk solvent. Structural motions of the protein are controlled by the alpha fluctuations in the solvent surrounding the protein. Some internal motions most likely involving side chains are controlled electrostatically by beta fluctuations in the hydration shell. The dynamics of proteins is consequently dominated by the environment (H. Frauenfelder et al. PNAS 106, 5129 (2009). One can speculate that this organization permits exchange of information among biomolecules. The energy landscape is not just organized into two tiers, alpha and beta, but cryogenic experiments have revealed more tiers and protein more properties similar to that of glasses. While proteins function at ambient temperatures, cryogenic studies are necessary to understand the physics relevant for biology.

  8. 48 CFR 1815.403 - Obtaining cost or pricing data.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Obtaining cost or pricing... ADMINISTRATION CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Contract Pricing 1815.403 Obtaining cost or pricing data....

  9. 48 CFR 1815.403 - Obtaining cost or pricing data.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Obtaining cost or pricing... ADMINISTRATION CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Contract Pricing 1815.403 Obtaining cost or pricing data....

  10. 48 CFR 1815.403 - Obtaining cost or pricing data.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 6 2014-10-01 2014-10-01 false Obtaining cost or pricing... ADMINISTRATION CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Contract Pricing 1815.403 Obtaining cost or pricing data....

  11. 48 CFR 1815.403 - Obtaining cost or pricing data.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 6 2013-10-01 2013-10-01 false Obtaining cost or pricing... ADMINISTRATION CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Contract Pricing 1815.403 Obtaining cost or pricing data....

  12. 48 CFR 1815.403 - Obtaining cost or pricing data.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 6 2012-10-01 2012-10-01 false Obtaining cost or pricing... ADMINISTRATION CONTRACTING METHODS AND CONTRACT TYPES CONTRACTING BY NEGOTIATION Contract Pricing 1815.403 Obtaining cost or pricing data....

  13. How You Can Obtain Dollars for Needed Shop Facilities

    ERIC Educational Resources Information Center

    Levenson, Harold E.

    1976-01-01

    Tells where to get more information and application forms for grants and how to write proposals to obtain aid in expanding or building school shop facilities. Useful tips which may enable educators to obtain funding are also offered. (HD)

  14. Interfacial Protein-Protein Associations

    PubMed Central

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2014-01-01

    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 longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage. PMID:24274729

  15. 49 CFR 375.519 - Must I obtain weight tickets?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false Must I obtain weight tickets? 375.519 Section 375... Household Goods Weighing the Shipment § 375.519 Must I obtain weight tickets? (a) You must obtain weight... separate weight ticket for each weighing. The weigh master must sign each weight ticket. Each weight...

  16. 49 CFR 375.519 - Must I obtain weight tickets?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 5 2011-10-01 2011-10-01 false Must I obtain weight tickets? 375.519 Section 375... Household Goods Weighing the Shipment § 375.519 Must I obtain weight tickets? (a) You must obtain weight... separate weight ticket for each weighing. The weigh master must sign each weight ticket. Each weight...

  17. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may obtain, at their own expense, official...

  18. 32 CFR 806b.8 - Obtaining law enforcement records.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Obtaining law enforcement records. 806b.8... ADMINISTRATION PRIVACY ACT PROGRAM Obtaining Law Enforcement Records and Confidentiality Promises § 806b.8 Obtaining law enforcement records. The Commander, Air Force Office of Special Investigation; the...

  19. 5 CFR 2422.33 - Relief obtainable under part 2423.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 3 2011-01-01 2011-01-01 false Relief obtainable under part 2423. 2422... obtainable under part 2423. Remedial relief that was or could have been obtained as a result of a motion, objection, or challenge filed or raised under this subpart, may not be the basis for similar relief if...

  20. 47 CFR 1.8002 - Obtaining an FRN.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 1 2010-10-01 2010-10-01 false Obtaining an FRN. 1.8002 Section 1.8002 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE FCC Registration Number § 1.8002 Obtaining an FRN. (a) The FRN must be obtained by anyone doing business with the Commission, see 31 U.S.C. 7701(c)(2), including but...

  1. 47 CFR 1.8002 - Obtaining an FRN.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Obtaining an FRN. 1.8002 Section 1.8002 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE FCC Registration Number § 1.8002 Obtaining an FRN. (a) The FRN must be obtained by anyone doing business with the Commission, see 31 U.S.C. 7701(c)(2), including but...

  2. 48 CFR 22.1007 - Requirement to obtain wage determinations.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Requirement to obtain wage determinations. 22.1007 Section 22.1007 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION... Amended 22.1007 Requirement to obtain wage determinations. The contracting officer shall obtain...

  3. 40 CFR 35.6315 - Alternative methods for obtaining property.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... document a cost comparison analysis to determine which of these methods of obtaining equipment is the most... comparison analysis to EPA for approval. The recipient must obtain the equipment through the most cost..., the recipient may hire the services of a contractor. (ii) The recipient must obtain award...

  4. 40 CFR 35.6315 - Alternative methods for obtaining property.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... document a cost comparison analysis to determine which of these methods of obtaining equipment is the most... comparison analysis to EPA for approval. The recipient must obtain the equipment through the most cost..., the recipient may hire the services of a contractor. (ii) The recipient must obtain award...

  5. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  6. 21 CFR 862.1635 - Total protein test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Total protein test system. 862.1635 Section 862....1635 Total protein test system. (a) Identification. A total protein test system is a device intended to measure total protein(s) in serum or plasma. Measurements obtained by this device are used in...

  7. Therapeutic proteins: A to Z.

    PubMed

    Ozgur, Aykut; Tutar, Yusuf

    2013-12-01

    In recent years, therapeutic proteins have become an important growing class of drugs in the pharmaceutics industry. The development of recombinant DNA technology has caused to appreciation of therapeutic value of many proteins and peptides in medicine. Currently, approximately 100 therapeutic proteins obtained approval from Food and Drug Administration (FDA) and they are widely used in the treatment of various diseases such as cancer, diabetes, anemia and infections. This paper will summarize the production processes, pharmaceuticals and physicochemical properties and important classes of therapeutic proteins with their potential use in clinical applications. PMID:24261980

  8. Immunoprofiling Using NAPPA Protein Microarrays

    PubMed Central

    Sibani, Sahar; LaBaer, Joshua

    2012-01-01

    Protein microarrays provide an efficient method to immunoprofile patients in an effort to rapidly identify disease immunosignatures. The validity of using autoantibodies in diagnosis has been demonstrated in type 1 diabetes, rheumatoid arthritis, and systemic lupus, and is now being strongly considered in cancer. Several types of protein microarrays exist including antibody and antigen arrays. In this chapter, we describe the immunoprofiling application for one type of antigen array called NAPPA (nucleic acids programmable protein array). We provide a guideline for setting up the screening study and designing protein arrays to maximize the likelihood of obtaining quality data. PMID:21370064

  9. Natural proteins: Sources, isolation, characterization and applications.

    PubMed

    Nehete, Jitendra Y; Bhambar, Rajendra S; Narkhede, Minal R; Gawali, Sonali R

    2013-07-01

    Worldwide, plant protein contributes substantially as a food resource because it contains essential amino acids for meeting human physiological requirements. However, many versatile plant proteins are used as medicinal agents as they are produced by using molecular tools of biotechnology. Proteins can be obtained from plants, animals and microorganism cells. The abundant economical proteins can be obtained from plant seeds. These natural proteins are obtained by isolation procedures depending on the physicochemical properties of proteins. Isolation and purification of single protein from cells containing mixtures of unrelated proteins is achievable due to the physical and chemical attributes of proteins. The following characteristics are unique to each protein: Amino acid composition, sequence, subunit structures, size, shape, net charge, isoelectric point, solubility, heat stability and hydrophobicity. Based on these properties, various methods of isolation exist, like salting out and isoionic precipitation. Purification of proteins is quiet challenging and, therefore, several approaches like sodium dodecyl sulfate gel electrophoresis and chromatography are available. Characterization of proteins can be performed by mass spectrometry/liquid chromatography-mass spectrometry (LC-MS). The amino acid sequence of a protein can be detected by using tandem mass spectrometry. In this article, a review has been made on the sources, isolation, purification and characterization of natural proteins. PMID:24347918

  10. Obtaining structural and functional information for GPCRs using the substituted-cysteine accessibility method (SCAM).

    PubMed

    Liapakis, George

    2014-01-01

    G-protein coupled receptors (GPCRs) are proteins of the plasma membrane, which are characterized by seven membrane-spanning segments (TMs). GPCRs play an important role in almost all of our physiological and pathophysiological conditions by interacting with a large variety of ligands and stimulating different G-proteins and signaling cascades. By playing a key role in the function of our body and being involved in the pathophysiology of many disorders, GPCRs are very important therapeutic targets. Determination of the structure and function of GPCRs could advance the design of novel receptor-specific drugs against various diseases. A powerful method to obtain structural and functional information for GPCRs is the cysteine substituted accessibility method (SCAM). SCAM is used to systematically map the TM residues of GPCRs and determine their functional role. SCAM can also be used to determine differences in the structures of the TMs in different functional states of GPCRs. PMID:25335535

  11. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  12. Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

    PubMed

    Wylie, Benjamin J; Dzikovski, Boris G; Pawsey, Shane; Caporini, Marc; Rosay, Melanie; Freed, Jack H; McDermott, Ann E

    2015-04-01

    We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces. PMID:25828256

  13. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Philip D.; Wander, Marc J.

    2012-09-11

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  14. Amphiphiles for protein solubilization and stabilization

    DOEpatents

    Gellman, Samuel Helmer; Chae, Pil Seok; Laible, Phillip D; Wander, Marc J

    2014-11-04

    The invention provides amphiphiles for manipulating membrane proteins. The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety and/or in a lipophilic moiety. Such amphiphiles are useful as detergents for solubilization and stabilization of membrane proteins, including photosynthetic protein superassemblies obtained from bacterial membranes.

  15. Protein Translocation across the Rough Endoplasmic Reticulum

    PubMed Central

    Mandon, Elisabet C.; Trueman, Steven F.; Gilmore, Reid

    2013-01-01

    The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration. PMID:23251026

  16. Collapse transition in proteins.

    PubMed

    Ziv, Guy; Thirumalai, D; Haran, Gilad

    2009-01-01

    The coil-globule transition, a tenet of the physics of polymers, has been identified in recent years as an important unresolved aspect of the initial stages of the folding of proteins. We describe the basics of the collapse transition, starting with homopolymers and continuing with proteins. Studies of denatured-state collapse under equilibrium are then presented. An emphasis is placed on single-molecule fluorescence experiments, which are particularly useful for measuring properties of the denatured state even under conditions of coexistence with the folded state. Attempts to understand the dynamics of collapse, both theoretically and experimentally, are then described. Only an upper limit for the rate of collapse has been obtained so far. Improvements in experimental and theoretical methodology are likely to continue to push our understanding of the importance of the denatured-state thermodynamics and dynamics for protein folding in the coming years. PMID:19081910

  17. Whey Protein

    MedlinePlus

    ... intolerance, for replacing or supplementing milk-based infant formulas, and for reversing weight loss and increasing glutathione ( ... allergic reactions compared to infants who receive standard formula. However, taking why protein might not be helpful ...

  18. Photosystem II based multilayers obtained by electrostatic layer-by-layer assembly on quartz substrates.

    PubMed

    Ventrella, Andrea; Catucci, Lucia; Placido, Tiziana; Longobardi, Francesco; Agostiano, Angela

    2014-06-01

    Photosystem II (PSII) proteins from spinach leaves were immobilized onto quartz substrates according to the Layer-by-Layer (LbL) procedure, alternating protein to polyethylenimine (PEI) layers by exploiting electrostatic interactions. The effects of several factors, such as storage conditions, ageing of the PSII-modified substrates, as well as PSII concentration in buffer, on the quality of the prepared multilayers, were investigated by UV-vis Absorption Spectroscopy and Atomic Force Microscopy (AFM). A number of 13 layers was found to be optimal to guarantee intense PSII optical signals with homogeneous morphological distributions of proteins. The multilayers resulted stable if stored in contact with air at 4 °C, as observed by UV-vis Absorption spectra recorded after 48 h. The best results in terms of AFM images and electron transfer efficiency (measured by Hill Reaction assays) were gained by using 5.6 × 10(-7) M chlorophyll concentration, obtaining multilayers with the most ordered protein distributions and the highest electron transfer efficiency, i.e. 85% of an iso-absorbing PSII suspension. The results highlight the possibility to successfully immobilize PSII proteins, without considerable loss of bioactivity, thanks to the mild nature of the electrostatic LbL technique, opening up possibilities of applications in the bioelectrochemical energy conversion and biosensoristic fields. PMID:24550146

  19. LEA proteins prevent protein aggregation due to water stress

    PubMed Central

    Goyal, Kshamata; Walton, Laura J.; Tunnacliffe, Alan

    2005-01-01

    LEA (late embryogenesis abundant) proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock. However, although various functions of LEA proteins have been proposed, their precise role has not been defined. Recent bioinformatics studies suggest that LEA proteins might behave as molecular chaperones, and the current study was undertaken to test this hypothesis. Recombinant forms of AavLEA1, a group 3 LEA protein from the anhydrobiotic nematode Aphelenchus avenae, and Em, a group 1 LEA protein from wheat, have been subjected to functional analysis. Heat-stress experiments with citrate synthase, which is susceptible to aggregation at high temperatures, suggest that LEA proteins do not behave as classical molecular chaperones, but they do exhibit a protective, synergistic effect in the presence of the so-called chemical chaperone, trehalose. In contrast, both LEA proteins can independently protect citrate synthase from aggregation due to desiccation and freezing, in keeping with a role in water-stress tolerance; similar results were obtained with lactate dehydrogenase. This is the first evidence of anti-aggregation activity of LEA proteins due to water stress. Again, a synergistic effect of LEA and trehalose was observed, which is significant given that non-reducing disaccharides are known to accumulate during dehydration in plants and nematodes. A model is proposed whereby LEA proteins might act as a novel form of molecular chaperone, or ‘molecular shield’, to help prevent the formation of damaging protein aggregates during water stress. PMID:15631617

  20. Thermal Effusivity of Vegetable Oils Obtained by a Photothermal Technique

    NASA Astrophysics Data System (ADS)

    Cervantes-Espinosa, L. M.; de L. Castillo-Alvarado, F.; Lara-Hernández, G.; Cruz-Orea, A.; Hernández-Aguilar, C.; Domínguez-Pacheco, A.

    2014-10-01

    Thermal properties of several vegetable oils such as soy, corn, and avocado commercial oils were obtained by using a photopyroelectric technique. The inverse photopyroelectric configuration was used in order to obtain the thermal effusivity of the oil samples. The theoretical equation for the photopyroelectric signal in this configuration, as a function of the incident light modulation frequency, was fitted to the experimental data in order to obtain the thermal effusivity of these samples. The obtained results are in good agreement with the thermal effusivity reported for other vegetable oils. All measurements were done at room temperature.

  1. Ready Reference. Publishers' Toll-Free Telephone Numbers and Web Sites; How To Obtain an ISBN; How To Obtain an ISSN; How To Obtain an SAN.

    ERIC Educational Resources Information Center

    Koltay, Emery

    2002-01-01

    Includes four articles: one lists publishers' toll-free telephone numbers and Web sites; and the others explain how to obtain an ISBN (International Standard Book Number), an ISSN (International Standard Serial Number), and an SAN (Standard Address Number) for organizations involved in the book industry. (LRW)

  2. Antithrombotic and Antioxidant Activity of Amaranth Hydrolysate Obtained by Activation of an Endogenous Protease.

    PubMed

    Sabbione, Ana Clara; Ibañez, Sabrina M; Martínez, E Nora; Añón, María Cristina; Scilingo, Adriana A

    2016-06-01

    Ingestion of diets with antithrombotic and antioxidant components offer a convenient and effective way to prevent and reduce the incidence of cardiovascular diseases. The aim of the present work was to obtain an amaranth hydrolysate by the activation of an endogenous aspartic protease, to establish adequate experimental conditions, and to evaluate its antithrombotic and antioxidant activity in order to assess its potential application as an ingredient in functional foods. The results obtained not only confirmed the presence of an endogenous protease in the amaranth isolate, but also allowed us to select an adequate incubation conditions (pH 2, 40 °C, 16 h). The hydrolysate obtained (degree of hydrolysis 5.3 ± 0.4 %) showed potential antithrombotic activity (IC50 = 5.9 ± 0.1 mg soluble protein/mL) and had more antioxidant activity than the isolate, indicating that the activation of the protease released bioactive peptides from amaranth proteins. Decreasing the pH is a simple and cheap process and is another way to obtain potential functional ingredients with bioactive compounds. PMID:27023251

  3. 25 CFR 175.50 - Obtaining rights-of-way.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Obtaining rights-of-way. 175.50 Section 175.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN ELECTRIC POWER UTILITIES... utility's facilities, the customer shall be responsible for obtaining all rights-of-way necessary to...

  4. 40 CFR 270.100 - Who must obtain a RAP?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 28 2013-07-01 2013-07-01 false Who must obtain a RAP? 270.100 Section...) EPA ADMINISTERED PERMIT PROGRAMS: THE HAZARDOUS WASTE PERMIT PROGRAM Remedial Action Plans (RAPs) Applying for A Rap § 270.100 Who must obtain a RAP? When a facility or remediation waste management site...

  5. 40 CFR 270.100 - Who must obtain a RAP?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 27 2014-07-01 2014-07-01 false Who must obtain a RAP? 270.100 Section...) EPA ADMINISTERED PERMIT PROGRAMS: THE HAZARDOUS WASTE PERMIT PROGRAM Remedial Action Plans (RAPs) Applying for A Rap § 270.100 Who must obtain a RAP? When a facility or remediation waste management site...

  6. 40 CFR 270.100 - Who must obtain a RAP?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 28 2012-07-01 2012-07-01 false Who must obtain a RAP? 270.100 Section...) EPA ADMINISTERED PERMIT PROGRAMS: THE HAZARDOUS WASTE PERMIT PROGRAM Remedial Action Plans (RAPs) Applying for A Rap § 270.100 Who must obtain a RAP? When a facility or remediation waste management site...

  7. 40 CFR 270.100 - Who must obtain a RAP?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Who must obtain a RAP? 270.100 Section...) EPA ADMINISTERED PERMIT PROGRAMS: THE HAZARDOUS WASTE PERMIT PROGRAM Remedial Action Plans (RAPs) Applying for A Rap § 270.100 Who must obtain a RAP? When a facility or remediation waste management site...

  8. 40 CFR 270.100 - Who must obtain a RAP?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 27 2011-07-01 2011-07-01 false Who must obtain a RAP? 270.100 Section...) EPA ADMINISTERED PERMIT PROGRAMS: THE HAZARDOUS WASTE PERMIT PROGRAM Remedial Action Plans (RAPs) Applying for A Rap § 270.100 Who must obtain a RAP? When a facility or remediation waste management site...

  9. 7 CFR 1421.14 - Obtaining peanut loans.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Obtaining peanut loans. 1421.14 Section 1421.14... peanut loans. (a) Peanuts loans to individual producers may be obtained through: (1) County offices; or... presented for disbursement unless the peanuts pledged as collateral for the marketing assistance loan...

  10. 7 CFR 1421.14 - Obtaining peanut loans.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Obtaining peanut loans. 1421.14 Section 1421.14... peanut loans. (a) Peanuts loans to individual producers may be obtained through: (1) County offices; or... presented for disbursement unless the peanuts pledged as collateral for the marketing assistance loan...

  11. 7 CFR 1421.14 - Obtaining peanut loans.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Obtaining peanut loans. 1421.14 Section 1421.14... peanut loans. (a) Peanuts loans to individual producers may be obtained through: (1) County offices; or... presented for disbursement unless the peanuts pledged as collateral for the marketing assistance loan...

  12. 7 CFR 1421.14 - Obtaining peanut loans.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Obtaining peanut loans. 1421.14 Section 1421.14... peanut loans. (a) Peanuts loans to individual producers may be obtained through: (1) County offices; or... presented for disbursement unless the peanuts pledged as collateral for the marketing assistance loan...

  13. 7 CFR 1421.14 - Obtaining peanut loans.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Obtaining peanut loans. 1421.14 Section 1421.14... peanut loans. (a) Peanuts loans to individual producers may be obtained through: (1) County offices; or... presented for disbursement unless the peanuts pledged as collateral for the marketing assistance loan...

  14. 16 CFR 4.8 - Costs for obtaining Commission records.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Costs for obtaining Commission records. 4.8 Section 4.8 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE MISCELLANEOUS RULES § 4.8 Costs for obtaining Commission records. (a) Definitions. For the purpose of...

  15. 25 CFR 175.50 - Obtaining rights-of-way.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Obtaining rights-of-way. 175.50 Section 175.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN ELECTRIC POWER UTILITIES Rights-of-Way § 175.50 Obtaining rights-of-way. Where there is no existing right(s)-of-way for...

  16. 25 CFR 175.50 - Obtaining rights-of-way.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Obtaining rights-of-way. 175.50 Section 175.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN ELECTRIC POWER UTILITIES Rights-of-Way § 175.50 Obtaining rights-of-way. Where there is no existing right(s)-of-way for...

  17. 25 CFR 175.50 - Obtaining rights-of-way.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Obtaining rights-of-way. 175.50 Section 175.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN ELECTRIC POWER UTILITIES Rights-of-Way § 175.50 Obtaining rights-of-way. Where there is no existing right(s)-of-way for...

  18. 25 CFR 175.50 - Obtaining rights-of-way.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Obtaining rights-of-way. 175.50 Section 175.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN ELECTRIC POWER UTILITIES Rights-of-Way § 175.50 Obtaining rights-of-way. Where there is no existing right(s)-of-way for...

  19. 36 CFR 905.735-504 - Procedures for obtaining statements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Procedures for obtaining statements. 905.735-504 Section 905.735-504 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION STANDARDS OF CONDUCT Statements of Employment and Financial Interests § 905.735-504 Procedures for obtaining statements. (a)...

  20. 20 CFR 327.15 - Reasonable efforts to obtain work.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... INSURANCE ACT AVAILABLE FOR WORK § 327.15 Reasonable efforts to obtain work. (a) Requirement. A claimant may... not covered by the act, who may reasonably be expected to have openings in work suitable for him; (4... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Reasonable efforts to obtain work....

  1. Obtaining the Electron Angular Momentum Coupling Spectroscopic Terms, jj

    ERIC Educational Resources Information Center

    Orofino, Hugo; Faria, Roberto B.

    2010-01-01

    A systematic procedure is developed to obtain the electron angular momentum coupling (jj) spectroscopic terms, which is based on building microstates in which each individual electron is placed in a different m[subscript j] "orbital". This approach is similar to that used to obtain the spectroscopic terms under the Russell-Saunders (LS) coupling…

  2. 24 CFR 3286.307 - Process for obtaining trainer's qualification.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Process for obtaining trainer's...-Administered States § 3286.307 Process for obtaining trainer's qualification. (a) Where to apply. An applicant for qualification as a trainer must provide the applicant's legal name, address, and telephone...

  3. 32 CFR 564.40 - Procedures for obtaining medical care.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... that failure to promptly report the occurrence of a disease or injury may result in the loss of medical... 32 National Defense 3 2010-07-01 2010-07-01 true Procedures for obtaining medical care. 564.40... RESERVES NATIONAL GUARD REGULATIONS Medical Attendance and Burial § 564.40 Procedures for obtaining...

  4. 22 CFR 92.80 - Obtaining American vital statistics records.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 22 Foreign Relations 1 2013-04-01 2013-04-01 false Obtaining American vital statistics records. 92... statistics records. Individuals who inquire as to means of obtaining copies of or extracts from American... Vital Statistics Office at the place where the record is kept, which is usually in the capital city...

  5. 22 CFR 92.80 - Obtaining American vital statistics records.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 22 Foreign Relations 1 2014-04-01 2014-04-01 false Obtaining American vital statistics records. 92... statistics records. Individuals who inquire as to means of obtaining copies of or extracts from American... Vital Statistics Office at the place where the record is kept, which is usually in the capital city...

  6. 22 CFR 92.80 - Obtaining American vital statistics records.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Obtaining American vital statistics records. 92... statistics records. Individuals who inquire as to means of obtaining copies of or extracts from American... Vital Statistics Office at the place where the record is kept, which is usually in the capital city...

  7. 48 CFR 209.105-1 - Obtaining information.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Performance Information Retrieval System (PPIRS), available at http://www.ppirs.gov. Information relating to... 48 Federal Acquisition Regulations System 3 2014-10-01 2014-10-01 false Obtaining information. 209....105-1 Obtaining information. (1) For guidance on using the Exclusions section of the System for...

  8. 48 CFR 209.105-1 - Obtaining information.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Information Retrieval System (PPIRS), available at http://www.ppirs.gov. Information relating to contract... 48 Federal Acquisition Regulations System 3 2012-10-01 2012-10-01 false Obtaining information. 209....105-1 Obtaining information. (1) For guidance on using the Excluded Parties List System, see PGI...

  9. 48 CFR 209.105-1 - Obtaining information.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Performance Information Retrieval System (PPIRS), available at http://www.ppirs.gov. Information relating to... 48 Federal Acquisition Regulations System 3 2013-10-01 2013-10-01 false Obtaining information. 209....105-1 Obtaining information. (1) For guidance on using the System for Award Management Exclusions,...

  10. 13 CFR 142.36 - Can I obtain judicial review?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Can I obtain judicial review? 142.36 Section 142.36 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION PROGRAM FRAUD CIVIL REMEDIES ACT REGULATIONS Decisions and Appeals § 142.36 Can I obtain judicial review? If the...

  11. 12 CFR 792.06 - Can I obtain other records?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Can I obtain other records? 792.06 Section 792.06 Banks and Banking NATIONAL CREDIT UNION ADMINISTRATION REGULATIONS AFFECTING THE OPERATIONS OF THE... Act Records Available Upon Request § 792.06 Can I obtain other records? Except with respect to...

  12. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  13. 9 CFR 2.52 - How to obtain tags.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false How to obtain tags. 2.52 Section 2.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE ANIMAL WELFARE REGULATIONS Identification of Animals § 2.52 How to obtain tags. Dealers or exhibitors may...

  14. 48 CFR 209.105-1 - Obtaining information.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Obtaining information. 209....105-1 Obtaining information. (1) For guidance on using the Excluded Parties List System, see PGI 209... Information Retrieval System (PPIRS), available at http://www.ppirs.gov. Information relating to...

  15. 48 CFR 509.105-1 - Obtaining information.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Obtaining information. 509.105-1 Section 509.105-1 Federal Acquisition Regulations System GENERAL SERVICES ADMINISTRATION... Obtaining information. (a) From a prospective contractor. FAR 9.105-1 lists a number of sources...

  16. 48 CFR 9.105-1 - Obtaining information.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Obtaining information. 9... information. (a) Before making a determination of responsibility, the contracting officer shall possess or obtain information sufficient to be satisfied that a prospective contractor currently meets...

  17. A method for obtaining coefficients of compositional inverse generating functions

    NASA Astrophysics Data System (ADS)

    Kruchinin, Dmitry V.; Shablya, Yuriy V.; Kruchinin, Vladimir V.; Shelupanov, Alexander A.

    2016-06-01

    The aim of this paper is to show how to obtain expressions for coefficients of compositional inverse generating functions in explicit way. The method is based on the Lagrange inversion theorem and composita of generating functions. Also we give a method of obtaining expressions for coefficients of reciprocal generating functions and consider some examples.

  18. A New Method for Obtaining Russell-Saunders Terms

    ERIC Educational Resources Information Center

    Liu, Ying; Liu, Yue; Liu, Bihui

    2011-01-01

    A new method for obtaining Russell-Saunders terms of atomic configurations is reported. This new method is significantly different from, while at the same time complementary to, previously published methods for obtaining atomic terms. This novel procedure is elicited by the method used to determine the splitting of S, P, D terms in weak ligand…

  19. 32 CFR 806b.8 - Obtaining law enforcement records.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Obtaining law enforcement records. The Commander, Air Force Office of Special Investigation; the Commander... 32 National Defense 6 2010-07-01 2010-07-01 false Obtaining law enforcement records. 806b.8 Section 806b.8 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR...

  20. 46 CFR 70.35-5 - Where obtainable.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...: For Federal Register citations affecting § 70-35-5, see the List of CFR Sections Affected, which... 46 Shipping 3 2012-10-01 2012-10-01 false Where obtainable. 70.35-5 Section 70.35-5 Shipping COAST... of Shipping's Standards § 70.35-5 Where obtainable. (a) The standards established by the...

  1. 46 CFR 188.35-5 - Where obtainable.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Where obtainable. 188.35-5 Section 188.35-5 Shipping... PROVISIONS American Bureau of Shipping's Standards § 188.35-5 Where obtainable. (a) The standards established... also be examined at the Office of the Commandant (CG-CVC), U.S. Coast Guard, 2100 2nd St. SW.,...

  2. 46 CFR 70.35-5 - Where obtainable.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 3 2011-10-01 2011-10-01 false Where obtainable. 70.35-5 Section 70.35-5 Shipping COAST... of Shipping's Standards § 70.35-5 Where obtainable. (a) The standards established by the American... Office of the Commandant (CG-52), 2100 2nd St., SW., Stop 7126, Washington, DC 20593-7126, or at...

  3. 46 CFR 188.35-5 - Where obtainable.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Where obtainable. 188.35-5 Section 188.35-5 Shipping... PROVISIONS American Bureau of Shipping's Standards § 188.35-5 Where obtainable. (a) The standards established... also be examined at the Office of the Commandant (CG-543), U.S. Coast Guard, 2100 2nd St. SW.,...

  4. 46 CFR 70.35-5 - Where obtainable.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 3 2010-10-01 2010-10-01 false Where obtainable. 70.35-5 Section 70.35-5 Shipping COAST... of Shipping's Standards § 70.35-5 Where obtainable. (a) The standards established by the American... Office of the Commandant (CG-52), 2100 2nd St., SW., Stop 7126, Washington, DC 20593-7126, or at...

  5. 46 CFR 90.35-5 - Where obtainable.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Where obtainable. 90.35-5 Section 90.35-5 Shipping COAST... American Bureau of Shipping's Standards § 90.35-5 Where obtainable. The standards established by the... Office of the Commandant (CG-5PS), 2100 2nd St. SW., Stop 7126, Washington, DC 20593-7126, or at...

  6. 46 CFR 90.35-5 - Where obtainable.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Where obtainable. 90.35-5 Section 90.35-5 Shipping COAST... American Bureau of Shipping's Standards § 90.35-5 Where obtainable. (a) The standards established by the... Office of the Commandant (CG-52), 2100 2nd St. SW., Stop 7126, Washington, DC 20593-7126, or at...

  7. 46 CFR 90.35-5 - Where obtainable.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Where obtainable. 90.35-5 Section 90.35-5 Shipping COAST... American Bureau of Shipping's Standards § 90.35-5 Where obtainable. (a) The standards established by the... Office of the Commandant (CG-52), 2100 2nd St. SW., Stop 7126, Washington, DC 20593-7126, or at...

  8. 46 CFR 188.35-5 - Where obtainable.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Where obtainable. 188.35-5 Section 188.35-5 Shipping... PROVISIONS American Bureau of Shipping's Standards § 188.35-5 Where obtainable. (a) The standards established... also be examined at the Office of the Commandant (CG-543), U.S. Coast Guard, 2100 2nd St. SW.,...

  9. Full complex spectrum simultaneous obtaining SD-OCT

    NASA Astrophysics Data System (ADS)

    Gelikonov, Grigory V.; Gelikonov, Valentin M.; Shilyagin, Pavel A.

    2011-03-01

    An efficient technique of simultaneous obtaining of quadrature spectral components of interference signal in spectrometer-based OCT is proposed. The components are obtained in air-spaced non-polarization interferometer by partition of reference beam onto two parts and using an achromatic phase shifter. Several setups of phase sifter are described and compared.

  10. 5 CFR 1501.12 - Obtaining further information.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 3 2010-01-01 2010-01-01 false Obtaining further information. 1501.12 Section 1501.12 Administrative Personnel THE INTERNATIONAL ORGANIZATIONS EMPLOYEES LOYALTY BOARD OPERATIONS OF THE INTERNATIONAL ORGANIZATIONS EMPLOYEES LOYALTY BOARD § 1501.12 Obtaining further...