WorldWideScience

Sample records for tandem affinity purified

  1. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

    Science.gov (United States)

    Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

    2009-06-01

    In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

  2. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    Directory of Open Access Journals (Sweden)

    Renata Angeli

    2009-01-01

    Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

  3. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...

  4. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture...... of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

  5. Profiling of Parkin-binding partners using tandem affinity purification.

    Directory of Open Access Journals (Sweden)

    Alessandra Zanon

    Full Text Available Parkinson's disease (PD is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2 are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells, we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

  6. Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

    Science.gov (United States)

    Blankenburg, Hagen; Doncheva, Nadezhda T.; Schwienbacher, Christine; Serafin, Alice; Alexa, Adrian; Weichenberger, Christian X.; Albrecht, Mario; Klein, Christine; Hicks, Andrew A.; Pramstaller, Peter P.

    2013-01-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function. PMID:24244333

  7. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    International Nuclear Information System (INIS)

    Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

    1987-01-01

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific 32 P-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect

  8. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    Directory of Open Access Journals (Sweden)

    Chin-Soon Chee

    2014-01-01

    Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

  9. Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

    Science.gov (United States)

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.

  10. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  11. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Directory of Open Access Journals (Sweden)

    Hua-zhen Wang

    Full Text Available Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP could greatly increase the soluble expression level of Glucokinase (GlcK, α-Amylase (Amy and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  12. An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes

    OpenAIRE

    Cipak, Lubos; Spirek, Mario; Novatchkova, Maria; Chen, Zhiming; Rumpf, Cornelia; Lugmayr, Wolfgang; Mechtler, Karl; Ammerer, Gustav; Csaszar, Edina; Gregan, Juraj

    2009-01-01

    Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP-tagging of predicted Schizosaccharomyces pombe genes (http://mendel.imp.ac.at/Pombe_tagging/). To validate the approach...

  13. Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These e......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets....... These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play...

  14. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

    DEFF Research Database (Denmark)

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan

    2010-01-01

    lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice...... channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation....

  15. Immobilized metal affinity chromatography co-purifies TGF-β1 with histidine-tagged recombinant extracellular proteins.

    Directory of Open Access Journals (Sweden)

    Jasvir Kaur

    Full Text Available Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC. Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls.

  16. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

    Science.gov (United States)

    Labriola, Jonathan M; daCosta, Corrie J B; Wang, Shuzhi; Figeys, Daniel; Smith, Jeffrey C; Sturgeon, R Michel; Baenziger, John E

    2010-04-02

    Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

  17. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against α-transforming growth factor

    International Nuclear Information System (INIS)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-01-01

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human α-transforming growth factor (α-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting α-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native α-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of α-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of α-TGF has a cellular role beyond that of an autocrine growth factor

  18. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against. cap alpha. -transforming growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-04-07

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human ..cap alpha..-transforming growth factor (..cap alpha..-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting ..cap alpha..-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native ..cap alpha..-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of ..cap alpha..-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of ..cap alpha..-TGF has a cellular role beyond that of an autocrine growth factor.

  19. Probing the Structure of Affinity-Purified and Lipid-Reconstituted Torpedo Nicotinic Acetylcholine Receptor†

    Science.gov (United States)

    Hamouda, Ayman K.; Chiara, David C.; Blanton, Michael P.; Cohen, Jonathan B.

    2010-01-01

    The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand gated ion channels (LGIC) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical/biophysical techniques, detergent-solubilization, purification, and lipid-reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to compare the state-dependent photolabeling of Torpedo nAChR before and after purification and reincorporation into lipid. For purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9 and M2-13, the agonist-enhanced labeling of δThr274 (δM2-18) within the δ subunit helix bundle, and the labeling at the lipid-protein interface (αM4) were the same as for nAChR in native membranes. However, addition of agonist did not enhance [125I]TID photolabeling of δIle288 within the δM2-M3 loop. These results indicate that after purification and reconstitution of Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the δM2-M3 loop. To further characterize the pharmacology of [125I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [125I]TID photolabeling. PCP inhibited [125I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6), while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the δ subunit helix bundle. PMID:18991407

  20. Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

    Science.gov (United States)

    Hamouda, Ayman K; Chiara, David C; Blanton, Michael P; Cohen, Jonathan B

    2008-12-02

    The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

  1. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Rodero M

    2002-01-01

    Full Text Available In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3 has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis. The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 mg/ml of antigenic concentration.

  3. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    International Nuclear Information System (INIS)

    Lazarovici, P.; Yavin, E.

    1986-01-01

    The pharmacokinetic interaction of an affinity-purified 125 I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10 -9 M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 0 C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 0 C but not at 37 0 C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin

  4. Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

    Science.gov (United States)

    Cai, Bingjing; Wang, Haiying; Zheng, Huiru; Wang, Hui

    2015-01-01

    Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

  5. Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

    Science.gov (United States)

    Fairburn, B; Muthana, M; Hopkinson, K; Slack, L K; Mirza, S; Georgiou, A S; Espigares, E; Wong, C; Pockley, A G

    2006-09-01

    The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and

  6. Interdomain Linker Determines Primarily the Structural Stability of Dystrophin and Utrophin Tandem Calponin-Homology Domains Rather than Their Actin-Binding Affinity.

    Science.gov (United States)

    Bandi, Swati; Singh, Surinder M; Mallela, Krishna M G

    2015-09-08

    Tandem calponin-homology (CH) domains are the most common actin-binding domains in proteins. However, structural principles underlying their function are poorly understood. These tandem domains exist in multiple conformations with varying degrees of inter-CH-domain interactions. Dystrophin and utrophin tandem CH domains share high sequence similarity (∼82%), yet differ in their structural stability and actin-binding affinity. We examined whether the conformational differences between the two tandem CH domains can explain differences in their stability and actin binding. Dystrophin tandem CH domain is more stable by ∼4 kcal/mol than that of utrophin. Individual CH domains of dystrophin and utrophin have identical structures but differ in their relative orientation around the interdomain linker. We swapped the linkers between dystrophin and utrophin tandem CH domains. Dystrophin tandem CH domain with utrophin linker (DUL) has similar stability as that of utrophin tandem CH domain. Utrophin tandem CH domain with dystrophin linker (UDL) has similar stability as that of dystrophin tandem CH domain. Dystrophin tandem CH domain binds to F-actin ∼30 times weaker than that of utrophin. After linker swapping, DUL has twice the binding affinity as that of dystrophin tandem CH domain. Similarly, UDL has half the binding affinity as that of utrophin tandem CH domain. However, changes in binding free energies due to linker swapping are much lower by an order of magnitude compared to the corresponding changes in unfolding free energies. These results indicate that the linker region determines primarily the structural stability of tandem CH domains rather than their actin-binding affinity.

  7. Discovery of protein complexes with core-attachment structures from Tandem Affinity Purification (TAP) data.

    Science.gov (United States)

    Wu, Min; Li, Xiao-Li; Kwoh, Chee-Keong; Ng, See-Kiong; Wong, Limsoon

    2012-09-01

    Many cellular functions involve protein complexes that are formed by multiple interacting proteins. Tandem Affinity Purification (TAP) is a popular experimental method for detecting such multi-protein interactions. However, current computational methods that predict protein complexes from TAP data require converting the co-complex relationships in TAP data into binary interactions. The resulting pairwise protein-protein interaction (PPI) network is then mined for densely connected regions that are identified as putative protein complexes. Converting the TAP data into PPI data not only introduces errors but also loses useful information about the underlying multi-protein relationships that can be exploited to detect the internal organization (i.e., core-attachment structures) of protein complexes. In this article, we propose a method called CACHET that detects protein complexes with Core-AttaCHment structures directly from bipartitETAP data. CACHET models the TAP data as a bipartite graph in which the two vertex sets are the baits and the preys, respectively. The edges between the two vertex sets represent bait-prey relationships. CACHET first focuses on detecting high-quality protein-complex cores from the bipartite graph. To minimize the effects of false positive interactions, the bait-prey relationships are indexed with reliability scores. Only non-redundant, reliable bicliques computed from the TAP bipartite graph are regarded as protein-complex cores. CACHET constructs protein complexes by including attachment proteins into the cores. We applied CACHET on large-scale TAP datasets and found that CACHET outperformed existing methods in terms of prediction accuracy (i.e., F-measure and functional homogeneity of predicted complexes). In addition, the protein complexes predicted by CACHET are equipped with core-attachment structures that provide useful biological insights into the inherent functional organization of protein complexes. Our supplementary material can

  8. TANDEM

    Data.gov (United States)

    Federal Laboratory Consortium — The Tandem Van de Graaff facility provides researchers with beams of more than 40 different types of ions - atoms that have been stripped of their electrons. One of...

  9. Recent relaxation of purifying selection on the tandem-repetitive early-stage histone H3 gene in brooding sea stars.

    Science.gov (United States)

    Foltz, David W; Mah, Christopher L

    2009-06-01

    Patterns of nucleotide substitution differ between marine species that have a pelagic feeding (planktotrophic) larval stage and related species that lack such a stage, for both adaptive and non-adaptive reasons. Here, patterns of nucleotide and inferred amino acid substitution are analyzed for the tandem-repetitive early-stage histone H3 gene in 36 sea star species of the order Forcipulatida with documented larval habitat. The relative rate of nonsynonymous substitution (expressed as ω=d(N)/d(S)) was significantly higher in lineages with a brooded non-feeding (lecithotrophic) larval form than in lineages with a planktotrophic larval form. There was also a significant excess of conservative over radical substitutions. The increase in ω for brooders as compared to non-brooders was much greater than for previously analyzed mitochondrial sequences in echinoderms. These data are consistent with the hypothesis that purifying selection on this gene has been relaxed in brooding lineages compared to non-brooding lineages. The hypotheses of adaptive or neutral evolution are less plausible, although recent pseudogenization following a period of relaxed purifying selection could also explain the results.

  10. [Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification].

    Science.gov (United States)

    Shestakova, E A; Boutin, M; Bourassa, S; Bonneil, E; Bijl, J J

    2017-01-01

    Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9

  11. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    Science.gov (United States)

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  12. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

    Science.gov (United States)

    Shamashkin, Michael; Godavarti, Ranga; Iskra, Timothy; Coffman, Jon

    2013-10-01

    A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation. Copyright © 2013 Wiley Periodicals, Inc.

  13. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Methods for Purifying Enzymes for Mycoremediation

    Science.gov (United States)

    Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

    2014-01-01

    A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

  15. Purifying Nanomaterials

    Science.gov (United States)

    Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

    2014-01-01

    A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

  16. Ultra-fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up.

    Science.gov (United States)

    Yang, Xihui; Hu, Yichen; Kong, Weijun; Chu, Xianfeng; Yang, Meihua; Zhao, Ming; Ouyang, Zhen

    2014-11-01

    A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ≥ 0.9993), low limit of detection (0.5-0.8 μg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. A highly selective dispersive liquid-liquid microextraction approach based on the unique fluorous affinity for the extraction and detection of per- and polyfluoroalkyl substances coupled with high performance liquid chromatography tandem-mass spectrometry.

    Science.gov (United States)

    Wang, Juan; Shi, Yali; Cai, Yaqi

    2018-04-06

    In the present study, a highly selective fluorous affinity-based dispersive liquid-liquid microextraction (DLLME) technique was developed for the extraction and analysis of per- and polyfluoroalkyl substances (PFASs) followed by high performance liquid chromatography tandem-mass spectrometry. Perfluoro-tert-butanol with multiple C-F bonds was chosen as the extraction solvent, which was injected into the aqueous samples with a dispersive solvent (acetonitrile) in a 120:800 (μL, v/v) mixture for PFASs enrichment. The fluorous affinity-based extraction mechanism was confirmed by the significantly higher extraction recoveries for PFASs containing multiple fluorine atoms than those for compounds with fewer or no fluorine atoms. The extraction recoveries of medium and long-chain PFASs (CF 2  > 5) exceeded 70%, except perfluoroheptanoic acid, while those of short-chain PFASs were lower than 50%, implying that the proposed DLLME may not be suitable for their extraction due to weak fluorous affinity. This highly fluoroselective DLLME technique can greatly decrease the matrix effect that occurs in mass spectrometry detection when applied to the analysis of urine samples. Under the optimum conditions, the relative recoveries of PFASs with CF 2  > 5 ranged from 80.6-121.4% for tap water, river water and urine samples spiked with concentrations of 10, 50 and 100 ng/L. The method limits of quantification for PFASs in water and urine samples were in the range of 0.6-8.7 ng/L. Furthermore, comparable concentrations of PFASs were obtained via DLLME and solid-phase extraction, confirming that the developed DLLME technique is a promising method for the extraction of PFASs in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography.

    Science.gov (United States)

    Aono, Hiromasa; Wen, Dingy; Zang, Li; Houde, Damian; Pepinsky, R Blake; Evans, David R H

    2010-08-06

    Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG(1) monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG(1) mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG(1) mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG(1) molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture. Copyright 2010 Elsevier B.V. All rights reserved.

  19. Evaluation of immobilized metal-ion affinity chromatography and electrospray ionization tandem mass spectrometry for recovery and identification of copper(II-binding ligands in seawater using the model ligand 8-hydroxyquinoline

    Directory of Open Access Journals (Sweden)

    Richard L Nixon

    2016-11-01

    Full Text Available Complexation by organic ligands dominates the speciation of iron (Fe, copper (Cu, and other bioactive trace metals in seawater, controlling their bioavailability and distribution in the marine environment. Several classes of high-affinity Fe-binding ligands (siderophores have been identified in seawater but the chemical structures of marine Cu-complexing ligands remain unknown. Immobilized metal-ion affinity chromatography (IMAC allows Cu ligands to be isolated from bulk dissolved organic matter (DOM in seawater and separated into fractions which can be characterized independently using electrochemical and spectroscopic techniques. Attempts have been made to combine IMAC with electrospray ionization mass spectrometry (ESI-MS to characterize marine Cu ligands, but results have proven inconclusive due to the lack of tandem mass spectrometry (MS/MS data to confirm ligand recovery. We used 8-hydroxyquinoline (8-HQ, a well-characterized model ligand that forms strong 1:2 metal:ligand complexes with Cu2+ at pH 8 (log β2 = 18.3, to evaluate Cu(II-IMAC and ESI-MS/MS for recovery and identification of copper(II-complexing ligands in seawater. One-litre samples of 0.45µm-filtered surface seawater were spiked with 8-HQ at low concentrations (up to 100 nM and fractionated by IMAC. Fractions eluted with acidified artificial seawater were desalted and re-suspended in methanol via solid-phase extraction (SPE to obtain extracts suitable for ESI-MS analysis. Recovery of 8-HQ by Cu(II-IMAC was confirmed unambiguously by MS/MS and found to average 81% based upon accurate quantitation via multiple reaction monitoring (MRM. Cu(II-IMAC fractionation of unspiked seawater using multiple UV detection wavelengths suggests an optimal fraction size of 2 mL for isolating and analyzing Cu ligands with similar properties.

  20. Handbook of purified gases

    CERN Document Server

    Schoen, Helmut

    2015-01-01

    Technical gases are used in almost every field of industry, science and medicine and also as a means of control by government authorities and institutions and are regarded as indispensable means of assistance. In this complete handbook of purified gases the physical foundations of purified gases and mixtures as well as their manufacturing, purification, analysis, storage, handling and transport are presented in a comprehensive way. This important reference work is accompanied with a large number of Data Sheets dedicated to the most important purified gases.  

  1. Evaluation of Immobilized Metal-Ion Affinity Chromatography and Electrospray Ionization Tandem Mass Spectrometry for Recovery and Identification of Copper(II)-Binding Ligands in Seawater Using the Model Ligand 8-Hydroxyquinoline

    OpenAIRE

    Nixon, Richard L.; Ross, Andrew R. S.

    2016-01-01

    Complexation by organic ligands dominates the speciation of iron (Fe), copper (Cu), and other bioactive trace metals in seawater, controlling their bioavailability and distribution in the marine environment. Several classes of high-affinity Fe-binding ligands (siderophores) have been identified in seawater but the chemical structures of marine Cu-complexing ligands remain unknown. Immobilized metal-ion affinity chromatography (IMAC) allows Cu ligands to be isolated from bulk dissolved organic...

  2. Purified water quality study

    International Nuclear Information System (INIS)

    Spinka, H.; Jackowski, P.

    2000-01-01

    Argonne National Laboratory (HEP) is examining the use of purified water for the detection medium in cosmic ray sensors. These sensors are to be deployed in a remote location in Argentina. The purpose of this study is to provide information and preliminary analysis of available water treatment options and associated costs. This information, along with the technical requirements of the sensors, will allow the project team to determine the required water quality to meet the overall project goals

  3. Individual Water Purifier Study

    Science.gov (United States)

    2007-11-01

    study participants (Appendix A) who contributed their time and expertise to the analysis described in this report. 5 Blank 6 CONTENTS 1. INTRODUCTION ...and Weaknesses of Individual Devices ....................... 44 8 INDIVIDUAL WATER PURIFIER STUDY 1. INTRODUCTION This study was performed to evaluate...Data were gathered on each device and documented in a database. A Multi-Criteria Decision Model ( MCDM ) was developed, and a panel of experts assessed

  4. Process for purifying gases

    Energy Technology Data Exchange (ETDEWEB)

    Nogami, Y.; Ooka, I.; Hamamoto, T.; Shibano, H.; Miyao, K.

    1977-12-13

    An improved process is described for purifying a gas containing at least one of hydrogen cyanide and hydrogen sulfide as a harmful component by fixing the harmful component in water as an aqueous solution of nitrogen-containing sulfur compound so as to render the gas free of the harmful component. The improvement consists of: (1) burning the aqueous solution of nitrogen-containing sulfur compound to obtain an exhaust gas containing sulfur dioxide; (2) separating the sulfur dioxide from the exhaust gas for recovery; and (3) exhausting the gas after the separation of the sulfur dioxide.

  5. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  6. Process for purifying molybdenum

    International Nuclear Information System (INIS)

    Cheresnowsky, J.

    1989-01-01

    This patent describes a process for purifying molybdenum containing arsenic and phosphorus. The process comprising: adding to an acidic slurry of molybdenum trioxide, a source of magnesium ions in a solid form, with the amount of magnesium and the magnesium ion concentration in the subsequently formed ammonium molybdate solution being sufficient to subsequently form insoluble compounds containing greater than about 80% by weight of the arsenic and greater than about 80% by weight of the phosphorus, and ammonia in an amount sufficient to subsequently dissolve the molybdenum and subsequently form the insoluble compounds, with the source of magnesium ions being added prior to the addition of the ammonia; digesting the resulting ammoniated slurry at a temperature sufficient to dissolve the molybdenum and form an ammonium molybdate solution while the pH is maintained at from bout 9 to about 10 to form a solid containing the insoluble compounds; and separating the solid from the ammonium molybdate solution

  7. Report: Affinity Chromatography.

    Science.gov (United States)

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  8. Tandem transformation of glycerol to esters.

    Science.gov (United States)

    Sotenko, Maria V; Rebroš, Martin; Sans, Victor S; Loponov, Konstantin N; Davidson, Matthew G; Stephens, Gill; Lapkin, Alexei A

    2012-12-31

    Tandem transformation of glycerol via microbial fermentation and enzymatic esterification is presented. The reaction can be performed with purified waste glycerol from biodiesel production in a continuous mode, combining continuous fermentation with membrane-supported enzymatic esterification. Continuous anaerobic fermentation was optimized resulting in the productivity of 2.4 g L⁻¹ h⁻¹ of 1,3-propanediol. Biphasic esterification of 1,3-propanediol was optimized to achieve ester yield of up to 75%. A hollow fibre membrane contactor with immobilized Rhizomucor miehei lipase was demonstrated for the continuous tandem fermentation-esterification process. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Large-scale analysis of in Vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N

    2003-01-01

    Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor...... specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from...... plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage...

  10. Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

    Science.gov (United States)

    Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

    2012-01-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

  11. Specific proliferative response of human lymphocytes to purified soluble antigens from Plasmodium falciparum in vitro cultures and to antigens from malaria patients' sera

    DEFF Research Database (Denmark)

    Bygbjerg, I C; Jepsen, S; Theander, T G

    1985-01-01

    Antigens of Plasmodium falciparum, in supernatants of in vitro cultures of the parasite were affinity purified on columns prepared with the IgG fraction of the serum of an immune individual. The purified antigens induced proliferation of lymphocytes from persons who had recently had malaria. The ...... purified antigen preparations from malaria patients' sera indicating that significant amounts of non-specific mitogens were not present.......Antigens of Plasmodium falciparum, in supernatants of in vitro cultures of the parasite were affinity purified on columns prepared with the IgG fraction of the serum of an immune individual. The purified antigens induced proliferation of lymphocytes from persons who had recently had malaria...

  12. Mine water purify from radium

    International Nuclear Information System (INIS)

    Lebecka, J.

    1996-01-01

    The article describes purification of radium containing water in coal mines. Author concludes that water purification is relatively simple and effective way to decrease environmental pollution caused by coal mining. The amount of radium disposed with type A radium water has been significantly decreased. The results of investigations show that it will be soon possible to purify also type B radium water. Article compares the amounts of radium disposed by coal mines in 1990, 1995 and forecast for 2000

  13. Home drinking-water purifiers

    International Nuclear Information System (INIS)

    Pizzichini, Massimo; Pozio, Alfonso; Russo, Claudio

    2005-01-01

    To salve the widespread problem of contaminated drinking water, home purifiers are now sold in Italy as well as other countries. This article describes how these devices work, how safe they are to use and how safe the water they produce, in the broad context of regulations on drinking water and mineral water. A new device being developed by ENEA to treat municipal water and ground water could provide greater chemical and bacteriological safety. However, the appearance of these new systems makes it necessary to update existing regulations [it

  14. Process for purifying zirconium sponge

    International Nuclear Information System (INIS)

    Abodishish, H.A.M.; Kimball, L.S.

    1992-01-01

    This patent describes a Kroll reduction process wherein a zirconium sponge contaminated with unreacted magnesium and by-product magnesium chloride is produced as a regulus, a process for purifying the zirconium sponge. It comprises: distilling magnesium and magnesium chloride from: a regulus containing a zirconium sponge and magnesium and magnesium chloride at a temperature above about 800 degrees C and at an absolute pressure less than about 10 mmHg in a distillation vessel to purify the zirconium sponge; condensing the magnesium and the magnesium chloride distilled from the zirconium sponge in a condenser; and then backfilling the vessel containing the zirconium sponge and the condenser containing the magnesium and the magnesium chloride with a gas; recirculating the gas between the vessel and the condenser to cool the zirconium sponge from above about 800 degrees C to below about 300 degrees C; and cooling the recirculating gas in the condenser containing the condensed magnesium and the condensed magnesium chloride as the gas cools the zirconium sponge to below about 300 degrees C

  15. Purification of a Mycoplasma pneumoniae adhesin by monoclonal antibody affinity chromatography.

    OpenAIRE

    Leith, D K; Baseman, J B

    1984-01-01

    A 165,000-dalton surface protein of Mycoplasma pneumoniae, designated protein P1, appears to be the major attachment ligand of the pathogen. We employed monoclonal antibody affinity chromatography to obtain purified protein P1.

  16. Affine Grassmann codes

    DEFF Research Database (Denmark)

    Høholdt, Tom; Beelen, Peter; Ghorpade, Sudhir Ramakant

    2010-01-01

    We consider a new class of linear codes, called affine Grassmann codes. These can be viewed as a variant of generalized Reed-Muller codes and are closely related to Grassmann codes.We determine the length, dimension, and the minimum distance of any affine Grassmann code. Moreover, we show that af...

  17. Continuous affine processes

    DEFF Research Database (Denmark)

    Buchardt, Kristian

    2016-01-01

    Affine processes possess the property that expectations of exponential affine transformations are given by a set of Riccati differential equations, which is the main feature of this popular class of processes. In this paper we generalise these results for expectations of more general transformati...

  18. Endurance Pump Test with MIL-PRF-83282 Hydraulic Fluid, Purified with Malabar Purifier

    National Research Council Canada - National Science Library

    Sharma, Shashi

    2004-01-01

    .... Endurance aircraft hydraulic pump tests under carefully controlled conditions were previously conducted using hydraulic fluid purified with a rotating-disk and vacuum type purifier, the portable...

  19. Robust Affine Invariant Descriptors

    Directory of Open Access Journals (Sweden)

    Jianwei Yang

    2011-01-01

    Full Text Available An approach is developed for the extraction of affine invariant descriptors by cutting object into slices. Gray values associated with every pixel in each slice are summed up to construct affine invariant descriptors. As a result, these descriptors are very robust to additive noise. In order to establish slices of correspondence between an object and its affine transformed version, general contour (GC of the object is constructed by performing projection along lines with different polar angles. Consequently, affine in-variant division curves are derived. A slice is formed by points fall in the region enclosed by two adjacent division curves. To test and evaluate the proposed method, several experiments have been conducted. Experimental results show that the proposed method is very robust to noise.

  20. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  1. Purification of sequence-specific DNA-binding proteins by affinity chromatography.

    Science.gov (United States)

    Kerrigan, L A; Kadonaga, J T

    2001-05-01

    Affinity chromatography is a very effective and straightforward means of purifying a protein based on its sequence-specific DNA-binding properties. The affinity chromatography procedure described in this unit uses DNA containing specific recognition sites for the desired protein that has been covalently linked to a solid support. The first basic protocol describes preparation of a DNA affinity resin, including cyanogen bromide (CNBr) activation of the agarose support. An provides a method to couple DNA to commercially available CNBr-activated Sepharose, and a support protocol describes how to purify crude synthetic oligonucleotides by gel electrophoresis prior to preparation of the affinity resin. The second basic protocol outlines the affinity chromatography procedure. A second support protocol describes determination of the appropriate type and quantity of nonspecific competitor DNA that should be used in the procedure and its preparation. Parameters essential to the success of an affinity chromatography experiment are discussed in detail in the Commentary.

  2. Negative ion sources for tandem accelerator

    International Nuclear Information System (INIS)

    Minehara, Eisuke

    1980-08-01

    Four kinds of negative ion sources (direct extraction Duoplasmatron ion source, radial extraction Penniing ion source, lithium charge exchange ion source and Middleton-type sputter ion source) have been installed in the JAERI tandem accelerator. The ion sources can generate many negative ions ranging from Hydrogen to Uranium with the exception of Ne, Ar, Kr, Xe and Rn. Discussions presented in this report include mechanisms of negative ion formation, electron affinity and stability of negative ions, performance of the ion sources and materials used for negative ion production. Finally, the author will discuss difficult problems to be overcome in order to get any negative ion sufficiently. (author)

  3. Deciphering Modifications in Swine Cardiac Troponin I by Top-Down High-Resolution Tandem Mass Spectrometry

    Science.gov (United States)

    Zhang, Jiang; Dong, Xintong; Hacker, Timothy A.; Ge, Ying

    2011-01-01

    Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the “gold-standard” serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylation sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications including labile phosphorylation and unexpected sequence variants. PMID:20223681

  4. Deciphering modifications in swine cardiac troponin I by top-down high-resolution tandem mass spectrometry.

    Science.gov (United States)

    Zhang, Jiang; Dong, Xintong; Hacker, Timothy A; Ge, Ying

    2010-06-01

    Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle, and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the "gold-standard" serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein, we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylated sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications, including labile phosphorylation and unexpected sequence variants. Copyright 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

  5. Induced affine inflation

    Science.gov (United States)

    Azri, Hemza; Demir, Durmuş

    2018-02-01

    Induced gravity, metrical gravity in which gravitational constant arises from vacuum expectation value of a heavy scalar, is known to suffer from Jordan frame vs Einstein frame ambiguity, especially in inflationary dynamics. Induced gravity in affine geometry, as we show here, leads to an emergent metric and gravity scale, with no Einstein-Jordan ambiguity. While gravity is induced by the vacuum expectation value of the scalar field, nonzero vacuum energy facilitates generation of the metric. Our analysis shows that induced gravity results in a relatively large tensor-to-scalar ratio in both metrical and affine gravity setups. However, the fact remains that the induced affine gravity provides an ambiguity-free framework.

  6. Hydrogen purifier module with membrane support

    Science.gov (United States)

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

    2012-07-24

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

  7. Affine stochastic mortality

    NARCIS (Netherlands)

    Schrager, D.F.

    2006-01-01

    We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing

  8. Experimental Affinities in Music

    OpenAIRE

    de Assis, Paulo

    2015-01-01

    Experimental Affinities in Music brings together diverse artistic, musicological, historical, and philosophical essays, enhancing a broad discourse on artistic experimentation, and exploring various experimental attitudes in music composed between the thirteenth and twentieth centuries. The golden thread running through the different chapters is the quest for inherently experimental musical practices, a quest pursued from interrogating, descriptive, or challenging perspectives, and always...

  9. Affine field theories

    International Nuclear Information System (INIS)

    Cadavid, A.C.

    1989-01-01

    The author constructs a non-Abelian field theory by gauging a Kac-Moody algebra, obtaining an infinite tower of interacting vector fields and associated ghosts, that obey slightly modified Feynman rules. She discusses the spontaneous symmetry breaking of such theory via the Higgs mechanism. If the Higgs particle lies in the Cartan subalgebra of the Kac-Moody algebra, the previously massless vectors acquire a mass spectrum that is linear in the Kac-Moody index and has additional fine structure depending on the associated Lie algebra. She proceeds to show that there is no obstacle in implementing the affine extension of supersymmetric Yang-Mills theories. The result is valid in four, six and ten space-time dimensions. Then the affine extension of supergravity is investigated. She discusses only the loop algebra since the affine extension of the super-Poincare algebra appears inconsistent. The construction of the affine supergravity theory is carried out by the group manifold method and leads to an action describing infinite towers of spin 2 and spin 3/2 fields that interact subject to the symmetries of the loop algebra. The equations of motion satisfy the usual consistency check. Finally, she postulates a theory in which both the vector and scalar fields lie in the loop algebra of SO(3). This theory has an expanded soliton sector, and corresponding to the original 't Hooft-Polyakov solitonic solutions she now finds an infinite family of exact, special solutions of the new equations. She also proposes a perturbation method for obtaining an arbitrary solution of those equations for each level of the affine index

  10. Magnetic porous corn starch for the affinity purifi cation of cyclodextrin glucanotransferase produced by Bacillus circulans

    Czech Academy of Sciences Publication Activity Database

    Šafaříková, Miroslava; Horská, Kateřina; Maděrová, Zdeňka; Tonkova, A.; Ivanova-Pashkoulova, V.; Šafařík, Ivo

    2012-01-01

    Roč. 30, č. 1 (2012), s. 96-101 ISSN 1024-2422 Institutional support: RVO:67179843 Keywords : Bacillus circulans * cyclodextrin glucanotransferase * magnetic starch * magnetic separation Subject RIV: EH - Ecology, Behaviour Impact factor: 0.895, year: 2012

  11. Affinity driven social networks

    Science.gov (United States)

    Ruyú, B.; Kuperman, M. N.

    2007-04-01

    In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.

  12. Tandem mirror reactor

    International Nuclear Information System (INIS)

    Moir, R.W.; Barr, W.L.; Carlson, G.A.

    1977-01-01

    A parametric analysis and a preliminary conceptual design for a 1000 MWe Tandem Mirror Reactor (TMR) are described. The concept is sufficiently attractive to encourage further work, both for a pure fusion TMR and a low technology TMR Fusion-Fission Hybrid

  13. Tandem mirror reactors

    International Nuclear Information System (INIS)

    Logan, B.G.; Barr, W.L.; Bender, D.J.

    1978-01-01

    We have made preliminary designs of tandem mirror fusion reactors burning D-T fuel and of fusion-fission (hybrid) tandem mirrors producing both fissile fuel and electricity. For the hybrid reactor, we find that by using stream-stabilized, 2XIIB-like plugs and by injecting 200-keV deuterium beams into a tritium-plasma target confined electrostatically in the solenoid (two-component operation), we obtain a useful Q (fusion power/injection power) near unity. The D-T tandem reactor parameters are optimized to obtain the minimum capital cost per kW(e) net. For $200/kW(e) of 1200-keV neutral beam injection power in the plugs and a solenoid cost of about $3 million per metre length, the optimum Q is near 5. To allow for more expensive injector costs, a higher D-T reactor Q of 10 is obtainable with either increased power output or decreased neutron wall loading. Fokker--Planck calculations show steady-state Q approximately 5 for D-D tandem reactors burning only deuterium fuel and its reaction products, with most of the charged-particle fusion power recovered in a direct converter

  14. Immunogenicity of a purified fragment of 17D yellow fever envelope protein.

    Science.gov (United States)

    Brandriss, M W; Schlesinger, J J; Walsh, E E

    1990-06-01

    Information on the immunogenic properties of purified flavivirus proteins may be useful in the development of recombinant or synthetic peptide vaccines. Using a monoclonal antibody, an attempt was made to purify the envelope (E) protein of 17D yellow fever virus (17D YF) by affinity chromatography. The purified material could not be identified as intact E protein but it did bear antigenic determinants of E as determined by selective reactivity with anti-E monoclonal antibodies. Rabbits immunized with this material produced antibodies that neutralized 17D YF and dengue-2 viruses in comparable titers, indicating that cross-reactive antigenic determinants were preserved. Immunization of mice resulted in protection against intracerebral challenge with 17D YF.

  15. A quantitative assay to measure the interaction between immunogenic peptides and purified class I major histocompatibility complex molecules

    DEFF Research Database (Denmark)

    Olsen, A C; Pedersen, L O; Hansen, A S

    1994-01-01

    A direct and sensitive biochemical assay to measure the interaction in solution between peptides and affinity-purified major histocompatibility complex (MHC) class I molecules has been generated. Specific binding reflecting the known class I restriction of cytotoxic T cell responses was obtained...

  16. Purified azure B as a reticulocyte stain.

    Science.gov (United States)

    Marshall, P N; Bentley, S A; Lewis, S M

    1976-01-01

    A comparison has been made between reticulocyte preparations stained with purified azure B and with several commerically available batches of brilliant cresyl blue and new methylene blue. Marked variations were observed in the composition and staining performances of the various batches of the two commerically available dyes. Although there were no significant differences in reticulocyte counts obtained with these two dyes, varying amounts of an extraneous, particulate dye deposit were present in these preparations, making accuracte counting both tedious and timeconsuming. Purified azure B, on the other hand, gave reproducibly stained, deposit-free preparations. Reticulocyte counts obtained from azure B preparations correlated almost exactly with those determined using new methylene blue. Purified azure B is therefore recommended as a convenient reticulocyte stain for routine use. Images PMID:64475

  17. Enzyme catalysed tandem reactions

    OpenAIRE

    Oroz-Guinea, Isabel; García-Junceda, Eduardo

    2013-01-01

    To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a r...

  18. Phase change liquid purifier and pump

    Science.gov (United States)

    Steinhour, Leif Alexi

    2017-05-23

    Systems, methods, and apparatus are provided for purifying and pumping liquids, and more particularly, for purifying and pumping water. The apparatus includes a chamber including a top portion and a bottom portion. A surface configured to be heated is proximate the bottom portion of the chamber. A baffle is disposed within the chamber and above the surface. The baffle is disposed at an angle relative to a vertical direction. The chamber further includes an inlet and a first outlet. The surface heats a liquid in the chamber, causing the liquid to boil. In operation, bubbles rise from the surface and are forced in a horizontal direction by the baffle disposed in the chamber.

  19. Method for purifying bidentate organophosphorous compounds

    International Nuclear Information System (INIS)

    Krupa, J.F.; Mcisaac, L.D.; Schroeder, N.C.

    1981-01-01

    Bidentate organophosphorous compounds are purified of undesirable impurities by contacting a solution of the compounds with a mercuric nitrate solution to form an insoluble mercuric bidentate compound which precipitates while the impurities remain in solution. The precipitate is washed and then contacted with a mixture of an aqueous solution of a strong mercuric ion complexing agent and an organic solvent to complex the mercuric ion away from the bidentate compound which then dissolves in the solvent. The purified bidentate compounds are useful for extracting the actinide elements from aqueous acidic nuclear waste solutions

  20. The tandem betatron accelerator

    International Nuclear Information System (INIS)

    Keinigs, R.

    1991-01-01

    This paper reports that the tandem betatron is a compact, high-current induction accelerator that has the capability to accelerate electrons to an energy of order one gigavolt. Based upon the operating principle of a conventional betatron, the tandem betatron employs two synchronized induction cores operating 180 degrees out of phase. Embedded within the cores are the vacuum chambers, and these are connected by linear transport sections to allow for moving the beam back and forth between the two betatrons. The 180 degree phase shift between the core fluxes permits the circumvention of the flux swing constraint that limits the maximum energy gain of a conventional betatron. By transporting the beam between the synchronized cores, an electron can access more than one acceleration cycle, and thereby continue to gain energy. This added degree of freedom also permits a significant decrease in the size of the magnet system. Biasing coils provide independent control of the confining magnetic field. Provided that efficient beam switching can be performed, it appears feasible that a one gigavolt electron beam can be generated and confined. At this energy, a high current electron beam circulating in a one meter radius orbit could provide a very intense source of short wavelength (λ < 10 nm) synchrotron radiation. This has direct application to the emerging field of x-ray lithography. At more modest energies (10 MeV-30 MEV) a compact tandem betatron could be employed in the fields of medical radiation therapy, industrial radiography, and materials processing

  1. Selection of ligands for affinity chromatography using quartz crystal biosensor.

    Science.gov (United States)

    Liu, Yang; Tang, Xiaoling; Liu, Feng; Li, Ke'an

    2005-07-01

    This paper described a new strategy for rapid selecting ligands for application in affinity chromatography using a quartz crystal microbalance (QCM) biosensor. An aminoglycoside antibiotic drug, kanamycin (KM), was immobilized on the gold electrodes of the QCM sensor chip. The binding interactions of the immobilized KM with various proteins in solution were monitored as the variations of the resonant frequency of the modified sensor. Such a rapid screen analysis of interactions indicated clearly that KM-immobilized sensor showed strong specific interaction only with lysozyme (LZM). The resultant sensorgrams were rapidly analyzed by using a kinetic analysis software based on a genetic algorithm to derive both the kinetic rate constants (k(ass) and k(diss)) and equilibrium dissociation constants (K(D)) for LZM-KM interactions. The immobilized KM showed higher affinity to LZM with a dissociation constant on the order of 10(-5) M, which is within the range of 10(-4)-10(-8) M and suitable for an affinity ligand. Therefore, KM was demonstrated for the first time as a novel affinity ligand for purification of LZM and immobilized onto the epoxy-activated silica in the presence of a high potassium phosphate concentration. The KM immobilized affinity column has proved useful for a very convenient purification of LZM from chicken egg white. The purity of LZM obtained was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction. These results confirmed that the selected KM ligand is indeed a valuable affinity ligand for purification of LZM. The new screening strategy based on a QCM biosensor is expected to be a promising way for rapid selecting specific ligands for purifying other valuable proteins.

  2. Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

    1987-01-01

    The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

  3. Home Air Purifiers Eradicate Harmful Pathogens

    Science.gov (United States)

    2014-01-01

    Marshall Space Flight Center funded the University of Madison-Wisconsin to develop ethylene scrubbers to keep produce fresh in space. Akida Holdings of Jacksonville, Florida, licensed the technology and developed Airocide, an air purifier that can kill airborne pathogens. Previously designed for industrial spaces, there is now a specially designed unit for home use.

  4. Antimicrobial action of purified raspberry flavonoid

    African Journals Online (AJOL)

    user

    2012-02-07

    Feb 7, 2012 ... In this paper, the bacteriostatic action of purified raspberry flavonoid was explored. A study was conducted on the effect of raspberry flavonoid on Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Saccharomyces cerevisiae, Rhizopus, Mucor and Penicillium. Raspberry flavonoid had the best.

  5. Adjoint affine fusion and tadpoles

    Energy Technology Data Exchange (ETDEWEB)

    Urichuk, Andrew, E-mail: andrew.urichuk@uleth.ca [Physics and Astronomy Department, University of Lethbridge, Lethbridge, Alberta T1K 3M4 (Canada); Walton, Mark A., E-mail: walton@uleth.ca [Physics and Astronomy Department, University of Lethbridge, Lethbridge, Alberta T1K 3M4 (Canada); International School for Advanced Studies (SISSA), via Bonomea 265, 34136 Trieste (Italy)

    2016-06-15

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  6. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  7. Endurance Pump Test with MIL-PRF-83282 Hydraulic Fluid, Purified with Malabar Purifier

    National Research Council Canada - National Science Library

    Sharma, Shashi

    2004-01-01

    The possible deterioration of the fluid during use or the potential for the purifier to remove some of the performance improving additives from the fluid as well as the contaminants was investigated...

  8. in affine space A^6

    Directory of Open Access Journals (Sweden)

    Ofelya Arabyan

    2017-10-01

    Full Text Available A three-dimensional submanifold M in affine space A^6 is studied by the method of exterior forms. It is proven that the structure of total space generates a special type of affine connection on this submanifold; the structure equations of M are found.

  9. Steroidogenesis in amlodipine treated purified Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Latif, Rabia, E-mail: rabialatif08@hotmail.com [Department of Physiology, Army Medical College, National University of Sciences and Technology, Islamabad (Pakistan); Lodhi, Ghulam Mustafa, E-mail: drmustafa786@gmail.com [Department of Physiology, Wah Medical College, Wah (Pakistan); Hameed, Waqas, E-mail: waqham@hotmail.com [Department of Physiology, Rehman Medical College, Peshawar (Pakistan); Aslam, Muhammad, E-mail: professormaslam@yahoo.com [Department of Physiology, Shifa College of Medicine, Islamabad (Pakistan)

    2012-01-01

    Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

  10. Hemoglobin affinity in Andean rodents

    Directory of Open Access Journals (Sweden)

    HRVOJ OSTOJIC

    2002-01-01

    Full Text Available Blood hemoglobin oxygen affinity (P50 was measured in three Andean species and in the laboratory rat (control, all raised near sea level. Chinchilla lanigera (Molina, 1792 has an altitudinal habitat range from low Andean slopes up to 3000 m., while Chinchilla brevicaudata (Waterhouse, 1848 has an altitudinal range from 3000 to 5000 m. The laboratory type guinea pig, wild type guinea pig (Cavia porcellus, (Waterhouse, 1748, and laboratory rat (Rattus norvegicus were also raised at sea level. The Andean species had high hemoglobin oxygen affinities (low P50 compared with the rat. Chinchilla brevicaudata had a higher affinity than Chinchilla lanigera. The wild type guinea pig had a higher affinity than the laboratory type. As has been shown in other species, this is another example of an inverse correlation between the altitude level and the P50 values. This is the first hemoglobin oxygen affinity study in Chinchilla brevicaudata.

  11. Modification of the Argonne tandem

    International Nuclear Information System (INIS)

    Yntema, J.L.

    1978-01-01

    For nuclear structure experiments with heavy ions it is necessary to have ion energies in excess of 5 MeV per nucleon. At the Argonne tandem FN accelerator this was accomplished by the addition of a superconducting linac. Modifications of the FN tandem to improve the performance of the pair is described

  12. Tandems as injectors for synchrotrons

    International Nuclear Information System (INIS)

    Ruggiero, A.G.

    1993-01-01

    This is a review on the use of tandem electrostatic accelerators for injection and fitting of synchrotrons to accelerate intense beams of heavy ions to relativistic energies. The paper emphasizes the need of operating the tandems in pulsed mode for this application. It has been experimentally demonstrated that at present this type of accelerator still provides the most reliable and best performance. (orig.)

  13. MHD stability of tandem mirrors

    International Nuclear Information System (INIS)

    Poulsen, P.; Molvik, A.; Shearer, J.

    1982-01-01

    The TMX-Upgrade experiment was described, and the manner in which various plasma parameters could be affected was discussed. The initial analysis of the MHD stability of the tandem mirror was also discussed, with emphasis on the negative tandem configuration

  14. Mapping Affinities in Academic Organizations

    Directory of Open Access Journals (Sweden)

    Dario Rodighiero

    2018-02-01

    Full Text Available Scholarly affinities are one of the most fundamental hidden dynamics that drive scientific development. Some affinities are actual, and consequently can be measured through classical academic metrics such as co-authoring. Other affinities are potential, and therefore do not leave visible traces in information systems; for instance, some peers may share interests without actually knowing it. This article illustrates the development of a map of affinities for academic collectives, designed to be relevant to three audiences: the management, the scholars themselves, and the external public. Our case study involves the School of Architecture, Civil and Environmental Engineering of EPFL, hereinafter ENAC. The school consists of around 1,000 scholars, 70 laboratories, and 3 institutes. The actual affinities are modeled using the data available from the information systems reporting publications, teaching, and advising scholars, whereas the potential affinities are addressed through text mining of the publications. The major challenge for designing such a map is to represent the multi-dimensionality and multi-scale nature of the information. The affinities are not limited to the computation of heterogeneous sources of information; they also apply at different scales. The map, thus, shows local affinities inside a given laboratory, as well as global affinities among laboratories. This article presents a graphical grammar to represent affinities. Its effectiveness is illustrated by two actualizations of the design proposal: an interactive online system in which the map can be parameterized, and a large-scale carpet of 250 square meters. In both cases, we discuss how the materiality influences the representation of data, in particular the way key questions could be appropriately addressed considering the three target audiences: the insights gained by the management and their consequences in terms of governance, the understanding of the scholars’ own

  15. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  16. JAERI tandem-accelerator and tandem-booster

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Tadashi [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-03-01

    In 1982, aiming at the new development of atomic energy research, the tandem accelerator of Japan Atomic Energy Research Institute (JAERI) was installed. In fiscal year 1993, the superconducting boosters which can increase the ion energy by up to 4 times were added, and the research in the region below 1000 MeV became possible. Those are electrostatic type accelerators which are easy to be used especially in basic research field, and are useful for future research. The tandem accelerator has been operated while maintaining the first class performance as the accelerator for various kinds of heavy ion beam. It has the special shape among electrostatic type accelerators, and is excellent in the easiness of control and stability. The main particulars of the tandem accelerator are shown. As for the ion sources of the tandem accelerator, three cesium sputter type ion sources are installed on two high voltage stands. The kinds of the ions which can be accelerated are mainly negative ions. As the improvement, electron cyclotron resonance (ECR) ion sources are expected to be adopted. As for the tandem boosters, the 1/4 wavelength type resonance hollow cylinder was adopted. The constitution of the tandem boosters is explained. The way of utilizing the tandem accelerator system and the aim for hereafter are reported. (K.I.)

  17. Tandem accelerator operation

    International Nuclear Information System (INIS)

    Rummel, R.L.; Carter, E.P.; Westerfeldt, C.R.

    1985-01-01

    For the period July 1, 1984 through July 31, 1985 the FN tandem accelerator was operated for 6760 hours. This represents 72% utilization of the maximum available machine time for the past 13 months. The total running time for this machine is now 104,470 hours, and the age of two of the accelerator tubes exceeds 100,000 hours. In August of last year the authors removed the liquid-nitrogen trapped mercury diffusion pump on the low energy end of the accelerator. In its place the authors installed a Varian Cryostack cryopump along with a new NEC beam scanner, a new set of beam chopper plates, and new vacuum gauges and protection circuits. The vacuum in the low energy tube has been improved from 5 x 10 -6 to 5 x 10 -7 Torr

  18. Enzyme catalysed tandem reactions.

    Science.gov (United States)

    Oroz-Guinea, Isabel; García-Junceda, Eduardo

    2013-04-01

    To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. 2017 Guralp Affinity Digitizer Evaluation.

    Energy Technology Data Exchange (ETDEWEB)

    Merchant, Bion J.

    2018-03-01

    Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

  20. The utility of affine variables and affine coherent states

    International Nuclear Information System (INIS)

    Klauder, John R

    2012-01-01

    Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when appropriate. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’. (review)

  1. Investigation of hydrazide derivatives inhibitory effect on peroxidase enzyme purified from turnip roots

    Science.gov (United States)

    Almaz, Züleyha; Öztekin, Aykut; Özdemir, Hasan

    2017-04-01

    Peroxidases (EC: 1.11.1.7) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group [1]. They are found in all cells and play a critical role in many biological processes, such as the host-defense mechanism [2]. Peroxidases (PODs) are widely used in clinical biochemistry, enzyme immunoassays, synthesis of various aromatic chemicals, treatment of waste water containing phenolic compounds [3, 4]. In this study, peroxidase enzyme was purified with Para amino benzohydrazide (PABH)-L-Tyrosine Sepharose 4B affinity chromatography to investigate the inhibitory effect of hydrazide derivatives on Turnip (Brassica rapa L.). IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzyme and inhibition type of these molecules were determined.

  2. Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers.

    Directory of Open Access Journals (Sweden)

    Myungsun Kang

    Full Text Available Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens. However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964. Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality, we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal.

  3. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Hitoshi; Akazawa, Daisuke [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Kato, Takanobu; Date, Tomoko [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Shirakura, Masayuki [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Nakamura, Noriko; Mochizuki, Hidenori [Toray Industries, Inc., Kanagawa (Japan); Tanaka-Kaneko, Keiko; Sata, Tetsutaro [Department of Pathology, National Institute of Infectious Diseases, Tokyo (Japan); Tanaka, Yasuhito [Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medicine, Nagoya (Japan); Mizokami, Masashi [Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba (Japan); Suzuki, Tetsuro [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Wakita, Takaji, E-mail: wakita@nih.go.jp [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan)

    2010-05-14

    To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

  4. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

    International Nuclear Information System (INIS)

    Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

    2010-01-01

    To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

  5. Structure/activity Relationship of Thapsigargin Inhibition on the Purified Golgi/secretory Pathway Ca2+/Mn2+ Transport ATPase (SPCA1a)

    DEFF Research Database (Denmark)

    Jialin, Chen; de Raeymaecker, Joren; Hovgaard, Jannik Brøndsted

    2017-01-01

    The Golgi/secretory pathway Ca2+/Mn2+ transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+ dependent ATPase activity following reconstitution in proteoliposomes. The purified...... SPCA1a displays a higher apparent Ca2+ affinity and lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linole-/oleamide and phosphatidyl ethanolamine inhibit, whereas phosphatidic acid and sphingomyelin enhance SPCA1a...

  6. Representations of affine Hecke algebras

    CERN Document Server

    Xi, Nanhua

    1994-01-01

    Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

  7. Thermogelling properties of purified poloxamer 407

    Directory of Open Access Journals (Sweden)

    Amir Fakhari

    2017-08-01

    Full Text Available Poloxamers are triblock copolymers with a center block of hydrophobic polypropylene oxide (PPO flanked by two hydrophilic polyethyleneoxide (PEO blocks. Among this family of copolymers, poloxamer 407 is a non-ionic surfactant with reversible gelation properties above a particular polymer concentration and a particular temperature. Easy preparation of poloxamer 407 based sterile injectable formulations have made this copolymer a good candidate for drug delivery, specifically when controlled release of the drug is required. Previously, the applications of compendial poloxamer 407 preparations were demonstrated; however, low viscosity, poor elasticity, and sol-to-gel transition temperature (Tsol-gel over a wide temperature range were observed. A purification process was introduced to eliminate impurities and low molecular weight copolymer molecules from the compendial poloxamer 407 resulting in higher viscosity values with Tsol-gel in a narrow temperature range. Here, poloxamer 407 was purified based on the proposed process and the rheological and analytical evaluation of the purified poloxamer 407 was conducted and compared to unpurified, compendial poloxamer 407. Then, the impact of poloxamer 407 concentration on gel formation was evaluated. For drug delivery applications, the effect of relevant buffer salts and the effect of addition of ethanol to the poloxamer 407 solutions were rheologically evaluated.

  8. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  9. Ozone Air Purifiers: Can They Improve Asthma Symptoms?

    Science.gov (United States)

    ... daughter has asthma. Would she benefit from an ozone air purifier in her room? Answers from James ... Li, M.D., Ph.D. Despite manufacturers' claims, ozone air purifiers don't remove asthma triggers from ...

  10. Purified human liver acid beta-D-galactosidases possessing activity towards G(M1)-ganglioside and lactosylceramide.

    Science.gov (United States)

    Miller, A L; Frost, R G; O'Brien, J S

    1977-01-01

    Our studies with purified human liver acid beta-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl beta-D-galactosidase and G(M1)-ganglioside beta-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincident elution from Sepharose 6B of all three enzyme activities. Images Fig. 1. PMID:411490

  11. Tandem Van de Graaff facility

    Data.gov (United States)

    Federal Laboratory Consortium — Completed in 1970, the Tandem Van de Graaff facility was for many years the world's largest electrostatic accelerator facility. It can provide researchers with beams...

  12. Superconducting linacs used with tandems

    International Nuclear Information System (INIS)

    Ben-Zvi, I.

    1984-01-01

    The main features of superconducting linacs used as post-accelerators of tandems are reviewed. Various aspects of resonators, cryogenics and electronics are discussed, and recent advances in the field are presented. (orig.)

  13. Characterization of α-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Palittapongarnpim Prasit

    2009-06-01

    Full Text Available Abstract Background Alpha-isopropylmalate synthase (α-IPMS is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding α-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR. The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two α-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His6-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units. Results The recombinant His6-α-IPMS proteins with two and 14 copies (α-IPMS-2CR and α-IPMS-14CR, respectively of the repeat units were purified by immobilized metal ion affinity chromatography and gel filtration. Both enzymes were found to be dimers by gel filtration. Both enzymes work well at pH values of 7–8.5 and temperatures of 37–42°C. However, α-IPMS-14CR tolerates pH values and temperatures outside of this range better than α-IPMS-2CR does. α-IPMS-14CR has higher affinity than α-IPMS-2CR for the two substrates, α-ketoisovalerate and acetyl CoA. Furthermore, α-IPMS-2CR was feedback inhibited by the end product l-leucine, whereas α-IPMS-14CR was not. Conclusion The differences in the kinetic properties and the l-leucine feedback inhibition between the two M. tuberculosis α-IPMS proteins containing low and high numbers of VNTR indicate that a large VNTR insertion affects protein structure and function. Demonstration of l-leucine binding to α-IPMS-14CR would confirm whether or not α-IPMS-14CR responds to end-product feedback inhibition.

  14. Proposed Lucas Heights tandem accelerator

    International Nuclear Information System (INIS)

    1985-01-01

    An 8 million volt tandem accelerator (LHTA - Lucas Heights Tandem Accelerator) is proposed to be installed at the Australian Atomic Energy Commission's Lucas Heights Research Laboratories. The new facility is expected to cost $6 million and will have applications such as ultrasensitive trace element analysis for management of groundwater resources, salinity in soil, soil erosion and Antarctic research. Applied physics applications include nuclear safeguards, energy sources, study of material surfaces, archaeology, archaeometry and occupational health

  15. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase......Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na......(+),K(+)-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na(+) affinity was higher (K(m) lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na......(+),K(+)-ATPase isoform analysis implied that heterodimers containing the beta(1) isoform have a higher Na(+) affinity than heterodimers containing the beta(2) isoform. Immunoprecipitation experiments demonstrated that dimers with alpha(1) are responsible for approximately 36% of the total Na,K-ATPase activity. Selective...

  16. Tandem Terminal Ion Source

    International Nuclear Information System (INIS)

    None

    2000-01-01

    OAK-B135 Tandem Terminal Ion Source. The terminal ion source (TIS) was used in several experiments during this reporting period, all for the(sup 7)Be((gamma))(sup 8)B experiment. Most of the runs used(sup 1)H(sup+) at terminal voltages from 0.3 MV to 1.5 MV. One of the runs used(sup 2)H(sup+) at terminal voltage of 1.4 MV. The other run used(sup 4)He(sup+) at a terminal voltage of 1.37 MV. The list of experiments run with the TIS to date is given in table 1 below. The tank was opened four times for unscheduled source repairs. On one occasion the tank was opened to replace the einzel lens power supply which had failed. The 10 kV unit was replaced with a 15 kV unit. The second time the tank was opened to repair the extractor supply which was damaged by a tank spark. On the next occasion the tank was opened to replace a source canal which had sputtered away. Finally, the tank was opened to replace the discharge bottle which had been coated with aluminum sputtered from the exit canal

  17. Apparatus and methods for purifying lead

    Science.gov (United States)

    Tunison, Harmon M.

    2016-01-12

    Disclosed is an exemplary method of purifying lead which includes the steps of placing lead and a fluoride salt blend in a container; forming a first fluid of molten lead at a first temperature; forming a second fluid of the molten fluoride salt blend at a second temperature higher than the first temperature; mixing the first fluid and the second fluid together; separating the two fluids; solidifying the molten fluoride salt blend at a temperature above a melting point of the lead; and removing the molten lead from the container. In certain exemplary methods the molten lead is removed from the container by decanting. In still other exemplary methods the molten salt blend is a Lewis base fluoride eutectic salt blend, and in yet other exemplary methods the molten salt blend contains sodium fluoride, lithium fluoride, and potassium fluoride.

  18. Kinetics and Thermal Properties of Crude and Purified β-Galactosidase with Potential for the Production of Galactooligosaccharides

    Directory of Open Access Journals (Sweden)

    Anna Rafaela Cavalcante Braga

    2013-01-01

    Full Text Available β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two β-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-β-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.

  19. Tandem Cylinder Noise Predictions

    Science.gov (United States)

    Lockard, David P.; Khorrami, Mehdi R.; CHoudhari, Meelan M.; Hutcheson, Florence V.; Brooks, Thomas F.; Stead, Daniel J.

    2007-01-01

    In an effort to better understand landing-gear noise sources, we have been examining a simplified configuration that still maintains some of the salient features of landing-gear flow fields. In particular, tandem cylinders have been studied because they model a variety of component level interactions. The present effort is directed at the case of two identical cylinders spatially separated in the streamwise direction by 3.7 diameters. Experimental measurements from the Basic Aerodynamic Research Tunnel (BART) and Quiet Flow Facility (QFF) at NASA Langley Research Center (LaRC) have provided steady surface pressures, detailed off-surface measurements of the flow field using Particle Image Velocimetry (PIV), hot-wire measurements in the wake of the rear cylinder, unsteady surface pressure data, and the radiated noise. The experiments were conducted at a Reynolds number of 166 105 based on the cylinder diameter. A trip was used on the upstream cylinder to insure a fully turbulent shedding process and simulate the effects of a high Reynolds number flow. The parallel computational effort uses the three-dimensional Navier-Stokes solver CFL3D with a hybrid, zonal turbulence model that turns off the turbulence production term everywhere except in a narrow ring surrounding solid surfaces. The current calculations further explore the influence of the grid resolution and spanwise extent on the flow and associated radiated noise. Extensive comparisons with the experimental data are used to assess the ability of the computations to simulate the details of the flow. The results show that the pressure fluctuations on the upstream cylinder, caused by vortex shedding, are smaller than those generated on the downstream cylinder by wake interaction. Consequently, the downstream cylinder dominates the noise radiation, producing an overall directivity pattern that is similar to that of an isolated cylinder. Only calculations based on the full length of the model span were able to

  20. Alternative Affinity Ligands for Immunoglobulins.

    Science.gov (United States)

    Kruljec, Nika; Bratkovič, Tomaž

    2017-08-16

    The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

  1. Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry.

    Science.gov (United States)

    Andon, Nancy L; Hollingworth, Sarah; Koller, Antonius; Greenland, Andrew J; Yates, John R; Haynes, Paul A

    2002-09-01

    We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

  2. Study of hot corrosion of flakes of non purified graphite and of purified graphite

    International Nuclear Information System (INIS)

    Boule, Michel

    1967-01-01

    The author reports the study of hot corrosion of the Ticonderoga graphite. He reports the study of the defects of graphite flakes (structure defects due to impurities), the dosing of these impurities, and then their removal by purification. Flakes have then been oxidised by means of a specially designed apparatus. Based on photographs taken by optical and electronic microscopy, the author compares the oxidation features obtained in dry air and in humid air, between purified and non purified flakes. He also reports the study of the evolution of oxidation with respect to the initial rate of impurities, and the study of the evolution of oxidation features in humid air during oxidation. All these comparisons are made while taking the oxidation rate into account [fr

  3. Affinity biosensors: techniques and protocols

    National Research Council Canada - National Science Library

    Rogers, Kim R; Mulchandani, Ashok

    1998-01-01

    ..., and government to begin or expand their biosensors research. This volume, Methods in Biotechnology vol. 7: Affinity Biosensors: Techniques and Protocols, describes a variety of classical and emerging transduction technologies that have been interfaced to bioaffinity elements (e.g., antibodies and receptors). Some of the reas...

  4. Introduction to tandem mirror physics

    International Nuclear Information System (INIS)

    Kesner, J.; Gerver, M.J.; Lane, B.G.; McVey, B.D.; Catto, P.J.; D'Ippolito, D.A.; Myra, J.R.

    1983-09-01

    This monograph, prepared jointly by the MIT Plasma Fusion Center Mirror Fusion group and SAI, Boulder, Colorado, presents a review of the development of mirror fusion theory from its conception some thirty years ago to the present. Pertinent historic experiments and their contribution are discussed to set the stage for a detailed analysis of current experiments and the problems which remain to be solved in bringing tandem mirror magnetic confinement fusion to fruition. In particular, Chapter III discusses in detail the equilibrium and stability questions which must be dealt with before tandem mirror reactors become feasible, while Chapters IV and V discuss some of the current machines and those under construction which will help to resolve critical issues in both physics and engineering whose solutions are necessary to the commercialization of tandem mirror fusion

  5. Fragments of protein A eluted during protein A affinity chromatography.

    Science.gov (United States)

    Carter-Franklin, Jayme N; Victa, Corazon; McDonald, Paul; Fahrner, Robert

    2007-09-07

    Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

  6. Decontamination of radioactive materials using a household water purifier

    International Nuclear Information System (INIS)

    Yanaga, Makoto; Oishi, Ayumi; Wada, Hideki

    2013-01-01

    A home water purifier was used to remove radioactive iodine and cesium from rainwater contaminated with the radioactive materials released by the Fukushima Daiichi Nuclear Power Plant accident following the earthquake and tsunami in 2011. To prevent internal radiation exposure, the rainwater was satisfactorily decontaminated by a simple method using the pitcher type water purifier, which is widely available to the general public. However, the results indicate that the ability for purification by the home water purifier will be reduced significantly if the water is not filtered by some method before the water purifier is used, especially in the case of natural water containing suspension matter. (author)

  7. JAERI Tandem annual report 1983

    International Nuclear Information System (INIS)

    Harada, Kichinosuke; Maruyama, Michio; Okashita, Hiroshi; Ozawa, Kunio; Shikazono, Naomoto; Tanaka, Shigeya

    1984-07-01

    This annual report describes research activities which have been performed with JAERI tandem accelerator from April 1, 1983 to March 31, 1984. Summary reports of 32 papers, publications, personnel and a list of co-operative reserches with universities are contained. (author)

  8. JAERI tandem annual report, 1982

    International Nuclear Information System (INIS)

    Harada, Kichinosuke; Maruyama, Michio; Ozawa, Kunio; Shikazono, Naomoto; Tamura, Tsutomu; Tanaka, Shigeya

    1983-06-01

    This annual report describes research activities which have been performed with JAERI tandem accelerator from September 1, 1981 to March 31, 1983. Summary reports of 38 papers, publications, personnel and a list of co-operative researches with universities are contained. (author)

  9. The affine quantum gravity programme

    CERN Document Server

    Klauder, J R

    2002-01-01

    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix left brace g-hat sub a sub b (x)right brace composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that sti...

  10. Affinity purification of native glycodelin from amniotic fluid for biological investigations and development of a glycodelin ELISA for clinical studies

    DEFF Research Database (Denmark)

    Sørensen, Steen; Myrhøj, Vibeke; Nguyen, Thanh Ha

    2017-01-01

    for functional studies because the carbohydrate part can be lacking or be insufficient in recombinant glycodelin from prokaryotic and eukaryotic cell systems. METHODS AND RESULTS: Native glycodelin was purified from amniotic fluid by a series of affinity chromatography steps and had many glycosylated forms...

  11. Scaling Analysis of Affinity Propagation

    OpenAIRE

    Furtlehner , Cyril; Sebag , Michèle; Xiangliang , Zhang

    2010-01-01

    14 pages, 11 figures; International audience; We analyze and exploit some scaling properties of the {\\em Affinity Propagation} (AP) clustering algorithm proposed by Frey and Dueck (2007). Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces t...

  12. Electron affinity of liquid water

    Energy Technology Data Exchange (ETDEWEB)

    Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; Paesani, Francesco; Galli, Giulia

    2018-01-16

    Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1–0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential of the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.

  13. Spectral affinity in protein networks.

    Science.gov (United States)

    Voevodski, Konstantin; Teng, Shang-Hua; Xia, Yu

    2009-11-29

    Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to quickly find nodes closest to a queried vertex in any protein

  14. Spectral affinity in protein networks

    Directory of Open Access Journals (Sweden)

    Teng Shang-Hua

    2009-11-01

    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to

  15. Assay of partially purified glutamate dehydrogenase isolated from ...

    African Journals Online (AJOL)

    Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

  16. Isoforms of purified methyltransferase from human blood platelets ...

    African Journals Online (AJOL)

    ... purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation ...

  17. Glucoamylase isoform (GAII) purified from a thermophilic fungus ...

    African Journals Online (AJOL)

    Scytalidium thermophilum 15.8 produced two extracellular glucoamylases. Using a DEAE-Cellulose chromatographic column glucoamylases form II (GAII) was separated and purified from glucoamylases form I (GAI) that was previously purified and characterised (Cereia et al., 2000) when the filtrate of the culture medium ...

  18. Reproducible in vitro regeneration system for purifying sugarcane ...

    African Journals Online (AJOL)

    This procedure may be considered as one of the best ever published report on regeneration from in vitro grown plants to purify clones without subjecting the plants to field conditions and harvesting the mature cane. This technique was used to purify transgenic sugarcane plants carrying Bacillus thuringiensis gene.

  19. Respirators: Air Purifying, Self-Study, Course 40723

    Energy Technology Data Exchange (ETDEWEB)

    Chochoms, Michael [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-12-21

    Respirators: Air Purifying Self-Study (COURSE 40723) is designed for Los Alamos National Laboratory (LANL) workers, support services subcontractors, and other LANL subcontractors who work under the LANL Respiratory Protection Program (RPP). This course also meets the air-purifying respirators (APRs) retraining requirement.

  20. Partially purified polygalacturonase from Aspergillus niger (SA6 ...

    African Journals Online (AJOL)

    Polygalacturonase (PG) was isolated from Aspergillus niger (A. niger) (SA6), partially purified and characterized. The PG showed two bands on SDS-PAGE suggesting an “endo and exo PG with apparent molecular weights of 35 and 40 KDa, respectively. It was purified 9-fold with a yield of 0.18% and specific activity of 246 ...

  1. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  2. Manifolds with integrable affine shape operator

    Directory of Open Access Journals (Sweden)

    Daniel A. Joaquín

    2005-05-01

    Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

  3. Topological conjugacy classes of affine maps

    OpenAIRE

    Tetiana, Budnytska

    2008-01-01

    A map $f: \\ff^n \\to \\ff^n$ over a field $\\ff$ is called affine if it is of the form $f(x)=Ax+b$, where the matrix $A \\in \\ff^{n\\times n}$ is called the linear part of affine map and $b \\in \\ff^n$. The affine maps over $\\ff=\\rr$ or $\\cc$ are investigated. We prove that affine maps having fixed points are topologically conjugate if and only if their linear parts are topologically conjugate. If affine maps have no fixed points and $n=1$ or 2, then they are topologically conjugate if and only if ...

  4. Nanocrystal assembly for tandem catalysis

    Science.gov (United States)

    Yang, Peidong; Somorjai, Gabor; Yamada, Yusuke; Tsung, Chia-Kuang; Huang, Wenyu

    2014-10-14

    The present invention provides a nanocrystal tandem catalyst comprising at least two metal-metal oxide interfaces for the catalysis of sequential reactions. One embodiment utilizes a nanocrystal bilayer structure formed by assembling sub-10 nm platinum and cerium oxide nanocube monolayers on a silica substrate. The two distinct metal-metal oxide interfaces, CeO.sub.2--Pt and Pt--SiO.sub.2, can be used to catalyze two distinct sequential reactions. The CeO.sub.2--Pt interface catalyzed methanol decomposition to produce CO and H.sub.2, which were then subsequently used for ethylene hydroformylation catalyzed by the nearby Pt--SiO.sub.2 interface. Consequently, propanal was selectively produced on this nanocrystal bilayer tandem catalyst.

  5. Tandem electrostatic accelerators for BNCT

    International Nuclear Information System (INIS)

    Ma, J.C.

    1994-01-01

    The development of boron neutron capture therapy (BNCT) into a viable therapeutic modality will depend, in part, on the availability of suitable neutron sources compatible with installation in a hospital environment. Low-energy accelerator-based intense neutron sources, using electrostatic or radio frequency quadrupole proton accelerators have been suggested for this purpose and are underdevelopment at several laboratories. New advances in tandem electrostatic accelerator technology now allow acceleration of the multi-milliampere proton beams required to produce therapeutic neutron fluxes for BNCT. The relatively compact size, low weight and high power efficiency of these machines make them particularly attractive for installation in a clinical or research facility. The authors will describe the limitations on ion beam current and available neutron flux from tandem accelerators relative to the requirements for BNCT research and therapy. Preliminary designs and shielding requirements for a tandern accelerator-based BNCT research facility will also be presented

  6. MR-Tandem: parallel X!Tandem using Hadoop MapReduce on Amazon Web Services.

    Science.gov (United States)

    Pratt, Brian; Howbert, J Jeffry; Tasman, Natalie I; Nilsson, Erik J

    2012-01-01

    MR-Tandem adapts the popular X!Tandem peptide search engine to work with Hadoop MapReduce for reliable parallel execution of large searches. MR-Tandem runs on any Hadoop cluster but offers special support for Amazon Web Services for creating inexpensive on-demand Hadoop clusters, enabling search volumes that might not otherwise be feasible with the compute resources a researcher has at hand. MR-Tandem is designed to drop in wherever X!Tandem is already in use and requires no modification to existing X!Tandem parameter files, and only minimal modification to X!Tandem-based workflows. MR-Tandem is implemented as a lightly modified X!Tandem C++ executable and a Python script that drives Hadoop clusters including Amazon Web Services (AWS) Elastic Map Reduce (EMR), using the modified X!Tandem program as a Hadoop Streaming mapper and reducer. The modified X!Tandem C++ source code is Artistic licensed, supports pluggable scoring, and is available as part of the Sashimi project at http://sashimi.svn.sourceforge.net/viewvc/sashimi/trunk/trans_proteomic_pipeline/extern/xtandem/. The MR-Tandem Python script is Apache licensed and available as part of the Insilicos Cloud Army project at http://ica.svn.sourceforge.net/viewvc/ica/trunk/mr-tandem/. Full documentation and a windows installer that configures MR-Tandem, Python and all necessary packages are available at this same URL. brian.pratt@insilicos.com

  7. Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

    Czech Academy of Sciences Publication Activity Database

    Majerská, J.; Schrumpfová, P.; Dokládal, Ladislav; Schorová, Š.; Stejskal, K.; Obořil, M.; Honys, D.; Kozáková, L.; Polanská, P.; Sýkorová, Eva

    2017-01-01

    Roč. 254, č. 4 (2017), s. 1547-1562 ISSN 0033-183X R&D Projects: GA ČR GA13-06943S Institutional support: RVO:68081707 Keywords : single-stranded-dna * genome-wide screen * arabidopsis-thaliana * reverse-transcriptase Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Genetics and heredity (medical genetics to be 3) Impact factor: 2.870, year: 2016

  8. "Nanocrystal bilayer for tandem catalysis"

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Yusuke; Tsung, Chia Kuang; Huang, Wenyu; Huo, Ziyang; E.Habas, Susan E; Soejima, Tetsuro; Aliaga, Cesar E; Samorjai, Gabor A; Yang, Peidong

    2011-01-24

    Supported catalysts are widely used in industry and can be optimized by tuning the composition and interface of the metal nanoparticles and oxide supports. Rational design of metal-metal oxide interfaces in nanostructured catalysts is critical to achieve better reaction activities and selectivities. We introduce here a new class of nanocrystal tandem catalysts that have multiple metal-metal oxide interfaces for the catalysis of sequential reactions. We utilized a nanocrystal bilayer structure formed by assembling platinum and cerium oxide nanocube monolayers of less than 10 nm on a silica substrate. The two distinct metal-metal oxide interfaces, CeO2-Pt and Pt-SiO2, can be used to catalyse two distinct sequential reactions. The CeO2-Pt interface catalysed methanol decomposition to produce CO and H2, which were subsequently used for ethylene hydroformylation catalysed by the nearby Pt-SiO2 interface. Consequently, propanal was produced selectively from methanol and ethylene on the nanocrystal bilayer tandem catalyst. This new concept of nanocrystal tandem catalysis represents a powerful approach towards designing high-performance, multifunctional nanostructured catalysts

  9. Affinity Purification and Comparative Biosensor Analysis of Citrulline-Peptide-Specific Antibodies in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Eszter Szarka

    2018-01-01

    Full Text Available Background: In rheumatoid arthritis (RA, anti-citrullinated protein/peptide antibodies (ACPAs are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: KD values of affinity-purified ACPA IgGs varied between 10−6 and 10−8 M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.

  10. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

    Science.gov (United States)

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-11-14

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

  11. Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography.

    Science.gov (United States)

    Dissing, J; Dahl, O; Svensmark, O

    1979-08-15

    1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

  12. The affine quantum gravity programme

    International Nuclear Information System (INIS)

    Klauder, John R

    2002-01-01

    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix { g-hat ab (x)} composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that still retain some basic characteristics of gravity, specifically a partial second-class constraint operator structure. Although perturbatively nonrenormalizable, gravity may possibly be understood nonperturbatively from a hard-core perspective that has proved valuable for specialized models. Finally, developing a procedure to pass to the genuine physical Hilbert space involves several interconnected steps that require careful coordination

  13. Affine-projective field laws

    International Nuclear Information System (INIS)

    Murphy, G.L.

    1975-01-01

    The general topic of geometric unified field theories is discussed in the first section. Some reasons are given for pursuing such theories, and some criticisms are considered. The second section develops the fundamental equations of a purely affine theory which is invariant under projective transformations of the affine connection. This theory is a generalization of that of Schrodinger. Possible identifications for the space-time metric are considered in Sec. III. Sections IV and V deal with the limits of pure gravitation and electrodynamics. In the symmetric limit, Einstein's vacuum equations with cosmological term are recovered. The theory also contains a generalized electrodynamic set of equations which is very similar to the Born-Infeld set. In the weak-field approximation, a finite mass must be attributed to the photon. The problem of motion for charges is discussed here, and it is argued that criticisms of unified field theories because of a supposed inability to produce the Lorentz force law are probably not justified. Three more speculative sections deal with possible explanations of nuclear forces, the spin-torsion relation, and particle structure

  14. Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

    Science.gov (United States)

    Hsu, Chuan-Chih; Xue, Liang; Arrington, Justine V.; Wang, Pengcheng; Paez Paez, Juan Sebastian; Zhou, Yuan; Zhu, Jian-Kang; Tao, W. Andy

    2017-06-01

    Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. [Figure not available: see fulltext.

  15. Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells.

    Science.gov (United States)

    Stachowiak, Radosław; Łyżniak, Marcin; Grabowska, Maja; Roeske, Katarzyna; Jagielski, Tomasz; Bielecki, Jacek; Budziszewska, Bożena K; Hoser, Grażyna; Kawiak, Jerzy

    2014-08-18

    Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells. A purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells. LLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.

  16. Immunodiffusion Studies of Purified Equine Infectious Anemia Virus

    Science.gov (United States)

    Nakajima, Hideo; Ushimi, Chuzo

    1971-01-01

    Antigenicity of purified equine infectious anemia (EIA) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with EIA virus. The purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. Furthermore, it was found that purified EIA virus reacted against the serum of horses infected with all strains of EIA virus which were antigenically different from one another. From the result, group-specific components of the virus rather than strain-specific ones were considered to be involved in the reaction. Serological reactivity was lost by adding antiserum from the infected horse to the antigen. The precipitating antibody usually appeared in the serum 1 to 2 weeks after the first febrile attack of EIA and remained for a longer period. Some characteristics of the purified antigen and specificity of the reaction for EIA are described. Images PMID:16557982

  17. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Directory of Open Access Journals (Sweden)

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  18. Properties of Folate Binding Protein Purified from Cow’s Milk

    Directory of Open Access Journals (Sweden)

    SUBANDRATE

    2012-09-01

    Full Text Available Folic acid played an important role in the metabolism of the body. To measure the serum folic acid levels could use the folate binding protein (FBP from cow’s milk with a technique analogous to ELISA. The aims of this study were to identify characteristics of FBP from cow’s milk and binding capacity of FBP to folic acid and to purify FBP from other whey protein passed through DEAE-cellulose chromatography column. Each of DEAE-cellulose peaks was passed in affinity chromatography column. FBP was released from affinity column with sodium acetate buffer pH 3.5. The purity of obtained FBP was demonstrated by a single spot in SDS-PAGE analysis and the estimated molecular weight of FBP was around 31 kDa. Our study indicated that 1 mol FBP bound 1 mol folic acid. Alkylation with iodoacetic acid decreased the binding capacity of FBP which suggested the presence of a–SH or imidazol group in its active site. The importance of disulfide bridge was proven by decreasing of folate binding capacity of FBP after -mercaptoethanol treatment. In contrary, the folate binding didn need Ca2+ ion, as indicated by EDTA test which gave the same result as control.

  19. Electrophoresis and electro-affinity transfer with specific antibodies to alpha-fetoprotein for detection of circulating immune complexes of alpha-fetoprotein.

    OpenAIRE

    Taketa, Kazuhisa; Ichikawa, Eriko; Taga, Hiroko; Hirai, Hidematsu

    1984-01-01

    A combination of agarose gel electrophoresis and a newly developed technique of electro-affinity transfer was applied to the detection of circulating immune complexes of human alpha-fetoprotein (AFP) and anti-AFP. After electrophoretic transfer to nitrocellulose membrane, to which affinity-purified polyclonal horse antibodies to human AFP were bound, the membranes were treated with or without rabbit immunoglobulins to human AFP, followed by overlaying with horseradish peroxidase-labeled goat ...

  20. Naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein partially purified from rat liver and rat brain membranes: an opioid/sigma receptor?

    Science.gov (United States)

    Tsao, L I; Su, T P

    1997-02-01

    A naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein was partially purified from rat liver and rat brain membranes in an affinity chromatography originally designed to purify sigma receptors. Detergent-solubilized extracts from membranes were adsorbed to Sephadex G-25 resin containing an affinity ligand for sigma receptors: N-(2- 3,4-dichlorophenyl]ethyl)-N-(6-aminohexyl)-(2-[1-pyrrolidinyl]) ethylamine (DAPE). After eluting the resin with haloperidol, a protein that bound [3H](+)SKF-10047 was detected in the eluates. However, the protein was not the sigma receptor. [3H](+)SKF-10047 binding to the protein was inhibited by the following compounds in the order of decreasing potency: (+)pentazocine > (-) pentazocine > (+/-)cyclazocine > (-)morphine > (-)naloxone > haloperidol > (+)SKF-10047 > DADLE > (-)SKF-10047. Further, the prototypic sigma receptor ligands, such as 1,3-di-o-tolylguanidine (DTG), (+)3-PPP, and progesterone, bound poorly to the protein. Tryptic digestion and heat treatment of the affinity-purified protein abolished radioligand binding. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of the partially-purified protein from the liver revealed a major diffuse band with a molecular mass of 31 kDa, a polypeptide of 65 kDa, and another polypeptide of > 97 kDa. This study demonstrates the existence of a novel protein in the rat liver and rat brain which binds opioids, benzomorphans, and haloperidol with namomolar affinity. The protein resembles the opioid/sigma receptor originally proposed by Martin et al. [(1976): J. Pharmacol. Exp. Ther., 197:517-532.]. A high degree of purification of this protein has been achieved in the present study.

  1. Antisymmetric tensor generalizations of affine vector fields.

    Science.gov (United States)

    Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-02-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

  2. Tandem mirror technology demonstration facility

    International Nuclear Information System (INIS)

    1983-10-01

    This report describes a facility for generating engineering data on the nuclear technologies needed to build an engineering test reactor (ETR). The facility, based on a tandem mirror operating in the Kelley mode, could be used to produce a high neutron flux (1.4 MW/M 2 ) on an 8-m 2 test area for testing fusion blankets. Runs of more than 100 h, with an average availability of 30%, would produce a fluence of 5 mW/yr/m 2 and give the necessary experience for successful operation of an ETR

  3. Tandem mirror technology demonstration facility

    Energy Technology Data Exchange (ETDEWEB)

    1983-10-01

    This report describes a facility for generating engineering data on the nuclear technologies needed to build an engineering test reactor (ETR). The facility, based on a tandem mirror operating in the Kelley mode, could be used to produce a high neutron flux (1.4 MW/M/sup 2/) on an 8-m/sup 2/ test area for testing fusion blankets. Runs of more than 100 h, with an average availability of 30%, would produce a fluence of 5 mW/yr/m/sup 2/ and give the necessary experience for successful operation of an ETR.

  4. Linac postaccelerators for tandem machines

    International Nuclear Information System (INIS)

    Jaeschke, E.

    1984-01-01

    Linear accelerators as postaccelerators have become an accepted total for increasing the final energy of tandem accelerators. This special application of linacs requires an extreme flexibility to cope with a wide velocity and specific charge range of ions while maintaining the excellent beam quality of the electrostatic machines. These requirements can best be fulfilled by choosing independently phased accelerator resonators of the spiral, splitring or quarter wave type in either normal or superconducting technology. Basic design considerations for postaccelerators are discussed and a survey about the major projects, operational or planned, is given. (orig.)

  5. A Novel Vertex Affinity for Community Detection

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  6. A thermal responsive affinity ligand for precipitation of sialylated proteins

    Directory of Open Access Journals (Sweden)

    Lindsay Arnold

    2016-01-01

    Full Text Available We report here the development of a thermal responsive affinity ligand specific to sialic acid, sialic acid containing oligosaccharides, glycoproteins, and other sialylated glycoconjugates. The ligand is a fusion protein of 40 repeats of pentapeptide of an elastin like polymer (ELP and the 21 kD sialic acid binding domain of a Vibrio cholera neuraminidase (VCNA. For cost-effective synthesis, the fusion protein was targeted to the periplasmic space of an E. coli lpp deletion mutant, resulting in its secretion to the growth medium. A pre-induction heat-shock step at 42 ˚C for 20 minutes was necessary to achieve high level expression of the ligand. Under optimized induction condition (18 ˚C, 0.1 mM IPTG and 48 hours of post-induction cultivation, the ligand was produced to about 100 mg/L. The ligand exhibited a transition temperature of 52 ˚C, which could be depressed to 37 ˚C with the addition of 0.5 M NaCl. Using fetuin as a model sialylated protein, the ligand was applied in an affinity precipitation process to illustrate its potential application in glycoprotein isolation. The ligand captured 100% fetuin from an aqueous solution when the molar ratio of ligand to fetuin was 10 to 1, which was lower than the expected for full titration of sialic acid on the glycoprotein by the lectin. Elution of fetuin from ligand was achieved with PBS buffer containing 2 mM sialic acid. To evaluate how protein and other contaminants influence the recovery of sialylated proteins, CHO medium was spiked into the fetuin solution. The predominant protein species in CHO medium was found to be albumin. Although its removal of over 94% was evident, purified fetuin contained some albumin due to its over-abundance. Additional experiments with albumin contaminant of varying concentrations showed that below 1 mg/L, albumin had no impact on the affinity precipitation, whereas above 10 mg/L, some albumin was co-purified with fetuin. However, even at 50 mg/ml, fetuin

  7. Robust sparse image reconstruction of radio interferometric observations with PURIFY

    Science.gov (United States)

    Pratley, Luke; McEwen, Jason D.; d'Avezac, Mayeul; Carrillo, Rafael E.; Onose, Alexandru; Wiaux, Yves

    2018-01-01

    Next-generation radio interferometers, such as the Square Kilometre Array, will revolutionize our understanding of the Universe through their unprecedented sensitivity and resolution. However, to realize these goals significant challenges in image and data processing need to be overcome. The standard methods in radio interferometry for reconstructing images, such as CLEAN, have served the community well over the last few decades and have survived largely because they are pragmatic. However, they produce reconstructed interferometric images that are limited in quality and scalability for big data. In this work, we apply and evaluate alternative interferometric reconstruction methods that make use of state-of-the-art sparse image reconstruction algorithms motivated by compressive sensing, which have been implemented in the PURIFY software package. In particular, we implement and apply the proximal alternating direction method of multipliers algorithm presented in a recent article. First, we assess the impact of the interpolation kernel used to perform gridding and degridding on sparse image reconstruction. We find that the Kaiser-Bessel interpolation kernel performs as well as prolate spheroidal wave functions while providing a computational saving and an analytic form. Secondly, we apply PURIFY to real interferometric observations from the Very Large Array and the Australia Telescope Compact Array and find that images recovered by PURIFY are of higher quality than those recovered by CLEAN. Thirdly, we discuss how PURIFY reconstructions exhibit additional advantages over those recovered by CLEAN. The latest version of PURIFY, with developments presented in this work, is made publicly available.

  8. Device operation of organic tandem solar cells

    NARCIS (Netherlands)

    Hadipour, A.; de Boer, B.; Blom, P. W. M.

    2008-01-01

    A generalized methodology is developed to obtain the current-voltage characteristic of polymer tandem solar cells by knowing the electrical performance of both sub cells. We demonstrate that the electrical characteristics of polymer tandem solar cells are correctly predicted for both the series and

  9. Folded tandem ion accelerator facility at Trombay

    Indian Academy of Sciences (India)

    Keywords. Folded tandem ion accelerator; carbon beam; magnets; electrostatic deflector; quadrupole lenses; foil/gas strippers; Rutherford back scattering. Abstract. The folded tandem ion accelerator (FOTIA) project at BARC has been commissioned. The analysed carbon beams of 40 nA(3+) and 25 nA(4+), at terminal ...

  10. Tandem Translation Classroom: A Case Study

    Science.gov (United States)

    Kim, Dohun; Koh, Taejin

    2018-01-01

    The transition to student-centred learning, advances in teleconferencing tools, and active international student exchange programmes have stimulated tandem learning in many parts of the world. This pedagogical model is based on a mutual language exchange between tandem partners, where each student is a native speaker in the language the…

  11. The Kyoto University tandem upgrading project

    International Nuclear Information System (INIS)

    Nakamura, Masanobu; Shimoura, Susumu; Takimoto, Kiyohiko; Sakaguchi, Harutaka; Kobayashi, Shinsaku

    1988-01-01

    A brief description on the Kyoto University tandem upgrading project. The project consists of replacing the old 5 MV tandem Van de Graaff by an 8UDH pelletron. The old pressure vessel and beam lines are used again without significant modification. The project is planned to be completed at the end of 1989. (orig.)

  12. Packet models revisited: tandem and priority systems

    NARCIS (Netherlands)

    M.R.H. Mandjes (Michel)

    2004-01-01

    textabstractWe examine two extensions of traditional single-node packet-scale queueing models: tandem networks and (strict) priority systems. Two generic input processes are considered: periodic and Poisson arrivals. For the two-node tandem, an exact expression is derived for the joint distribution

  13. Scaling analysis of affinity propagation

    Science.gov (United States)

    Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

    2010-06-01

    We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N(h+2)/(h+1)) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N(2-d)/(h+1)d . Finally, under some conditions we observe that there is a value s∗ of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss∗ ) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set.

  14. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Science.gov (United States)

    Bee, Christine; Abdiche, Yasmina N; Pons, Jaume; Rajpal, Arvind

    2013-01-01

    Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  15. On affine non-negative matrix factorization

    DEFF Research Database (Denmark)

    Laurberg, Hans; Hansen, Lars Kai

    2007-01-01

    We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate...

  16. Using Affinity Diagrams to Evaluate Interactive Prototypes

    DEFF Research Database (Denmark)

    Lucero, Andrés

    2015-01-01

    Affinity diagramming is a technique used to externalize, make sense of, and organize large amounts of unstructured, far-ranging, and seemingly dissimilar qualitative data. HCI and interaction design practitioners have adopted and used affinity diagrams for different purposes. This paper discusses...

  17. Global affine differential geometry of hypersurfaces

    CERN Document Server

    Li, An-Min; Zhao, Guosong; Hu, Zejun

    2015-01-01

    This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

  18. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...

  19. Improving image segmentation by learning region affinities

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  20. Development of a biogas purifier for rural areas in Japan

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Y.; Hinata, T. [Hokkaido Central Agricultural Experiment Station, Hokkaido (Japan); Yasui, S. [Zukosha Co. Ltd., Obihiro, Hokkaido (Japan); Noguchi, N. [Hokkaido Univ., Sapporo, Hokkaido (Japan); Tsukamoto, T. [IHI Shibaura. Co. Ltd., Obihiro, Hokkaido (Japan); Imai, T. [Green Plan Co. Ltd., Sapporo, Hokkaido (Japan); Kanai, M. [Air Water Co. Ltd, Sakai, Osaka (Japan); Matsuda, Z. [Hokuren Agricultural Research Center, Sapporo, Hokkaido (Japan)

    2010-07-01

    Although the biogas that is currently produced for dairy farms in Japan is a carbon-neutral energy, its use is restricted to farming areas only because there is no effective method of transporting unused biogas. There is a need for establishing practical methods for biogas removal from operating systems. In this study, a gas separation membrane was used in order to modify biogas to city gas 12A specifications, and to develop a biogas purifier equipped with a device to fill high pressure purified gas into cylinders to be taken outside the farming area. The objective was to expand the use of biogas produced from stand-alone gas plants. The amount of purified gas produced at a newly created refining-compression-filling (RCF) facility was approximately 97.0 Nm{sup 3}/day, for a raw material amount of about 216.0 Nm{sup 3}/day. The heat quantity of the purified gas was 38.9 MJ/Nm{sup 3}, which was within city gas 12A specifications. A total of 14.3 cylinders were filled each day with the manufactured purified gas. Test calculations along with a simulation exercise revealed that it would be possible to provide purified gas to approximately 6 per cent of common residences in a town in northern Japan. It was concluded that the newly created RCF facility allowed the modification of carbon-neutral biogas to conform to city gas 12A specifications, and allowed the transport of this gas out of the farming area.

  1. In vitro evolution and affinity-maturation with Coliphage qβ display.

    Directory of Open Access Journals (Sweden)

    Claudia Skamel

    Full Text Available The Escherichia coli bacteriophage, Qβ (Coliphage Qβ, offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV. DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

  2. Metal chelate affinity precipitation of RNA and purification of plasmid DNA

    Science.gov (United States)

    Balan, Sindhu; Murphy, Jason; Galaev, Igor; Kumar, Ashok; Fox, George E.; Mattiasson, Bo; Willson, Richard C.

    2003-01-01

    The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.

  3. Status of JAERI tandem accelerator

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Tadashi; Kanda, Susumu; Takeuchi, Suehiro [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment] (and others)

    2001-02-01

    JAERI Tandem Accelerator had been operated approximately 230 days in fiscal year of 1999. Meanwhile, we had three times of maintenance period with vent. Total operation-times were 5273 hours. We could not carry out the experiment using rare gas, due to malfunction of the RF power supply for the ECR ion source. The type of the RF power supply is peculiar and it is impossible to get spare parts for repair. We are now investigating the backup RF power supply. The power supply for the magnet became unstable due to degradation of insulation in the shunt resistance, which is used for feedback stabilization. Stability was recovered after cleaning. The acrylic resin shaft was cracked. This cracks have a potential for severe accidents. So far bearing of the shaft has no problem. The reason of cracks may be self-destruction by charge accumulation in the shaft. JAERI Tandem Accelerator is approximately 20 years old. There appear requirements on the higher ion currents for additional ion species. Therefore, authors are investigating cost effective improvement plans of RFQ (Radio Frequency Quadra-pole) and IH type accelerator based on KEK (High Energy Accelerator Research Organization) R and D. As a whole, maintenance services for the control system are increasing due to some changes of computer programs. There are some difficulties to keep skilled personnel for facilities operation. Authors are gradually increasing hired personnel with contract from 1993. However, loads for JAERI permanent staffs are still heavy. It takes much longer time to educate skilled persons especially for safety. (Y. Tanaka)

  4. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  5. A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography.

    Science.gov (United States)

    Qu, Jian-Bo; Huang, Yong-Dong; Jing, Guang-Lun; Liu, Jian-Guo; Zhou, Wei-Qing; Zhu, Hu; Lu, Jian-Ren

    2011-05-01

    Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. The Cutting Edge of Affinity Electrophoresis Technology

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  7. The Cutting Edge of Affinity Electrophoresis Technology.

    Science.gov (United States)

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-03-18

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.

  8. Air purification by cementitious materials : Evaluation of air purifying properties

    NARCIS (Netherlands)

    Hüsken, G.; Brouwers, H.J.H.; Al-Mattarneh, H.; Mustapha, K.N.; Nuruddin, M.F.

    2008-01-01

    This paper addresses the evaluation of the photocatalytic properties of concrete containing titanium dioxide (TiO2). Here, the assessment of the air purifying abilities of the hardened concrete regarding the degradation of nitric oxide (NO) is of major interest. A setup for measuring the performance

  9. Effect of partially purified angiotensin converting enzyme inhibitory ...

    African Journals Online (AJOL)

    This study evaluated the effect of partially-purified angiotensin converting enzyme (ACE) inhibitory proteins obtained from the leaves of Moringa oleifera on blood glucose, serum ACE activity and lipid profile of alloxaninduced diabetic rats. Twenty-five apparently healthy male albino rats were divided into five groups of five ...

  10. Predicting group of metabolites available in partially purified tomato ...

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture .... chemistry, organic chemistry, physical chemistry and biochemistry. Normally, isomers of a .... ced analytic capabilities and reduced analyzing time. When combined with infrared spectroscopy, the ...

  11. Delivery of metronidazole from purified Nigerian sheabutter in ...

    African Journals Online (AJOL)

    The drug delivery propensities of 1 %w/w metronidazole (MTZ) ointment formulations in purified Nigerian sheabutter base have been compared to those of similar formulations produced with standard or modified bases, with a view to determining bases capable of ensuring rapid release of MTZ from ointment formulations ...

  12. Influence of a highly purified senna extract on colonic epithelium

    NARCIS (Netherlands)

    van Gorkom, B A; Karrenbeld, A; van Der Sluis, T; Koudstaal, J; de Vries, E G; Kleibeuker, J H

    2000-01-01

    BACKGROUND: Chronic use of sennoside laxatives often causes pseudomelanosis coli. A recent study suggested that pseudomelanosis coli is associated with an increased colorectal cancer risk. A single high dose of highly purified senna extract increased proliferation rate and reduced crypt length in

  13. Method of purifying phosphoric acid after solvent extraction

    International Nuclear Information System (INIS)

    Kouloheris, A.P.; Lefever, J.A.

    1979-01-01

    A method of purifying phosphoric acid after solvent extraction is described. The phosphoric acid is contacted with a sorbent which sorbs or takes up the residual amount of organic carrier and the phosphoric acid separated from the organic carrier-laden sorbent. The method is especially suitable for removing residual organic carrier from phosphoric acid after solvent extraction uranium recovery. (author)

  14. Introducing a simple and economical method to purify Giardia ...

    African Journals Online (AJOL)

    To prepare anti-Giardia lamblia antibodies needed to design diagnostic kits as well as parasite culture and other molecular studies, we require purification of the parasite cysts. In this study, we designed a rapid, simple and inexpensive method to purify parasite cysts from fecal samples of the patients suffering from ...

  15. Mobile Technology Affinity in Renal Transplant Recipients.

    Science.gov (United States)

    Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

    Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Scaling laws in phytoplankton nutrient uptake affinity

    DEFF Research Database (Denmark)

    Lindemann, Christian; Fiksen, Øyvind; Andersen, Ken Haste

    2016-01-01

    Nutrient uptake affinity affects the competitive ability of microbial organisms at low nutrient concentrations. From the theory of diffusion limitation it follows that uptake affinity scales linearly with the cell radius. This is in conflict with some observations suggesting that uptake affinity...... scales to a quantity that is closer to the square of the radius, i.e. to cell surface area. We show that this apparent conflict can be resolved by nutrient uptake theory. Pure diffusion limitation assumes that the cell is a perfect sink which means that it is able to absorb all encountered nutrients...

  17. Affinity Strings: Enterprise Data for Resource Recommendations

    Directory of Open Access Journals (Sweden)

    Shane Nackerud

    2008-12-01

    Full Text Available The University of Minnesota Libraries have created a MyLibrary portal, with databases and e-journals targeted to users, based on their affiliations. The University's enterprise authentication system provides an "affinity string", now used to personalize the MyLibrary portal. This affinity string automates discovery of a user's relationship to the University--describing a user's academic department and degree program or position at the University. Affinity strings also provide the Libraries with an anonymized view of resource usage, allowing data collection that respects users' privacy and lays the groundwork for automated recommendation of relevant resources based on the practices and habits of their peers.

  18. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    Science.gov (United States)

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The N- and C-Terminal Domains Differentially Contribute to the Structure and Function of Dystrophin and Utrophin Tandem Calponin-Homology Domains.

    Science.gov (United States)

    Singh, Surinder M; Bandi, Swati; Mallela, Krishna M G

    2015-11-24

    Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions.

  20. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    Science.gov (United States)

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.

  1. Use of anionic denaturing detergents to purify insoluble proteins after overexpression

    Directory of Open Access Journals (Sweden)

    Schlager Benjamin

    2012-12-01

    Full Text Available Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.

  2. Automorphisms in Birational and Affine Geometry

    CERN Document Server

    Ciliberto, Ciro; Flenner, Hubert; McKernan, James; Prokhorov, Yuri; Zaidenberg, Mikhail

    2014-01-01

    The main focus of this volume is on the problem of describing the automorphism groups of affine and projective varieties, a classical subject in algebraic geometry where, in both cases, the automorphism group is often infinite dimensional. The collection covers a wide range of topics and is intended for researchers in the fields of classical algebraic geometry and birational geometry (Cremona groups) as well as affine geometry with an emphasis on algebraic group actions and automorphism groups. It presents original research and surveys and provides a valuable overview of the current state of the art in these topics. Bringing together specialists from projective, birational algebraic geometry and affine and complex algebraic geometry, including Mori theory and algebraic group actions, this book is the result of ensuing talks and discussions from the conference “Groups of Automorphisms in Birational and Affine Geometry” held in October 2012, at the CIRM, Levico Terme, Italy. The talks at the conference high...

  3. Biodiversity, zoogeography and affinity of Orissa sponges

    Digital Repository Service at National Institute of Oceanography (India)

    Thomas, P.A.; Sree, A.; Bapuji, M.; Rao, K.M.; Murthy, K.S.R.

    from these reefs. The diversity, bio-zeo-distribution and affinity of these sponges are discussed. The analysis of data is mainly arrived at by quasi-quantitative sampling supported by observations and video documentation. Community structure...

  4. Quantum deformation of the affine transformation algebra

    International Nuclear Information System (INIS)

    Aizawa, N.; Sato, Haru-Tada

    1994-01-01

    We discuss a quantum deformation of the affine transformation algebra in one-dimensional space. It is shown that the quantum algebra has a non-cocommutative Hopf algebra structure, simple realizations and quantum tensor operators. (orig.)

  5. Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents.

    Science.gov (United States)

    Katilius, Evaldas; Carmel, Andrew B; Koss, Heidi; O'Connell, Dan; Smith, Breanna C; Sanders, Glenn M; LaBerge, Greggory S

    2018-03-27

    We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (forensic casework. Copyright © 2018. Published by Elsevier B.V.

  6. Affine Moment Invariants Generated by Graph Method

    Czech Academy of Sciences Publication Activity Database

    Suk, Tomáš; Flusser, Jan

    2011-01-01

    Roč. 44, č. 9 (2011), 2047 – 2056 ISSN 0031-3203 R&D Projects: GA ČR(CZ) GA102/08/1593 Institutional research plan: CEZ:AV0Z10750506 Keywords : Image moments * Object recognition * Affine transformation * Affine moment invariants * Pseudoinvariants * Graph representation * Irreducibility * Independence Subject RIV: IN - Informatics, Computer Science Impact factor: 2.292, year: 2011 http://library.utia.cas.cz/separaty/2011/ZOI/suk-0359752.pdf

  7. Rapid purification of circular DNA by triplex-mediated affinity capture

    Science.gov (United States)

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  8. New tandem accelerator facility of Tokyo University

    International Nuclear Information System (INIS)

    Kobayashi, Koichi; Hatori, Satoshi; Nakano, Chuichiro

    1993-01-01

    An old tandem accelerator is being replaced by a new one at the University of Tokyo. The new facility consists of a vertical 5 MV Pelletron with two ion sources and dedicated beam lines for fine analytical studies. (orig.)

  9. Inverted 3J Tandem Thermophotovoltaic Modules Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Spire Semiconductor proposes to make an InGaAs-based three-junction (3J) tandem thermophotovoltaic (TPV) cell to utilize more of the blackbody spectrum (from a GPHS)...

  10. Different endothelin receptor affinities in dog tissues

    International Nuclear Information System (INIS)

    Loeffler, B.M.L.; Loehrer, W.

    1991-01-01

    Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here the authors report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl2, 1 mM EDTA, pH 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 micrograms of protein) were incubated at 25 degrees C for 180 min in the presence of 20 pM 125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 - 80 pM, Bmax 60 - 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 pM, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 pM, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 - 6 nM

  11. TASKA - Tandem Spiegelmaschine Karlsruhe. Vol. 1

    International Nuclear Information System (INIS)

    1982-06-01

    TASKA (Tandem Spiegelmaschine Karlsruhe) is a near term engineering test facility based on a tandem mirror concept with thermal barriers. The main objectives of this study were to develop a preconceptual design of a facility that could provide engineering design information for a Demonstration Fusion Power Reactor. Thus TASKA has to serve as testbed for technologies of plasma engineering, superconducting magnets, materials, plasma heating, breeding and test blankets, tritium technology, and remote handling. (orig.) [de

  12. Large deviations for tandem queueing systems

    Directory of Open Access Journals (Sweden)

    Roland L. Dobrushin

    1994-01-01

    Full Text Available The crude asymptotics of the large delay probability in a tandem queueing system is considered. The main result states that one of the two channels in the tandem system defines the crude asymptotics. The constant that determines the crude asymptotics is given. The results obtained are based on the large deviation principle for random processes with independent increments on an infinite interval recently established by the authors.

  13. Vortex shedding from tandem cylinders

    Science.gov (United States)

    Alam, Md. Mahbub; Elhimer, Mehdi; Wang, Longjun; Jacono, David Lo; Wong, C. W.

    2018-03-01

    An experimental investigation is conducted on the flow around tandem cylinders for ranges of diameter ratio d/ D = 0.25-1.0, spacing ratio L/ d = 5.5-20, and Reynolds number Re = 0.8 × 104-2.42 × 104, where d and D are the diameters of the upstream and downstream cylinders, respectively, L is the distance from the upstream cylinder center to the forward stagnation point of the downstream one. The focus is given on examining the effects of d/ D, L/ d and Re on Strouhal number St, flow structures and fluid forces measured using hotwire, particle image velocimetry (PIV) and load cell measurement techniques, respectively. Changes in d/ D and L/ d in the ranges examined lead to five flow regimes, namely lock-in, intermittent lock-in, no lock-in, subharmonic lock-in and shear-layer reattachment regimes. Time-mean drag coefficient ( C D) and fluctuating drag and lift coefficients ({C^'D} and {C^'L}) are more sensitive to L/ d than d/ D. The scenario is opposite for St where d/ D is more prominent than L/ d to change the St. The detailed facet of the dependence on d/ D and L/ d of C D, {C^'D}, {C^'L} and St is discussed based on shear-layer velocity, approaching velocity, vortex formation length, and wake width.

  14. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    International Nuclear Information System (INIS)

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na + , Cl - and K + to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na + . Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na + and Cl - , the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na + binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl - . Cl - enhances the transporters affinity for imipramine, as well as for Na + . At concentrations in the range of its K M for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na + -independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both [ 3 H]imipramine binding and [ 3 H]serotonin transport

  15. The metric-affine gravitational theory as the gauge theory of the affine group

    International Nuclear Information System (INIS)

    Lord, E.A.

    1978-01-01

    The metric-affine gravitational theory is shown to be the gauge theory of the affine group, or equivalently, the gauge theory of the group GL(4,R) of tetrad deformations in a space-time with a locally Minkowskian metric. The identities of the metric-affine theory, and the relationship between them and those of general relativity and Sciama-Kibble theory, are derived. (Auth.)

  16. Thermal inactivation kinetics of partially purified mango pectin methylesterase

    Directory of Open Access Journals (Sweden)

    Claudio Alonso DÍAZ-CRUZ

    2016-01-01

    Full Text Available Abstract Kinetic parameters of thermal inactivation of pectin methylesterase (PME in a partially purified mango enzyme extract were determined. The PME of mango partially purified by salting out showed different patterns of thermal inactivation, indicating the presence of a thermostable fraction at 70 °C and a thermolabile fraction at lower temperatures. The inactivation of the thermostable fraction exhibited a linear behavior that yielded a z-value of 9.44 °C and an activation energy (Ea of 245.6 kJ mol-1 K-1 using the Arrhenius model. The thermostable mango PME fraction represented 17% of total crude enzyme extract, which emphasizes the importance of residual enzyme activity after heat treatment.

  17. Ultrasonic-resonator-combined apparatus for purifying nuclear aerosol particles

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Suxia; Zhang, Quanhu; Li, Sufen; Chen, Chen; Su, Xianghua [Xi' an Hi-Tech Institute, Xi' an (China)

    2017-12-15

    The radiation hazards of radionuclides in the air arising from the storage room of nuclear devices to the operators cannot be ignored. A new ultrasonic-resonator-combined method for purifying nuclear aerosol particles is introduced. To remove particles with diameters smaller than 0.3 μm, an ultrasonic chamber is induced to agglomerate these submicron particles. An apparatus which is used to purify the nuclear aerosol particles is described in the article. The apparatus consists of four main parts: two filtering systems, an ultrasonic chamber and a high-pressure electrostatic precipitator system. Finally, experimental results demonstrated the effectiveness of the implementation of the ultrasonic resonators. The feasibility of the method is proven by its application to the data analysis of the experiments.

  18. Effect of Amphiphilic Alkyl Chain Length Upon Purified LATEX Stability

    International Nuclear Information System (INIS)

    Amira Amir Hassan; Amir Hashim Mohd Yatim

    2015-01-01

    Rubber particles in purified latex (PL) are stabilized by a film of protein and fatty acid soap (surfactant). Saturated straight-chain fatty acid soaps can assist an enhancement of latex stability. However, whether the alkyl chain length plays an important role in increasing the stability is still an issue. The aim of this study is to investigate the effect of alkyl chain length of anionic surfactant on the stability of purified latex. The fatty acid soap of decanoate (9), laurate (11), sodium dodecyl sulphate (SDS) (12) and palmitate (15) were used. The numbers in parentheses indicating the number of carbon present in alkyl chain of the soap. The results showed that the impact of alkyl chain length on the stability of latex is in the order of laurate > decanoate > SDS > palmitate > purified latex accordingly. The alkyl chain length does giving a significant effect on latex stability after longer stirring time. The particle size of latex with the presence of surfactant is greater compare to a single particle itself due to extension of particles diameter. Thus suitable interaction of the nonpolar tail of surfactant with the hydrophobic regions of latex surface played a major role in maintaining a stable latex system. (author)

  19. Proteomic analysis of purified coronavirus infectious bronchitis virus particles

    Directory of Open Access Journals (Sweden)

    Shu Dingming

    2010-06-01

    Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

  20. Physicochemical and kinetic properties of purified sheep's milk xanthine oxidoreductase.

    Science.gov (United States)

    Benboubetra, Mustapha; Baghiani, Abderahmene; Atmani, Djebbar; Harrison, Roger

    2004-06-01

    Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 +/- 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 +/- 0.04 micromole urate min(-1) mg(-1)) was low relative to that of the bovine milk enzyme (1.83 +/- 0.02 micromole urate min(-1) mg(-1)), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulpho-enzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.

  1. Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

    Directory of Open Access Journals (Sweden)

    Uldaeliz Trujillo

    Full Text Available The polyunsaturated fatty acid (PUFA synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect and in structural stabilization of the multidomain protein (synergistic effect. While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of

  2. Solution Structure of the Tandem Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase Reveals Beads-on-a-String Configuration

    KAUST Repository

    Trujillo, Uldaeliz

    2013-02-28

    The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP

  3. DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

    Science.gov (United States)

    Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

    2018-01-01

    Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Enzymatic Hydrolysis of Alginate to Produce Oligosaccharides by a New Purified Endo-Type Alginate Lyase

    Directory of Open Access Journals (Sweden)

    Benwei Zhu

    2016-06-01

    Full Text Available Enzymatic hydrolysis of sodium alginate to produce alginate oligosaccharides has drawn increasing attention due to its advantages of containing a wild reaction condition, excellent gel properties and specific products easy for purification. However, the efficient commercial enzyme tools are rarely available. A new alginate lyase with high activity (24,038 U/mg has been purified from a newly isolated marine strain, Cellulophaga sp. NJ-1. The enzyme was most active at 50 °C and pH 8.0 and maintained stability at a broad pH range (6.0–10.0 and temperature below 40 °C. It had broad substrate specificity toward sodium alginate, heteropolymeric MG blocks (polyMG, homopolymeric M blocks (polyM and homopolymeric G blocks (polyG, and possessed higher affinity toward polyG (15.63 mM as well as polyMG (23.90 mM than polyM (53.61 mM and sodium alginate (27.21 mM. The TLC and MS spectroscopy analysis of degradation products suggested that it completely hydrolyzed sodium alginate into oligosaccharides of low degrees of polymerization (DPs. The excellent properties would make it a promising tool for full use of sodium alginate to produce oligosaccharides.

  5. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

    Directory of Open Access Journals (Sweden)

    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  6. Classification of neocortical interneurons using affinity propagation

    Science.gov (United States)

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  7. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  8. The interaction between beta 2-microglobulin (beta 2m) and purified class-I major histocompatibility (MHC) antigen

    DEFF Research Database (Denmark)

    Pedersen, L O; Hansen, A S; Olsen, A C

    1994-01-01

    been generated recently and this paper reports on a similar assay for the interaction between beta 2m and class I. As a model system human beta 2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined...... by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nM. The rate of association at 37 degrees C is very fast (the ka is around 5 x 10(4)/M/s) whereas the dissociation is slow (the kd is around 8 x 10(-6)/s); the ratio of dissociation...

  9. Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

    Directory of Open Access Journals (Sweden)

    Luis F. Padilla-Morales

    2016-03-01

    Full Text Available The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR rich membrane solubilized with long chain (16 saturated carbons lysophospholipid with glycerol headgroup (LFG-16. The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique. Keywords: Detergents, Fluorescence recovery after photobleaching, Lipidic Cubic Phase, nAChR, Planar lipid bilayer, Two-electrode voltage clamp

  10. Biomimetic affinity ligands for protein purification.

    Science.gov (United States)

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  11. Monolithic media for applications in affinity chromatography.

    Science.gov (United States)

    Sproß, Jens; Sinz, Andrea

    2011-08-01

    Affinity chromatography presents a highly versatile analytical tool, which relies on exploiting highly specific interactions between molecules and their ligands. This review covers the most recent literature on the application of monoliths as stationary phases for various affinity-based chromatographic applications. Different affinity approaches as well as separations using molecularly imprinted monoliths are discussed. Hybrid stationary phases created by embedding of particles or nanoparticles into a monolithic stationary phase are also considered in this review article. The ease of preparation of monoliths and the multitude of functionalization techniques, which have matured during the past years, make monoliths interesting for an increasing number of biochemical and medical applications. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. On Affine Fusion and the Phase Model

    Directory of Open Access Journals (Sweden)

    Mark A. Walton

    2012-11-01

    Full Text Available A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n Wess-Zumino-Novikov-Witten (WZNW conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  13. Affine coherent states and Toeplitz operators

    Science.gov (United States)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  14. Tandem Catalysis Utilizing Olefin Metathesis Reactions.

    Science.gov (United States)

    Zieliński, Grzegorz K; Grela, Karol

    2016-07-04

    Since olefin metathesis transformation has become a favored synthetic tool in organic synthesis, more and more distinct non-metathetical reactions of alkylidene ruthenium complexes have been developed. Depending on the conditions applied, the same olefin metathesis catalysts can efficiently promote isomerization reactions, hydrogenation of C=C double bonds, oxidation reactions, and many others. Importantly, these transformations can be carried out in tandem with olefin metathesis reactions. Through addition of one portion of a catalyst, a tandem process provides structurally advanced products from relatively simple substrates without the need for isolation of the intermediates. These aspects not only make tandem catalysis very attractive from a practical point of view, but also open new avenues in (retro)synthetic planning. However, in the literature, the term "tandem process" is sometimes used improperly to describe other types of multi-reaction sequences. In this Concept, a number of examples of tandem catalysis involving olefin metathesis are discussed with an emphasis on their synthetic value. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Tandem mass spectrometry at low kinetic energy

    International Nuclear Information System (INIS)

    Cooks, R.G.; Hand, O.W.

    1987-01-01

    Recent progress in mass spectrometry, as applied to molecular analysis, is reviewed with emphasis on tandem mass spectrometry. Tandem instruments use multiple analyzers (sector magnets, quadrupole mass filters and time-of-flight devices) to select particular molecules in ionic form, react them in the gas-phase and then record the mass, momenta or kinetic energies of their products. The capabilities of tandem mass spectrometry for identification of individual molecules or particular classes of compounds in complex mixtures are illustrated. Several different types of experiments can be run using a tandem mass spectrometer; all share the feature of sifting the molecular mixture being analyzed on the basis of chemical properties expressed in terms of ionic mass, kinetic energy or charge state. Applications of mass spectrometry to biological problems often depend upon desorption methods of ionization in which samples are bombarded with particle beams. Evaporation of preformed charged species from the condensed phase into the vacuum is a particularly effective method of ionization. It is suggested that the use of accelerator mass spectrometers be extended to include problems of molecular analysis. In such experiments, low energy tandem mass spectrometry conducted in the eV or keV range of energies, would be followed by further characterization of the production ion beam using high selective MeV collision processes

  16. The dynamics of metric-affine gravity

    International Nuclear Information System (INIS)

    Vitagliano, Vincenzo; Sotiriou, Thomas P.; Liberati, Stefano

    2011-01-01

    Highlights: → The role and the dynamics of the connection in metric-affine theories is explored. → The most general second order action does not lead to a dynamical connection. → Including higher order invariants excites new degrees of freedom in the connection. → f(R) actions are also discussed and shown to be a non- representative class. - Abstract: Metric-affine theories of gravity provide an interesting alternative to general relativity: in such an approach, the metric and the affine (not necessarily symmetric) connection are independent quantities. Furthermore, the action should include covariant derivatives of the matter fields, with the covariant derivative naturally defined using the independent connection. As a result, in metric-affine theories a direct coupling involving matter and connection is also present. The role and the dynamics of the connection in such theories is explored. We employ power counting in order to construct the action and search for the minimal requirements it should satisfy for the connection to be dynamical. We find that for the most general action containing lower order invariants of the curvature and the torsion the independent connection does not carry any dynamics. It actually reduces to the role of an auxiliary field and can be completely eliminated algebraically in favour of the metric and the matter field, introducing extra interactions with respect to general relativity. However, we also show that including higher order terms in the action radically changes this picture and excites new degrees of freedom in the connection, making it (or parts of it) dynamical. Constructing actions that constitute exceptions to this rule requires significant fine tuned and/or extra a priori constraints on the connection. We also consider f(R) actions as a particular example in order to show that they constitute a distinct class of metric-affine theories with special properties, and as such they cannot be used as representative toy

  17. New unitary affine-Virasoro constructions

    International Nuclear Information System (INIS)

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M.; Yamron, J.P.

    1990-01-01

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g

  18. Control and estimation of piecewise affine systems

    CERN Document Server

    Xu, Jun

    2014-01-01

    As a powerful tool to study nonlinear systems and hybrid systems, piecewise affine (PWA) systems have been widely applied to mechanical systems. Control and Estimation of Piecewise Affine Systems presents several research findings relating to the control and estimation of PWA systems in one unified view. Chapters in this title discuss stability results of PWA systems, using piecewise quadratic Lyapunov functions and piecewise homogeneous polynomial Lyapunov functions. Explicit necessary and sufficient conditions for the controllability and reachability of a class of PWA systems are

  19. Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.

    Directory of Open Access Journals (Sweden)

    Indhu Kanakaraj

    Full Text Available Immobilized Metal Affinity Chromatography (IMAC has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+-iminodiacetic acid (IDA agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+-IDA agarose can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.

  20. Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA.

    Directory of Open Access Journals (Sweden)

    Andrew M Edwards

    2011-04-01

    Full Text Available Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5β(1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.

  1. Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila.

    Science.gov (United States)

    Yilmaz, Gürkan; Arslanyolu, Muhittin

    2015-03-25

    A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila. In vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2-4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting. We successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.

  2. Crystal structure of the three tandem FF domains of the transcription elongation regulator CA150.

    Science.gov (United States)

    Lu, Ming; Yang, Jun; Ren, Zhiyong; Sabui, Subir; Espejo, Alexsandra; Bedford, Mark T; Jacobson, Raymond H; Jeruzalmi, David; McMurray, John S; Chen, Xiaomin

    2009-10-23

    FF domains are small protein-protein interaction modules that have two flanking conserved phenylalanine residues. They are present in proteins involved in transcription, RNA splicing, and signal transduction, and often exist in tandem arrays. Although several individual FF domain structures have been determined by NMR, the tandem nature of most FF domains has not been revealed. Here we report the 2.7-A-resolution crystal structure of the first three FF domains of the human transcription elongation factor CA150. Each FF domain is composed of three alpha-helices and a 3(10) helix between alpha-helix 2 and alpha-helix 3. The most striking feature of the structure is that an FF domain is connected to the next by an alpha-helix that continues from helix 3 to helix 1 of the next. The consequent elongated arrangement allows exposure of many charged residues within the region that can be engaged in interaction with other molecules. Binding studies using a peptide ligand suggest that a specific conformation of the FF domains might be required to achieve higher-affinity binding. Additionally, we explore potential DNA binding of the FF construct used in this study. Overall, we provide the first crystal structure of an FF domain and insights into the tandem nature of the FF domains and suggest that, in addition to protein binding, FF domains might be involved in DNA binding.

  3. Crossing Chris: Some Markerian Affinities

    Directory of Open Access Journals (Sweden)

    Adrian Martin

    2010-01-01

    -pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

    Abstract (E: This essay creatively explores a group of artists, writers, and other special individuals whose work or life story can be described as having an intriguing affinity with the protean career of Chris Marker. Avoiding the ‘usual suspects’ (such as Godard or Sebald, it discusses gossip columnist Milt Machlin, record collector Harry Smith, painter Gianfranco Baruchello, writer-filmmaker Edgardo Cozarinsky, and several others. From this constellation, a particular view of Markerian poetics emerges, touching upon the meanings of anonymity, storytelling, history and archiving.

     

    Abstract (F: Cet essai brosse de manière créative le portrait d’un groupe d'artistes, d'écrivains et d'autres personnes particulières dont le travail ou la biographie peuvent être décrits comme montrant une étrange mais certaine connivence avec la carrière protéiforme de Chris Marker. Evitant les lieux communs (comme Godard ou Sebald, cet article trace des références moins attendues :

  4. PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTO

    Directory of Open Access Journals (Sweden)

    Edgar Antonio Reyes Montaño

    2011-12-01

    Full Text Available Producing polyclonal antibodies (IgY inchickens has advantages over those obtainedin other animal models, since theyhave been used as a tool for studyingdifferent proteins (NMDA glutamate receptorin our case, specifically the NR1subunit. We produced specific antibodiesagainst expression products by thealternative splicing of the gene encodingNMDA receptor NR1 subunit in adult ratbrain. Three peptides corresponding tothe splicing sites (N1, C1 and C2’ cassetteswere designed, synthesised and usedindividually as antigens in hens. Specificimmunoglobulins were purified fromyolks. The antibodies were then used forpurifying the NMDA receptor NR1 subunitusing affinity chromatography couplingthe three antibodies to the support.R

  5. Mass spectrometry by means of tandem accelerators

    International Nuclear Information System (INIS)

    Tuniz, C.

    1985-01-01

    Mass spectrometry based on an accelerator allows to measure rare cosmogenic isotopes found in natural samples with isotopic abundances up to 10E-15. The XTU Tandem of Legnaro National Laboratories can measure mean heavy isotopes (36Cl, 41Ca, 129I) in applications interesting cosmochronology and Medicine. The TTT-3 Tandem of the Naples University has been modified in view of precision studies of C14 in Archeology, Paleantology and Geology. In this paper a review is made of principles and methodologies and of some applicationy in the framework of the National Program for mass spectrametry research with the aid of accelerators

  6. A Purified Recombinant Lipopeptide as Adjuvant for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Ying-Chyi Song

    2014-01-01

    Full Text Available Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs, leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion. Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4. We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.

  7. Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins.

    Directory of Open Access Journals (Sweden)

    Pantelis Georgiades

    Full Text Available Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.

  8. Fan Affinity Laws from a Collision Model

    Science.gov (United States)

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  9. General super Virasoro construction on affine G

    International Nuclear Information System (INIS)

    Mohammedi, N.

    1990-10-01

    We consider a bosonic current algebra and a theory of free fermions and construct a general N = 1 super Virasoro current algebra. We obtain a master-set of equations which comprises the bosonic master equation for general Virasoro construction on affine G. As an illustration we study the case of the group SU(2). (author). 13 refs

  10. It's the peptide-MHC affinity, stupid.

    Science.gov (United States)

    Kammertoens, Thomas; Blankenstein, Thomas

    2013-04-15

    Adoptively transferred T cells can reject large established tumors, but recurrence due to escape variants frequently occurs. In this issue of Cancer Cell, Engels et al. demonstrate that the affinity of the target peptide to the MHC molecule determines whether large tumors will relapse following adoptive T cell therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Proton affinity measurements using ion mobility spectrometry

    International Nuclear Information System (INIS)

    Tabrizchi, Mahmoud.; Shooshtari, Saeed

    2003-01-01

    Relative proton affinities are usually measured by means of high pressure mass spectrometry. In this work ion mobility spectrometry was used to determine proton affinities. The standard molar enthalpy change ΔH 0 M for the reaction MH + +N[rlhar2]M+NH + was found to be: -(56.3±0.8) kJ·mol -1 , when M was ethyl acetate and N ethanol; -(27.8±0.5) kJ·mol -1 , when M was acetophenone and N ethyl acetate; -(10.6±0.2) kJ·mol -1 , when M was cycloheptanone and N ethyl acetate; -(66.1±1.2) kJ·mol -1 , when M was dimethyl methyl phosphonate and N ethyl acetate; and, -(56.7±0.9) kJ·mol -1 , when M was dimethyl methyl phosphonate and N cycloheptanone. These values are internally consistent and in good agreement with results obtained with high pressure mass spectrometry. On the basis of proton affinities of cycloheptanone and ethyl acetate, an absolute proton affinity of (902±1.2) kJ·mol -1 has been determined for dimethyl methyl phosphonate

  12. Classification of neocortical interneurons using affinity propagation

    Directory of Open Access Journals (Sweden)

    Roberto eSantana

    2013-12-01

    Full Text Available In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. Neuronal classification has been a difficult problem because it is unclear what a neuronal cell class actually is and what are the best characteristics are to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological or molecular characteristics, when applied to selected datasets, have provided quantitative and unbiased identification of distinct neuronal subtypes. However, better and more robust classification methods are needed for increasingly complex and larger datasets. We explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. In fact, using a combined anatomical/physiological dataset, our algorithm differentiated parvalbumin from somatostatin interneurons in 49 out of 50 cases. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  13. Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli.

    Science.gov (United States)

    Pearce, Lesley A; Yu, Meng; Waddington, Lynne J; Barr, Jennifer A; Scoble, Judith A; Crameri, Gary S; McKinstry, William J

    2015-12-01

    Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

  14. Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.

    Science.gov (United States)

    Longstaff, Colin; Hogwood, John; Gray, Elaine; Komorowicz, Erzsebet; Varjú, Imre; Varga, Zoltán; Kolev, Krasimir

    2016-03-01

    Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.

  15. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  16. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.

    Science.gov (United States)

    Ohrt, Thomas; Odenwälder, Peter; Dannenberg, Julia; Prior, Mira; Warkocki, Zbigniew; Schmitzová, Jana; Karaduman, Ramazan; Gregor, Ingo; Enderlein, Jörg; Fabrizio, Patrizia; Lührmann, Reinhard

    2013-07-01

    Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.

  17. Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

    DEFF Research Database (Denmark)

    Heegaard, N H; Sen, J W; Nissen, Mogens Holst

    2000-01-01

    The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of......The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red...

  18. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  19. The affinity purification and characterization of ATP synthase complexes from mitochondria.

    Science.gov (United States)

    Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

    2013-02-13

    The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

  20. Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

    Science.gov (United States)

    Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

    2017-11-01

    Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. A dual protease approach for expression and affinity purification of recombinant proteins.

    Science.gov (United States)

    Raran-Kurussi, Sreejith; Waugh, David S

    2016-07-01

    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  2. Liquid-liquid extraction of enzymes by affinity aqueous two-phase systems

    Directory of Open Access Journals (Sweden)

    Xu Yan

    2003-12-01

    Full Text Available From analytical to commercial scale, aqueous two-phase systems have their application in the purification, characterization and study of biomaterials. In order to improve the selectivity of the systems, the biospecific affinity ligands were introduced. In the affinity partitioning aqueous two-phase system, have many enzymes been purified. This review discusses the partitioning of some enzymes in the affinity aqueous two-phase systems in regard to the different ligands, including reactive dyes, metal ions and other ligands. Some integration of aqueous two-phase system with other techniques for more effective purification of enzymes are also presented.Tanto em escala de laboratório como industrial, os sistemas de duas fases aquosas podem ser utilizados para a purificação, caracterização e estudos de biomateriais. Para aumentar a seletividade desse sistema, ligantes de afinidade bioespecíficos podem ser utilizados. No sistema de duas fases aquosas por afinidade, muitas enzimas podem ser purificadas. Neste artigo de revisão, a partição de algumas enzimas por esse tipo de afinidade, utilizando diferentes ligantes como corantes e íons metálicos, são discutidas. Além disso, a integração desse sistema de duas fases aquosas com outras técnicas de purificação estão sendo apresentados, com o objetivo mostrar a melhoria da eficiência do processo.

  3. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (

    Directory of Open Access Journals (Sweden)

    P. Paengkoum

    2015-10-01

    Full Text Available The objectives of this study were to determine the molecular weight of condensed tannins (CT extracted from mangosteen (Garcinia mangostana L peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4 and volatile fatty acids (VFA production. The average molecular weight (Mw of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin. In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM compared to the control (p<0.05. The effects of CT from mangosteen-peel on in vitro DM degradability (IVDMD and in vitro N degradability was negative and linear (p<0.01. Total VFA, concentrations of acetic, propionic, butyric and isovaleric acids decreased linearly with increasing amount of CT. The aforementioned results show that protein binding affinity of CT from mangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations.

  4. Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

    DEFF Research Database (Denmark)

    Heegaard, N H; Sen, J W; Nissen, Mogens Holst

    2000-01-01

    The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red......-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red...... more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins....

  5. A tandem queue with delayed server release

    NARCIS (Netherlands)

    Nawijn, W.M.

    1997-01-01

    We consider a tandem queue with two stations. The rst station is an s-server queue with Poisson arrivals and exponential service times. After terminating his service in the rst station, a customer enters the second station to require service at an exponential single server, while in the meantime he

  6. Efficient simulation of a tandem Jackson network

    NARCIS (Netherlands)

    Kroese, Dirk; Nicola, V.F.

    2002-01-01

    The two-node tandem Jackson network serves as a convenient reference model for the analysis and testing of different methodologies and techniques in rare event simulation. In this paper we consider a new approach to efficiently estimate the probability that the content of the second buffer exceeds

  7. Edge diagnostics for tandem mirror machines

    International Nuclear Information System (INIS)

    Allen, S.L.

    1984-01-01

    The edge plasma in a tandem mirror machine shields the plasma core from cold neutral gas and impurities. A variety of diagnostics are used to measure the fueling, shielding, and confinement of the edge plasma in both the end plug and central cell regions. Fast ion gauges and residual gas analyzers measure the gas pressure and composition outside of the plasma. An array of Langmuir probes is used to measure the electron density and temperature. Extreme ultraviolet (euv) and visible spectroscopy are used to measure both the impurity and deuterium densities and to estimate the shielding factor for the core plasma. The linear geometry of a tandem mirror also allows direct measurements of the edge plasma by sampling the ions and electrons lost but the ends of the machine. Representative data obtained by these diagnostics during operation of the Tandem Mirror Experiment (TMX) and Tandem Mirror Experiment-Upgrade (TMX-U) experiments are presented. Diagnostics that are currently being developed to diagnose the edge plasma are also discussed

  8. Tandem queue with server slow-down

    NARCIS (Netherlands)

    Miretskiy, D.I.; Scheinhardt, W.R.W.; Mandjes, M.R.H.

    2007-01-01

    We study how rare events happen in the standard two-node tandem Jackson queue and in a generalization, the socalled slow-down network, see [2]. In the latter model the service rate of the first server depends on the number of jobs in the second queue: the first server slows down if the amount of

  9. The PSI/ETH tandem accelerator facility

    Energy Technology Data Exchange (ETDEWEB)

    Synal, H.A.; Doebeli, M.; Fuhrmann, H.; Kubik, P.W.; Nebiker, P.W. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)] [and others

    1997-09-01

    The 1996 operation of the PSI/ETH tandem accelerator at ETH Hoenggerberg is summarised with a detailed compilation of the beam time statistics and the statistics of AMS samples for the different radioisotopes and for the major fields of research. (author) 2 tab.

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

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

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

  11. STATIONARY DISTRIBUTION OF A TANDEM QUEUE WITH ADDITIONAL FLOWS ON THE STATIONS OF THE TANDEM

    Directory of Open Access Journals (Sweden)

    V. I. Klimenok

    2017-01-01

    Full Text Available A tandem queueing system consisting of a finite number of multi-server stations without buffers is analized. The input flow at the first station is a ???????????? (Markovian arrival process. The customers from this flow aim to be served at all stations of the tandem. For any station, besides transit customers proceeding from the previous station, an additional ???????????? flow of new customers arrives at this station directly. Customers from this flow aim to be served at this station and all subsequent stations of the tandem. The service times of customer at the stations are exponentially distributed with the service rate depending of number of the station. The algorithms for culculation of stationary distributions and the loss probabilities associated with the tandem are given.

  12. Interpretation of tandem mass spectra of posttranslationally modified peptides

    DEFF Research Database (Denmark)

    Bunkenborg, J.; Matthiesen, R.

    2013-01-01

    Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of rapidly generating many thousands of tandem mas...... and validating tandem mass spectra and gives some useful tables to aid this process....

  13. Extension of heuristics for simulating population overflow in Jackson tandem queuing networks to non-Markovian tandem queuing networks

    NARCIS (Netherlands)

    Zaburnenko, T.S.; de Boer, Pieter-Tjerk; Haverkort, Boudewijn R.H.M.

    In this paper we extend previously proposed state-dependent importance sampling heuristics for simulation of population overflow in Markovian tandem queuing networks to non-Markovian tandem networks, and experimentally demonstrate the asymptotic efficiency of the resulting heuristics.

  14. Measurement of Ozone Emission and Particle Removal Rates from Portable Air Purifiers

    Science.gov (United States)

    Mang, Stephen A.; Walser, Maggie L.; Nizkorodov, Sergey A.; Laux, John M.

    2009-01-01

    Portable air purifiers are popular consumer items, especially in areas with poor air quality. Unfortunately, most users of these air purifiers have minimal understanding of the factors affecting their efficiency in typical indoor settings. Emission of the air pollutant ozone (O[subscript 3]) by certain air purifiers is of particular concern. In an…

  15. Axial dispersion in a Kureha Crystal Purifier (KCP)

    Science.gov (United States)

    Otawara, K.; Matsuoka, T.

    2002-04-01

    It is widely accepted that the degree of axial back-mixing in the equipment affects the performance of a column crystallizer. Specifically, small such mixing is inevitable for obtaining highly pure crystal. In fact, it is believed that scale-up of some column crystallizers has been terminated due to the large axial back-mixing or maldistribution. Thus, experiments have been performed for estimating axial dispersion coefficients of liquid phase representing axial back-mixing in the column of a Kureha Crystal Purifier (KCP). The effect of the column diameter on such coefficients has been investigated and it has become evident that the axial back-mixing in the column is more significant in the larger column. Nevertheless, the results have also indicated that the axial back-mixing in KCPs of industrial sizes can be substantially smaller than those in other types of column crystallizers.

  16. A novel venom protein of the Asian bee (Apis cerana indica with an affinity to human α1-microglobulin

    Directory of Open Access Journals (Sweden)

    Rosdiana Natzir

    1999-01-01

    Full Text Available Bee stings are a common health problem throughout the world and can sometimes result in fatal anaphylactic reactions. We have studied Asian bee (Apis cerana indica, Apis cerana nigrocincta and Apis dorsata venoms and have discovered a novel protein with a molecular size of 50 kDa (p50, as shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which has not been reported in the venom of the Western honey-bee, Apis mellifera (AM. The p50 protein showed a unique affinity to human α1-microglobulin (α1-m. As a result, p50 was purified using an affinity column with α1-m. The p50 protein was further purified by an affinity column with a monoclonal antibody raised against p50 in mice. The p50 protein induced an inflammatory reaction following injection into mouse ear; that is, degranulation of mast cells, edema, hyperemia and hyperpermeation of the local capillaries were observed. The reaction was very similar to that seen when phospholipase A2 of AM, a representative bee venom, was administered by injection. The inflammatory reaction induced by p50 was completely inhibited by mixing p50 with α1-m prior to injection. These results indicate that p50 is a unique venom component of the Asian bee that induces the inflammatory reaction and that human α1-m may be involved as a protective mechanism against bee stings of at least some Asian bee species.

  17. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3.

    Science.gov (United States)

    Cheng, A; Le, T; Palacios, M; Bookbinder, L H; Wassarman, P M; Suzuki, F; Bleil, J D

    1994-05-01

    Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse.

  18. Altered glycosylation profile of purified plasma ACT from Alzheimer's disease.

    Science.gov (United States)

    Ianni, Manuela; Manerba, Marcella; Di Stefano, Giuseppina; Porcellini, Elisa; Chiappelli, Martina; Carbone, Ilaria; Licastro, Federico

    2010-12-16

    Alzheimer's disease (AD) is one of the most frequent cause of neurodegenerative disorder in the elderly. Inflammation has been implicated in brain degenerative processes and peripheral markers of brain AD related impairment would be useful. Plasma levels of alpha-1-antichymotrypsin (ACT), an acute phase protein and a secondary component of amyloid plaques, are often increased in AD patients and high blood ACT levels correlate with progressive cognitive deterioration. During inflammatory responses changes in the micro-heterogeneity of ACT sugar chains have been described. N-Glycanase digestion from Flavobacterium meningosepticum (PNGase F) was performed on both native and denatured purified ACT condition and resolved to Western blot with the purpose to revealed the ACT de-glycosylation pattern.Further characterization of the ACT glycan profile was obtained by a glycoarray; each lectin group in the assay specifically recognizes one or two glycans/epitopes. Lectin-bound ACT produced a glyco-fingerprint and mayor differences between AD and controls samples were assessed by a specific algorithms. Western blot analysis of purified ACT after PNGase F treatment and analysis of sugar composition of ACT showed significantly difference in "glyco-fingerprints" patterns from controls (CTR) and AD; ACT from AD showing significantly reduced levels of sialic acid. A difference in terminal GlcNac residues appeared to be related with progressive cognitive deterioration. Low content of terminal GlcNac and sialic acid in peripheral ACT in AD patients suggests that a different pattern of glycosylation might be a marker of brain inflammation. Moreover ACT glycosylation analysis could be used to predict AD clinical progression and used in clinical trials as surrogate marker of clinical efficacy.

  19. Magnetism for understanding catalyst analysis of purified carbon nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Bellouard, Christine; Mercier, Guillaume; Cahen, Sébastien; Ghanbaja, Jaafar; Medjahdi, Ghouti [Institut Jean Lamour, CNRS-Université de Lorraine, BP 70239, 54506 Vandoeuvre-lès-Nancy (France); Gleize, Jérôme [Laboratoire de Chimie Physique-Approche Multi-échelle de Milieux Complexes-Université de Lorraine, 1 Bd Arago, 57078 Metz (France); Lamura, Gianrico [CNR-SPIN – Dipartimento di Fisica, via Dodecaneso 33, 16146 Genova (Italy); Hérold, Claire [Institut Jean Lamour, CNRS-Université de Lorraine, BP 70239, 54506 Vandoeuvre-lès-Nancy (France); Vigolo, Brigitte, E-mail: Brigitte.Vigolo@univ-lorraine.fr [Institut Jean Lamour, CNRS-Université de Lorraine, BP 70239, 54506 Vandoeuvre-lès-Nancy (France)

    2016-08-01

    The precise quantification of catalyst residues in purified carbon nanotubes is often a major issue in view of any fundamental and/or applicative studies. More importantly, since the best CNTs are successfully grown with magnetic catalysts, their quantification becomes strictly necessary to better understand intrinsic properties of CNT. For these reasons, we have deeply analyzed the catalyst content remained in nickel–yttrium arc-discharge single walled carbon nanotubes purified by both a chlorine-gas phase and a standard acid-based treatment. The study focuses on Ni analysis which has been investigated by transmission electron microscopy, X-ray diffraction, thermogravimetry analysis, and magnetic measurements. In the case of the acid-based treatment, all quantifications result in a decrease of the nanocrystallized Ni by a factor of two. In the case of the halogen gas treatment, analysis and quantification of Ni content is less straightforward: a huge difference appears between X-ray diffraction and thermogravimetry results. Thanks to magnetic measurements, this disagreement is explained by the presence of Ni{sup 2+} ions, belonging to NiCl{sub 2} formed during the Cl-based purification process. In particular, NiCl{sub 2} compound appears under different magnetic/crystalline phases: paramagnetic or diamagnetic, or well intercalated in between carbon sheets with an ordered magnetic phase at low temperature. - Highlights: • Cl-gas treatment of Ni catalyst of carbon nanotubes leads to NiCl{sub 2} residue. • Magnetic measurements show the transformation of Ni{sup 0} in Ni{sup 2+}through a purification process. • High temperature Cl treatment removes 75% of metallic impurities. • Cl-purification yields to an amount of metal of 1.5% in arc-discharge CNT samples.

  20. Quality evaluation of tandem mass spectral libraries.

    Science.gov (United States)

    Oberacher, Herbert; Weinmann, Wolfgang; Dresen, Sebastian

    2011-06-01

    Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including "tandem-in-time" instruments; this is due to the coverage of a large range of different collision energy settings-including the very low range-which is an outstanding characteristics of the MSforID library.

  1. Affine Non-Local Means Image Denoising.

    Science.gov (United States)

    Fedorov, Vadim; Ballester, Coloma

    2017-05-01

    This paper presents an extension of the Non-Local Means denoising method, that effectively exploits the affine invariant self-similarities present in the images of real scenes. Our method provides a better image denoising result by grounding on the fact that in many occasions similar patches exist in the image but have undergone a transformation. The proposal uses an affine invariant patch similarity measure that performs an appropriate patch comparison by automatically and intrinsically adapting the size and shape of the patches. As a result, more similar patches are found and appropriately used. We show that this image denoising method achieves top-tier performance in terms of PSNR, outperforming consistently the results of the regular Non-Local Means, and that it provides state-of-the-art qualitative results.

  2. Staircase Models from Affine Toda Field Theory

    CERN Document Server

    Dorey, P; Dorey, Patrick; Ravanini, Francesco

    1993-01-01

    We propose a class of purely elastic scattering theories generalising the staircase model of Al. B. Zamolodchikov, based on the affine Toda field theories for simply-laced Lie algebras g=A,D,E at suitable complex values of their coupling constants. Considering their Thermodynamic Bethe Ansatz equations, we give analytic arguments in support of a conjectured renormalisation group flow visiting the neighbourhood of each W_g minimal model in turn.

  3. On the Lp affine isoperimetric inequalities

    Indian Academy of Sciences (India)

    Projection bodies have a long and complicated history which goes back to Minkowski. [3]. The classical Petty projection ... (1.1) where. ∗. K is the polar body of projection body K and equality holds if and only if K is an ellipsoid. ..... circumscribed to Bn. Then there is an affine image of ˜K of K satisfying. Sp( ˜K) ≤ Sp(△n) and.

  4. Levels of Distribution and the Affine Sieve

    OpenAIRE

    Kontorovich, Alex

    2014-01-01

    This article is an expanded version of the author's lecture in the Basic Notions Seminar at Harvard, September 2013. Our goal is a brief and introductory exposition of aspects of two topics in sieve theory which have received attention recently: (1) the spectacular work of Yitang Zhang, under the title "Level of Distribution," and (2) the so-called "Affine Sieve," introduced by Bourgain-Gamburd-Sarnak.

  5. Tannakian duality for affine homogeneous spaces

    OpenAIRE

    Banica, Teodor

    2017-01-01

    Associated to any closed quantum subgroup $G\\subset U_N^+$ and any index set $I\\subset\\{1,\\ldots,N\\}$ is a certain homogeneous space $X_{G,I}\\subset S^{N-1}_{\\mathbb C,+}$, called affine homogeneous space. We discuss here the abstract axiomatization of the algebraic manifolds $X\\subset S^{N-1}_{\\mathbb C,+}$ which can appear in this way, by using Tannakian duality methods.

  6. Automata and cells in affine Weyl groups

    OpenAIRE

    Gunnells, Paul E.

    2008-01-01

    Let W~ be an affine Weyl group, and let C be a left, right, or two-sided Kazhdan--Lusztig cell in W~. Let Reduced (C) be the set of all reduced expressions of elements of C, regarded as a formal language in the sense of the theory of computation. We show that Reduced (C) is a regular language. Hence the reduced expressions of the elements in any Kazhdan--Lusztig cell can be enumerated by a finite state automaton.

  7. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency.

    Directory of Open Access Journals (Sweden)

    Wen Ma

    Full Text Available Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs, belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP. However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5 was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed.

  8. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency.

    Science.gov (United States)

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua; Teissedre, Pierre-Louis

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed.

  9. Excited state electron affinity calculations for aluminum

    Science.gov (United States)

    Hussein, Adnan Yousif

    2017-08-01

    Excited states of negative aluminum ion are reviewed, and calculations of electron affinities of the states (3s^23p^2)^1D and (3s3p^3){^5}{S}° relative to the (3s^23p)^2P° and (3s3p^2)^4P respectively of the neutral aluminum atom are reported in the framework of nonrelativistic configuration interaction (CI) method. A priori selected CI (SCI) with truncation energy error (Bunge in J Chem Phys 125:014107, 2006) and CI by parts (Bunge and Carbó-Dorca in J Chem Phys 125:014108, 2006) are used to approximate the valence nonrelativistic energy. Systematic studies of convergence of electron affinity with respect to the CI excitation level are reported. The calculated value of the electron affinity for ^1D state is 78.675(3) meV. Detailed Calculations on the ^5S°c state reveals that is 1216.8166(3) meV below the ^4P state.

  10. In situ detection of tandem DNA repeat length

    Energy Technology Data Exchange (ETDEWEB)

    Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

    1996-11-01

    A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

  11. Magnetic Microbead Affinity Selection Screening (MagMass) of Botanical Extracts for Inhibitors of 15-Lipoxygenase

    Science.gov (United States)

    Rush, Michael D.; Walker, Elisabeth M.; Burton, Tristesse; van Breemen, Richard B.

    2016-01-01

    To expedite the identification of active natural products in complex mixtures such as botanical extracts, a Magnetic Microbead Affinity Selection Screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries. PMID:27802026

  12. Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase.

    Science.gov (United States)

    Rush, Michael D; Walker, Elisabeth M; Burton, Tristesse; van Breemen, Richard B

    2016-11-23

    To expedite the identification of active natural products in complex mixtures such as botanical extracts, a magnetic microbead affinity selection screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases, and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries.

  13. Affinity extraction of emerging contaminants from water based on bovine serum albumin as a binding agent.

    Science.gov (United States)

    Papastavros, Efthimia; Remmers, Rachael A; Snow, Daniel D; Cassada, David A; Hage, David S

    2018-03-01

    Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides, and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions than a traditional C 18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    Science.gov (United States)

    Pavoni, Emiliano; Flego, Michela; Dupuis, Maria Luisa; Barca, Stefano; Petronzelli, Fiorella; Anastasi, Anna Maria; D'Alessio, Valeria; Pelliccia, Angela; Vaccaro, Paola; Monteriù, Giorgia; Ascione, Alessandro; De Santis, Rita; Felici, Franco; Cianfriglia, Maurizio; Minenkova, Olga

    2006-01-01

    Background CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. Methods The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. Results The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. Conclusion The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance. PMID:16504122

  15. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    Directory of Open Access Journals (Sweden)

    De Santis Rita

    2006-02-01

    Full Text Available Abstract Background CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. Methods The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. Results The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. Conclusion The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

  16. Green fluorescent protein purification through Immobilized Metal Affinity Chromatografy (IMAC) and its relevance for Biomedical Science students during Biochemistry practical classes at La Trobe University – Australia

    OpenAIRE

    de Melo Silva, Alex Jose José; Alves, Lumar Lucena; Pakay, Julian

    2016-01-01

    This work was performed as an integrated practical of a Biomedical Science undergraduate course of Biochemistry subject, in order to demonstrate used techniques to purify of Green Fluorescent Protein (GFP). To perform the experiments the main methodology applied was the by immobilized metal affinity chromatography (IMAC).  The open reading frame for enhanced GFP was sub-cloned into the pQE30 expression vector. The subsequent production of protein tagged N-terminally with hexahistidine, facili...

  17. Affine fractal functions as bases of continuous funtions | Navascues ...

    African Journals Online (AJOL)

    The objective of the present paper is the study of affine transformations of the plane, which provide self-affine curves as attractors. The properties of these curves depend decisively of the coefficients of the system of affinities involved. The corresponding functions are continuous on a compact interval. If the scale factors are ...

  18. Comparative analysis of the affine scaling and Karmarkar's ...

    African Journals Online (AJOL)

    The affine scaling algorithm is a variant of the Karmarkar's algorithms. In this paper, we compare the affine scaling and the Karmarkar algorithms using the same test LP problem. Keywords: Polynomial-time, Complexity bound, Primal LP, Dual LP, Basic Solution, Degenerate Solution, Affine Space, Simplex and Polytope ...

  19. Duals of Affine Grassmann Codes and Their Relatives

    DEFF Research Database (Denmark)

    Beelen, P.; Ghorpade, S. R.; Hoholdt, T.

    2012-01-01

    Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine Grassm...

  20. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    Science.gov (United States)

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Operation of the tandem-linac accelerator

    International Nuclear Information System (INIS)

    Anon.

    1985-01-01

    The tandem-linac accelerator system is operated as a source of energetic heavy-ion projectiles for research in several areas of nuclear physics and occasionally in other areas of science. The accelerator system consists of a 9-MV tandem electrostatic accelerator and a superconducting-linac energy booster that can provide an additional 20 MV of acceleration. A figure shows the layout of this system, which will be operated in its present form until September 1985, when it will be incorporated into the larger ATLAS system. In both the present and future forms the accelerator is designed to provide the exceptional beam quality and overall versatility required for precision nuclear-structure research

  2. Determination of dapsone in meat and milk by liquid chromatography tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hadjigeorgiou, M. [State General Laboratory of Cyprus, Veterinary Drug Residues Lab, Kimonos 44, 1451 Nicosia (Cyprus)], E-mail: mhadjigeorgiou@sgl.moh.gov.cy; Papachrysostomou, Ch.; Theodorou, Z.; Kanari, P.; Constantinou, S. [State General Laboratory of Cyprus, Veterinary Drug Residues Lab, Kimonos 44, 1451 Nicosia (Cyprus)

    2009-04-01

    Within the EU the use of dapsone (4,4-diaminodiphenylsulfone) is prohibited in food-producing animals and consequently it's included in the Annex IV of the Directive 90/2377/EC. A quantitative confirmatory method has been developed and validated according to the criteria defined in the Commission Decision 2002/657/EC, for the determination of dapsone in meat and milk. Samples, after homogenization in alkaline conditions and organic solvent extraction, were purified on silica gel solid phase extraction cartridges. The eluate was evaporated and redissolved in mobile phase and was analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive electrospray ionisation (ESI) using deuterium labelled Sulphadimidine-d7 as internal standard. The calculated value for, decision limit, CC{alpha} is 0.12 {mu}g kg{sup -1}, and the detection capability; CC{beta} value is 0.16 {mu}g kg{sup -1}.

  3. Determination of dapsone in meat and milk by liquid chromatography tandem mass spectrometry

    International Nuclear Information System (INIS)

    Hadjigeorgiou, M.; Papachrysostomou, Ch.; Theodorou, Z.; Kanari, P.; Constantinou, S.

    2009-01-01

    Within the EU the use of dapsone (4,4-diaminodiphenylsulfone) is prohibited in food-producing animals and consequently it's included in the Annex IV of the Directive 90/2377/EC. A quantitative confirmatory method has been developed and validated according to the criteria defined in the Commission Decision 2002/657/EC, for the determination of dapsone in meat and milk. Samples, after homogenization in alkaline conditions and organic solvent extraction, were purified on silica gel solid phase extraction cartridges. The eluate was evaporated and redissolved in mobile phase and was analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive electrospray ionisation (ESI) using deuterium labelled Sulphadimidine-d7 as internal standard. The calculated value for, decision limit, CCα is 0.12 μg kg -1 , and the detection capability; CCβ value is 0.16 μg kg -1

  4. Tandem multimer expression and preparation of hypoglycemic peptide MC6 from Momordica charantia in Escherichia coli.

    Science.gov (United States)

    Wang, Fu-Jun; Song, Huai-Lei; Wang, Xiao-Meng; Zhang, Wen-Ji; Wang, Bi-Lian; Zhao, Jian; Hu, Zhi-Bi

    2012-02-01

    Tandem repeat multimers of Momordica charantia (MC) peptide MC6 were designed and the recombinant plasmid containing 10 copies of MC6 gene was constructed to improve the expression level of MC6 in Escherichia coli. Under the selected conditions of cultivation and induction, the expression level of recombinant TrxA-MC6(10) protein was above 25% of total bacteria protein. This fusion protein was purified and cleaved with HCl (13%, w/v). Either the un-cleaved or cleaved recombinant proteins was analyzed pharmacological activity by alloxan-induced diabetic mice and only the cleaved products of the recombinant protein showed significant hypoglycemic effects. The study provides a convenient and economical method for the large-scale production of anti-diabetic medicines for pharmaceutical applications.

  5. Common Wet Chemical Agents for Purifying Multiwalled Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Rasel Das

    2014-01-01

    Full Text Available Purification and functionalization of multiwalled carbon nanotubes (MWCNTs are challenging but vital for their effective applications in various fields including water purification technologies, optoelectronics, biosensors, fuel cells, and electrode arrays. The currently available purification techniques, often complicated and time consuming, yielded shortened and curled MWCNTs that are not suitable for applications in certain fields such as membrane technologies, hybrid catalysis, optoelectronics, and sensor developments. Here we described the H2O2 synergy on the actions of HCl and KOH in purifying and functionalizing pristine MWCNTs. The method (HCl/H2O2 showed 100% purification yield as compared to HCl and KOH/H2O2 with purification yields 93.46 and 3.92%, respectively. We probed the findings using transmission electron microscope, energy dispersive X-ray spectroscope, attenuated total reflectance infrared spectroscope, Raman spectroscope, thermal gravimetric analysis, and X-ray powder diffraction. The study is a new avenue for simple, rapid, low cost, and scalable purification of pristine MWCNTs for application in versatile fields.

  6. The purified colicin S8 is a multimeric protein.

    Science.gov (United States)

    Concepción Curbelo, J L; Garcia Diaz, M E

    2000-12-01

    Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.

  7. Synaptosomal protein synthesis in P2 and Ficoll purified fractions.

    Science.gov (United States)

    Eyman, Maria; Cefaliello, Carolina; Bruno, Anna Paola; Bruno, Annapaola; Crispino, Marianna; Giuditta, Antonio

    2012-01-30

    Cytoplasmic protein synthesis of brain synaptosomes has generally been determined in the Ficoll purified fraction which contains fewer contaminating mitochondria, microsomes and myelin fragments than the parent P2 fraction. Using a highly selective assay of this activity we have compared the total translation activity and the specific activity of the proteins synthesized by either fraction in control rats and in rats trained for a two-way active avoidance task. In control rats the specific activity remained essentially the same in both fractions but in trained rats the value of the Ficoll fraction was markedly lower (38.5%) than in the P2 fraction. Furthermore, the total translation activity of the Ficoll fraction was 30% lower than in the P2 fraction in control rats and 62% lower in trained rats. These decrements indicate that a large proportion of active synaptosomes present in the P2 fraction is not recovered in the Ficoll fraction, notably in rats undergoing plastic brain changes. We conclude that cytoplasmic protein synthesis of brain synaptosomes is better preserved in the P2 fraction. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Strong Purifying Selection at Synonymous Sites in D. melanogaster

    Science.gov (United States)

    Lawrie, David S.; Messer, Philipp W.; Hershberg, Ruth; Petrov, Dmitri A.

    2013-01-01

    Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in Drosophila melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated. PMID:23737754

  9. Anticoagulant activity of a natural protein purified from Hypomesus olidus.

    Science.gov (United States)

    Gou, Mengxing; Wang, Liyan; Liu, Xuejun

    2017-05-01

    A novel anticoagulant protein (E-II-1) was separated and purified from Hypomesus olidus, a unique freshwater fish in northern China. E-II-1 had a molecular mass of approximately 40 kDa with no subunits. The high content of hydrophobic amino acids and negatively charged amino acids in E-II-1 demonstrated that the amino acid compositions might contribute to the anticoagulant activity. E-II-1 contained α-helices 16.75%, β-sheets 42.67%, β-turn 25.58% and random coil 15.00%. In vitro blood coagulation time assay, E-II-1 significantly prolonged the activated partial thrombin time in a dose-dependent manner. Results indicated that E-II-1 acted as anticoagulants through the endogenous pathway with an inhibition of FXa. The specific activity of E-II-1 was 103.50 U/mg at a concentration of 1.00 mg/mL. Therefore, E-II-1 might be one of the promising anticoagulants originated from natural food sources with more safety and less side effects.

  10. PURIFIED WASTE FCC CATALYST AS A CEMENT REPLACEMENT MATERIAL

    Directory of Open Access Journals (Sweden)

    Danute Vaiciukyniene

    2015-06-01

    Full Text Available Zeolites are commonly used in the fluid catalytic cracking process. Zeolite polluted with oil products and became waste after some time used. The quantity of this waste inevitably rises by expanding rapidly oil industry. The composition of these catalysts depends on the manufacturer and on the process that is going to be used. The main factors retarding hydration process of cement systems and modifying them strength are organic compounds impurities in the waste FCC catalyst. The present paper shows the results of using purified waste FCC catalyst (pFCC from Lithuania oil refinery, as Portland cement replacement material. For this purpose, the purification of waste FCC catalyst (FCC samples was treated with hydrogen peroxide. Hydrogen peroxide (H2O2 is one of the most powerful oxidizers known. By acting of waste with H2O2 it can eliminate the aforementioned waste deficiency, and the obtained product becomes one of the most promising ingredients, in new advanced building materials. Hardened cement paste samples with FCC or pFCC were formed. It was observed that the pFCC blended cements developed higher strength, after 28 days, compared to the samples with FCC or reference samples. Typical content of Portland cement substituting does not exceed 30 % of mass of Portland cement in samples. Reducing the consumption of Portland cement with utilizing waste materials is preferred for reasons of environmental protection.

  11. Selective Biological Responses of Phagocytes and Lungs to Purified Histones.

    Science.gov (United States)

    Fattahi, Fatemeh; Grailer, Jamison J; Lu, Hope; Dick, Rachel S; Parlett, Michella; Zetoune, Firas S; Nuñez, Gabriel; Ward, Peter A

    2017-01-01

    Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1β was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4. © 2017 S. Karger AG, Basel.

  12. Carrier-mediated transport of metabolites in purified bean mitochondria

    International Nuclear Information System (INIS)

    DeSantis, A.; Arrigoni, O.; Palmieri, F.

    1976-01-01

    The mechanism of transport of Kreba cycle intermediates, phosphate and sulfur-containing compounds across the membrane of purified bean mitochondria was investigated by directly measuring the exchange between intramitochondrial labelled substrates and external anions and by testing the inhibitor sensitivity of these transport processes. 1) The exchange between intramitochondrial phosphate and external phosphate or sulfite is insensitive to N-ethylmaleimide or butylmalonate when either is added alone, but is completely inhibited by N-ethylmaleimide plus butylmalonate or by mersalyl. Internal phosphate is exchanged with malate, succinate, oxaloacetate, sulfate and thiosulfate; these reactions are inhibited by butylmalonate but not affected by N-ethyl-maleimide. 2) Internal sulfate is exchanged with malate, malonate, succinate, phosphate and sulfite in a butylmalonate- and mersalyl-sensitive reaction. Also the exchanges of malonate with phosphate, sulfate and sulfite are inhibited by butylmalonate and mersalyl. On the other hand, the exchange between intra- and extramitochondrial malonate is completely inhibited only by the combination of butylmalonate and 1,2,3-benzene-tricarboxylate. 3) We concluded that bean mitochondria contain the following transport systems: a phosphate carrier inhibited by N-ethylmalemide or mersalyl, a dicarboxylate carrier inhibited by butylmalonate or mersalyl, a citrate carrier inhibited by 1,2,3-benzene-tricarboxylate and an oxoglutarate carrier inhibited by phenylsuccinate or butylmalonate but insensitive to mersalyl. (auth.)

  13. Designs of tandem-mirror fusion reactors

    International Nuclear Information System (INIS)

    Carlson, G.A.; Barr, W.L.; Boghosian, B.M.

    1981-01-01

    We have completed a comparative evaluation of several end plug configurations for tandem mirror fusion reactors with thermal barriers. The axi-cell configuration has been selected for further study and will be the basis for a detailed conceptual design study to be carried out over the next two years. The axi-cell end plug has a simple mirror cell produced by two circular coils followed by a transition coil and a yin-yang pair, which provides for MHD stability

  14. Terminal ion source for an FN tandem

    International Nuclear Information System (INIS)

    Harper, G.C.

    1995-01-01

    An RF discharge source assembly has been developed for use in the terminal of the FN tandem van de Graaff accelerator at the Nuclear Physics Laboratory of the University of Washington. The primary motivation for developing the source was to provide a high intensity beam of 3 He + to produce 8 B from the reaction 6 Li( 3 He,n) 8 B. The design of the optics and the performance of the source are described here

  15. Moment approach to tandem mirror radial transport

    International Nuclear Information System (INIS)

    Siebert, K.D.; Callen, J.D.

    1986-02-01

    A moment approach is proposed for the study of tandem mirror radial transport in the resonant plateau regime. The salient features of the method are described with reference to axisymmetric tokamak transport theory. In particular, the importance of momentum conservation to the establishment of the azimuthal variations in the electrostatic potential is demonstrated. Also, an ad hoc drift kinetic equation is solved to determine parallel viscosity coefficients which are required to close the moment system

  16. Modulating uranium binding affinity in engineered Calmodulin EF-hand peptides: effect of phosphorylation

    International Nuclear Information System (INIS)

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Guilloreau, Luc; Berthomieu, Catherine; Delangle, Pascale; Adriano, Jean-Marc

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T 9 TKE 12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K d =25±6 nM to K d =5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the sub-nanomolar range (K d = 0.25±0.06 nM). FTIR analyses showed that the phospho-threonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν as (P-O) and ν s (P-O) IR modes of phospho-threonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν as (UO 2 ) 2+ vibration (from 923 cm -1 to 908 cm -1 ) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. (authors)

  17. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Romain Pardoux

    Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

  18. Affinity chromatography with pseudobiospecific ligands on high-performance supports for purification of proteins of biotechnological interest

    Directory of Open Access Journals (Sweden)

    N.B. Iannucci

    2003-03-01

    Full Text Available High-performance affinity matrices were obtained by attaching pseudobiospecific ligands to hollow-fibre membranes. The neutral protease contained in FlavourzymeTM was purified to homogeneity with Yellow 4R-HE affinity hollow-fibre membranes. Immobilisation of Red HE-3B allowed purification of a milk-clotting enzyme obtained by solid-state culture of Mucor bacilliformis. Copper immobilisation through iminodiacetic acid allowed fractionation of Biocon Bioconcentrated PlusTM to separate the pectinesterase-containing fraction. The productivity of the developed processes - 1900, 94 and 750 U/ml.min, respectively - was 10- to 15-fold higher than that achieved with the same ligands immobilised on agarose-based soft gels, mainly due to the shortening of the purification processes.

  19. ORNL 25 MV tandem accelerator control system

    International Nuclear Information System (INIS)

    Juras, R.C.; Biggerstaff, J.A.; Hoglund, D.E.

    1985-01-01

    The CAMAC-based control system for the 25 MV tandem electrostatic accelerator of the Holifield Heavy Ion Research Facility at Oak Ridge National Laboratory (ORNL) was specified by ORNL and built by the National Electrostatics Corporation. Two Perkin-Elmer 32-bit minicomputers are used in the system, a message switching computer and a supervisory computer. The message switching computer transmits and receives control information on six serial highways. This computer shares memory with the supervisory computer. Operator consoles are located on a serial highway; control is by means of a console CRT, trackball, and assignable shaft encoders and meters. Two identical consoles operate simultaneously: one is located in the tandem control room; the other is located in the cyclotron control room to facilitate operation during injection of tandem beams into the cyclotron or when beam lines under control of the cyclotron control system are used. The supervisory computer is used for accelerator parameter setup calculations, actual accelerator setup for new beams based on scaled, recorded parameters from previously run beams, and various other functions. Nearly seven years of control system operation and improvements will be discussed

  20. TandEM: Titan and Enceladus mission

    Science.gov (United States)

    Coustenis, A.; Atreya, S.K.; Balint, T.; Brown, R.H.; Dougherty, M.K.; Ferri, F.; Fulchignoni, M.; Gautier, D.; Gowen, R.A.; Griffith, C.A.; Gurvits, L.I.; Jaumann, R.; Langevin, Y.; Leese, M.R.; Lunine, J.I.; McKay, C.P.; Moussas, X.; Muller-Wodarg, I.; Neubauer, F.; Owen, T.C.; Raulin, F.; Sittler, E.C.; Sohl, F.; Sotin, Christophe; Tobie, G.; Tokano, T.; Turtle, E.P.; Wahlund, J.-E.; Waite, J.H.; Baines, K.H.; Blamont, J.; Coates, A.J.; Dandouras, I.; Krimigis, T.; Lellouch, E.; Lorenz, R.D.; Morse, A.; Porco, C.C.; Hirtzig, M.; Saur, J.; Spilker, T.; Zarnecki, J.C.; Choi, E.; Achilleos, N.; Amils, R.; Annan, P.; Atkinson, D.H.; Benilan, Y.; Bertucci, C.; Bezard, B.; Bjoraker, G.L.; Blanc, M.; Boireau, L.; Bouman, J.; Cabane, M.; Capria, M.T.; Chassefiere, E.; Coll, P.; Combes, M.; Cooper, J.F.; Coradini, A.; Crary, F.; Cravens, T.; Daglis, I.A.; de Angelis, E.; De Bergh, C.; de Pater, I.; Dunford, C.; Durry, G.; Dutuit, O.; Fairbrother, D.; Flasar, F.M.; Fortes, A.D.; Frampton, R.; Fujimoto, M.; Galand, M.; Grasset, O.; Grott, M.; Haltigin, T.; Herique, A.; Hersant, F.; Hussmann, H.; Ip, W.; Johnson, R.; Kallio, E.; Kempf, S.; Knapmeyer, M.; Kofman, W.; Koop, R.; Kostiuk, T.; Krupp, N.; Kuppers, M.; Lammer, H.; Lara, L.-M.; Lavvas, P.; Le, Mouelic S.; Lebonnois, S.; Ledvina, S.; Li, Ji; Livengood, T.A.; Lopes, R.M.; Lopez-Moreno, J. -J.; Luz, D.; Mahaffy, P.R.; Mall, U.; Martinez-Frias, J.; Marty, B.; McCord, T.; Salvan, C.M.; Milillo, A.; Mitchell, D.G.; Modolo, R.; Mousis, O.; Nakamura, M.; Neish, Catherine D.; Nixon, C.A.; Mvondo, D.N.; Orton, G.; Paetzold, M.; Pitman, J.; Pogrebenko, S.; Pollard, W.; Prieto-Ballesteros, O.; Rannou, P.; Reh, K.; Richter, L.; Robb, F.T.; Rodrigo, R.; Rodriguez, S.; Romani, P.; Bermejo, M.R.; Sarris, E.T.; Schenk, P.; Schmitt, B.; Schmitz, N.; Schulze-Makuch, D.; Schwingenschuh, K.; Selig, A.; Sicardy, B.; Soderblom, L.; Spilker, L.J.; Stam, D.; Steele, A.; Stephan, K.; Strobel, D.F.; Szego, K.; Szopa,

    2009-01-01

    TandEM was proposed as an L-class (large) mission in response to ESA’s Cosmic Vision 2015–2025 Call, and accepted for further studies, with the goal of exploring Titan and Enceladus. The mission concept is to perform in situ investigations of two worlds tied together by location and properties, whose remarkable natures have been partly revealed by the ongoing Cassini–Huygens mission. These bodies still hold mysteries requiring a complete exploration using a variety of vehicles and instruments. TandEM is an ambitious mission because its targets are two of the most exciting and challenging bodies in the Solar System. It is designed to build on but exceed the scientific and technological accomplishments of the Cassini–Huygens mission, exploring Titan and Enceladus in ways that are not currently possible (full close-up and in situ coverage over long periods of time). In the current mission architecture, TandEM proposes to deliver two medium-sized spacecraft to the Saturnian system. One spacecraft would be an orbiter with a large host of instruments which would perform several Enceladus flybys and deliver penetrators to its surface before going into a dedicated orbit around Titan alone, while the other spacecraft would carry the Titan in situ investigation components, i.e. a hot-air balloon (Montgolfière) and possibly several landing probes to be delivered through the atmosphere.

  1. Purified Betacyanins from Hylocereus undatus Peel Ameliorate Obesity and Insulin Resistance in High-Fat-Diet-Fed Mice.

    Science.gov (United States)

    Song, Haizhao; Chu, Qiang; Xu, Dongdong; Xu, Yang; Zheng, Xiaodong

    2016-01-13

    Natural bioactive compounds in food have been shown to be beneficial in preventing the development of obesity, diabetes, and other metabolic diseases. Increasing evidence indicates that betacyanins possess free-radical-scavenging and antioxidant activities, suggesting their beneficial effects on metabolic disorders. The main objective of this study was to isolate and identify the betaycanins from Hylocereus undatus (white-fleshed pitaya) peel and evaluate their ability to ameliorate obesity, insulin resistance, and hepatic steatosis in high-fat-diet (HFD)-induced obese mice. The purified pitaya peel betacyanins (PPBNs) were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and the male C57BL/6 mice were fed a low-fat diet, HFD, or HFD supplemented with PPBNs for 14 weeks. Our results showed that the white-fleshed pitaya peel contains 14 kinds of betacyanins and dietary PPBNs reduced HFD-induced body weight gain and ameliorated adipose tissue hypertrophy, hepatosteatosis, glucose intolerance, and insulin resistance. Moreover, the hepatic gene expression analysis indicated that PPBN supplementation increased the expression levels of lipid-metabolism-related genes (AdipoR2, Cpt1a, Cpt1b, Acox1, PPARγ, Insig1, and Insig2) and FGF21-related genes (β-Klotho and FGFR1/2) but decreased the expression level of Fads2, Fas, and FGF21, suggesting that the protective effect of PPBNs might be associated with the induced fatty acid oxidation, decreased fatty acid biosynthesis, and alleviated FGF21 resistance.

  2. High-throughput functional affinity purification of mannose binding proteins from Oryza sativa.

    Science.gov (United States)

    Andon, Nancy L; Eckert, Donna; Yates, John R; Haynes, Paul A

    2003-07-01

    We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.

  3. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Evans, D.M.; Williams, K.P.; McGuinness, B. [PerSeptive Biosystems, Framingham, MA (United States)] [and others

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  4. Molecular Affinity of Mabolo Extracts to an Octopamine Receptor of a Fruit Fly

    Directory of Open Access Journals (Sweden)

    Francoise Neil D. Dacanay

    2017-10-01

    Full Text Available Essential oils extracted from plants are composed of volatile organic compounds that can affect insect behavior. Identifying the active components of the essential oils to their biochemical target is necessary to design novel biopesticides. In this study, essential oils extracted from Diospyros discolor (Willd. were analyzed using gas chromatography mass spectroscopy (GC-MS to create an untargeted metabolite profile. Subsequently, a conformational ensemble of the Drosophila melanogaster octopamine receptor in mushroom bodies (OAMB was created from a molecular dynamics simulation to resemble a flexible receptor for docking studies. GC-MS analysis revealed the presence of several metabolites, i.e. mostly aromatic esters. Interestingly, these aromatic esters were found to exhibit relatively higher binding affinities to OAMB than the receptor’s natural agonist, octopamine. The molecular origin of this observed enhanced affinity is the π -stacking interaction between the aromatic moieties of the residues and ligands. This strategy, computational inspection in tandem with untargeted metabolomics, may provide insights in screening the essential oils as potential OAMB inhibitors.

  5. Exponentiality for the construct of affine sets

    Directory of Open Access Journals (Sweden)

    Veerle Claes

    2008-04-01

    Full Text Available The topological construct SSET of affine sets over the two-point set S contains many interesting topological subconstructs such as TOP, the construct of topological spaces, and CL, the construct of closure spaces. For this category and its subconstructs cartesian closedness is studied. We first give a classification of the subconstructs of SSET according to their behaviour with respect to exponenttiality. We formulate sufficient conditions implying that a subconstruct behaves similar to CL. On the other hand, we characterize a conglomerate of subconstructs with behaviour similar to TOP. Finally, we construct the cartesian closed topological hull of SSET.

  6. Thermodynamic characterization of tandem mismatches found in naturally occurring RNA

    Science.gov (United States)

    Christiansen, Martha E.; Znosko, Brent M.

    2009-01-01

    Although all sequence symmetric tandem mismatches and some sequence asymmetric tandem mismatches have been thermodynamically characterized and a model has been proposed to predict the stability of previously unmeasured sequence asymmetric tandem mismatches [Christiansen,M.E. and Znosko,B.M. (2008) Biochemistry, 47, 4329–4336], experimental thermodynamic data for frequently occurring tandem mismatches is lacking. Since experimental data is preferred over a predictive model, the thermodynamic parameters for 25 frequently occurring tandem mismatches were determined. These new experimental values, on average, are 1.0 kcal/mol different from the values predicted for these mismatches using the previous model. The data for the sequence asymmetric tandem mismatches reported here were then combined with the data for 72 sequence asymmetric tandem mismatches that were published previously, and the parameters used to predict the thermodynamics of previously unmeasured sequence asymmetric tandem mismatches were updated. The average absolute difference between the measured values and the values predicted using these updated parameters is 0.5 kcal/mol. This updated model improves the prediction for tandem mismatches that were predicted rather poorly by the previous model. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA duplexes containing tandem mismatches, and, furthermore, should allow for improved prediction of secondary structure from sequence. PMID:19509311

  7. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  8. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    International Nuclear Information System (INIS)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M.

    1989-01-01

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125 I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity

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

    Directory of Open Access Journals (Sweden)

    Gorbatiuk O. B.

    2015-04-01

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

  10. Connection between the Affine and conformal Affine Toda models and their Hirota's solution

    International Nuclear Information System (INIS)

    Constantinidis, C.P.; Ferreira, L.A.; Gomes, J.F.; Zimerman, A.H.

    1992-01-01

    It is shown that the Affine Toda models (AT) constitute a gauge fixed version of the Conformal Affine Toda model (CAT). This result enables one to map every solution of the AT models into an infinite number of solutions of the corresponding CAT models, each one associated to a point of the orbit of the conformal group. The Hirota's τ-function are introduced and soliton solutions for the AT and CAT models associated to SL (r+1) and SP (r) are constructed. (author)

  11. Heterogeneity among Mycobacterium ulcerans from French Guiana revealed by multilocus variable number tandem repeat analysis (MLVA).

    Science.gov (United States)

    Reynaud, Yann; Millet, Julie; Couvin, David; Rastogi, Nalin; Brown, Christopher; Couppié, Pierre; Legrand, Eric

    2015-01-01

    Buruli ulcer is an emerging and neglected tropical disease caused by Mycobacterium ulcerans. Few cases have been reported so far in the Americas. With 250 cases reported since 1969, French Guiana is the only Buruli ulcer endemic area in the continent. Thus far, no genetic diversity studies of strains of M. ulcerans from French Guiana have been reported. Our goal in the present study was to examine the genetic diversity of M. ulcerans strains in this region by using the Multilocus Variable Number Tandem Repeat Analysis (MLVA) approach. A total of 23 DNA samples were purified from ulcer biopsies or derived from pure cultures. MVLA was used in the study of six previously-described Variable Number of Tandem Repeat (VNTR) markers. A total of three allelic combinations were characterized in our study: genotype I which has been described previously, genotype III which is very similar to genotype I, and genotype II which has distinctly different characteristics in comparison with the other two genotypes. This high degree of genetic diversity appears to be uncommon for M. ulcerans. Further research based on complete genome sequencing of strains belonging to genotypes I and II is in progress and should lead soon to a better understanding of genetic specificities of M. ulcerans strains from French Guiana.

  12. Tandem Mass Spectrometry of Novel Ether-Linked Phospholipid Analogs of Anionic Pulmonary Surfactant Phospholipids

    Science.gov (United States)

    Fickes, Rachel; Voelker, Dennis R.; Berry, Karin Zemski; Murphy, Robert C.

    2016-01-01

    RATIONALE Structural analogs of the bioactive lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, were synthesized with a xylitol polar headgroup and both diacyl and diether radyl groups. Mass spectral characterization of xylitol phospholipids (PX) was carried out using collisional activation and high resolution mass measurements of positive molecular ion species and compared with the phosphatidylglycerol (PG) analogs. METHODS Xylitol phospholipids were synthesized using a transphosphatidylation reaction catalyzed by phospholipase D and purified by HPLC. Compounds were subjected to electrospray ionization and CID was performed using a tandem quadrupole mass spectrometer to generate positive and negative molecular ions. Diether phospholipids were additionally analyzed by high resolution mass spectrometry as protonated and sodiated molecular species in positive ion mode. RESULTS Ester linked xylitol phospholipid analogs behaved similar to PG after collisional activation of [M-H]−. The product ions formed by CID of the diether PG and PX negative ions only revealed information about the headgroup with no information about the aliphatic chains. In contrast, CID of protonated and sodiated diether phospholipid positive ions, revealed reactions corresponding to cleavage of the ether chain, likely occurring by charge driven reaction mechanisms. CONCLUSIONS Novel xylitol phospholipid analogs with diacyl and diether radyl substituents of the glycerol backbone were characterized by tandem mass spectrometry. These unique diether phospholipid analogs enabled exploration of ether cleavage reactions of the positive molecular ion species induced by collision induced decomposition. PMID:27689848

  13. Temperature effects on kinetic parameters and substrate affinity of Cel7A cellobiohydrolases

    DEFF Research Database (Denmark)

    Sørensen, Trine Holst; Cruys-Bagger, Nicolaj; Windahl, Michael Skovbo

    2015-01-01

    We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic...... Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased......, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect...

  14. Dye-Affinity Nanofibrous Membrane for Adsorption of Lysozyme: Preparation and Performance Evaluation

    Directory of Open Access Journals (Sweden)

    Steven Sheng-Shih Wang

    2018-01-01

    Full Text Available Polyacrylonitrile (PAN nanofibrous membrane was prepared by an electrospinning technique. After heat treatment and alkaline hydrolysis, the weak ion exchange membrane was grafted with chitosan molecule and then covalently immobilized with a Cibacron Blue F3GA (CB. Fibre diameter, porosity and pore size of the membrane and immobilized dye density were characterized. Furthermore, the membrane was applied to evaluate the binding performance of lysozyme under various operating parameters (pH, chitosan mass per volume ratio, dye concentration, ionic strength and temperature in batch mode. The experimental results were directly applied to purify lysozyme from chicken egg white by membrane chromatography. The results showed that the capture efficiency, recovery yield and purification factor were 90 and 87 %, and 47-fold, respectively, in a single step. The binding capacity remained consistent after five repeated cycles of adsorption-desorption operations. This work demonstrates that the dye-affinity nanofibrous membrane holds great potential for purification of lysozyme from real feedstock.

  15. Semi-purified glycerin in the meat quails feeding

    Directory of Open Access Journals (Sweden)

    Tiago Junior Pasquetti

    2014-12-01

    Full Text Available Three experiments were conducted to determine the nutritional value of semi-purified glycerin (SG, and its economic feasibility for use in meat quail feed. Its effect on the performance, body chemical composition, and carcass yield were assessed. In experiment 1, 22-day-old quails, were housed in metabolic cages, distributed in a completely randomized design (CRD, with three treatments, five replicates, and five birds per repetition, using 75 quails in total. Treatments consisted of a reference diet (RD and two test diets (RT in which 8% and 12% SG replaced the RD. The estimated value of nitrogen-corrected apparent metabolizable energy (AMEn for SG was 2,476 kcal/kg. In experiment 2, 1320 quails (55 quails in each box were distributed in a CRD with six treatments and four replicates. The treatments consisted of six diets containing increasing levels (0%, 3%, 6%, 9%, 12%, and 15% of SG. There were no effects of SG on the performance variables. The cost per kilogram of live weight produced increased linearly (P<0.05 with the percentage of SG in the diet. The performance data suggest that SG may be included in up to 15% of the diet in 1- to 14-day-old quails. In experiment 3, 1032 quail (43 quails in each box were distributed under the same conditions as in experiment 2. SG was positively associated with (P<0.05 litter moisture and body biomass accumulated (BBA weight. A quadratic effect on feed conversion and BBA (P<0.05 was observed. The cost per kilogram of live weight produced increased linearly (P<0.05 with the percentage of SG in the diet. Therefore, SG may be used to make-up 10% of the diet of 15–35-day-old quail.

  16. Filter system for purifying gas or air streams

    International Nuclear Information System (INIS)

    Ohlmeyer, M.; Wilhelm, J.

    1981-01-01

    A filter system is provided for purifying a gas stream by means of flowable or tricklable contact filter material, wherein the stream flows through the filter material and the filter material forms a movable bed. The system contains a filter chamber through which the filter material can flow and which is provided with an inlet opening and an outlet opening for the filter material between which the filter material is conveyed by gravity. The filter system includes deflection means for deflecting the stream , after a first passage of the stream through the filter bed to charge the filter bed for a first time, to a position above where the stream first passed through the filter bed and for conducting the stream at least once again transversely through the filter bed above the first charge so that the filter bed is charged a second time. The filter chamber contains a first opening where the stream enters the filter bed for the first time and is aligned with the deflection means, and a second opening aligned with the deflection means and above the first opening. The second opening is located where the stream leaves the filter bed for the second time, with a partial quantity of the gas stream being able to pass directly through the filter bed from the first opening to the second opening without going through the deflection means. The distance between the upper edge of the first opening and the lower edge of the second opening is at least twice the thickness of the filter chamber

  17. Development of a Microwave Regenerative Sorbent-Based Hydrogen Purifier

    Science.gov (United States)

    Wheeler, Richard R., Jr.; Dewberry, Ross H.; McCurry, Bryan D.; Abney, Morgan B.; Greenwood, Zachary W.

    2016-01-01

    This paper describes the design and fabrication of a Microwave Regenerative Sorbent-based Hydrogen Purifier (MRSHP). This unique microwave powered technology was developed for the purification of a hydrogen stream produced by the Plasma Pyrolysis Assembly (PPA). The PPA is a hydrogen recovery (from methane) post processor for NASA's Sabatier-based carbon dioxide reduction process. Embodied in the Carbon dioxide Reduction Assembly (CRA), currently aboard the International Space Station (ISS), the Sabatier reaction employs hydrogen to catalytically recover oxygen, in the form of water, from respiratory carbon dioxide produced by the crew. This same approach is base-lined for future service in the Air Revitalization system on extended missions into deep space where resupply is not practical. Accordingly, manned exploration to Mars may only become feasible with further closure of the air loop as afforded by the greater hydrogen recovery permitted by the PPA with subsequent hydrogen purification. By utilizing the well-known high sorbate loading capacity of molecular sieve 13x, coupled with microwave dielectric heating phenomenon, MRSHP technology is employed as a regenerative filter for a contaminated hydrogen gas stream. By design, freshly regenerated molecular sieve 13x contained in the MRSHP will remove contaminants from the effluent of a 1-CM scale PPA for several hours prior to breakthrough. By reversing flow and pulling a relative vacuum the MRSHP prototype then uses 2.45 GHz microwave power, applied through a novel coaxial antenna array, to rapidly heat the sorbent bed and drive off the contaminants in a short duration vacuum/thermal contaminant desorption step. Finally, following rapid cooling via room temperature cold plates, the MRSHP is again ready to serve as a hydrogen filter.

  18. Positive and purifying selection on the Drosophila Y chromosome.

    Science.gov (United States)

    Singh, Nadia D; Koerich, Leonardo B; Carvalho, Antonio Bernardo; Clark, Andrew G

    2014-10-01

    Y chromosomes, with their reduced effective population size, lack of recombination, and male-limited transmission, present a unique collection of constraints for the operation of natural selection. Male-limited transmission may greatly increase the efficacy of selection for male-beneficial mutations, but the reduced effective size also inflates the role of random genetic drift. Together, these defining features of the Y chromosome are expected to influence rates and patterns of molecular evolution on the Y as compared with X-linked or autosomal loci. Here, we use sequence data from 11 genes in 9 Drosophila species to gain insight into the efficacy of natural selection on the Drosophila Y relative to the rest of the genome. Drosophila is an ideal system for assessing the consequences of Y-linkage for molecular evolution in part because the gene content of Drosophila Y chromosomes is highly dynamic, with orthologous genes being Y-linked in some species whereas autosomal in others. Our results confirm the expectation that the efficacy of natural selection at weakly selected sites is reduced on the Y chromosome. In contrast, purifying selection on the Y chromosome for strongly deleterious mutations does not appear to be compromised. Finally, we find evidence of recurrent positive selection for 4 of the 11 genes studied here. Our results thus highlight the variable nature of the mode and impact of natural selection on the Drosophila Y chromosome. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Measles virus polypeptides in purified virions and in infected cells

    International Nuclear Information System (INIS)

    Vainionpaeae, R.; Ziola, B.; Salmi, A.

    1978-01-01

    A wild-type measles virus was radiolabeled during growth in VERO cells and purified by two successive potassium tartrate gradient centrifugations. The virion polypeptide composition was determined by SDS-polyacrylamide gel electrophoresis employing two different buffer systems. Six virus-specific polypeptides were consistently detected. The largest (L) had a molecular weight (MW) of greater than 150,000. The second largest polypeptide, G (MW 79,000), was the only glycoprotein found. The proteins designated polypeptide 2 (MW 66 to 70,000) and nucleocapsid protein or NP (MW 61,000) were phosphorylated. The remaining virus-coded proteins were polypeptide 5 (MW 40,000) and the matrix or M protein (MW 37,000). Measles virions also contained a polypeptide (MW 42,000) thought to be actin due to co-migration with this component of uninfected cells. Analysis of in vitro 3 H-acetic anhydride radiolabeled virions confirmed the presence of these seven polypeptides. Acetic anhydride also labeled a protein designated polypeptide 4 (MW 53,000) which was not consistently radiolabeled in vivo, as well as several other minor proteins believed to be cellular in origin. Synthesis of the six virus-specific structural polypeptides was detected in lysates of infected cells by SDS-polyacrylamide slab gel electrophoresis. Virus specificity of polypeptide 4 could not be confirmed due to the similar MW of several cellular polypeptides. Two non-virion, but virus-specified polypeptides, of MW 38,000 and 18,000 were also detected. Synthesis of the virus structural proteins was in the same proportions as the polypeptides found in virions except for under production of polypeptide G and over production of polypeptide 2. (author)

  20. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    Science.gov (United States)

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  1. Therapeutic effects of antigen affinity-purified polyclonal anti-receptor of advanced glycation end-product (RAGE) antibodies on cholestasis-induced liver injury in rats.

    Science.gov (United States)

    Xia, Peng; Deng, Qing; Gao, Jin; Yu, Xiaolan; Zhang, Yang; Li, Jingjing; Guan, Wen; Hu, Jianjun; Tan, Quanhui; Zhou, Liang; Han, Wei; Yuan, Yunsheng; Yu, Yan

    2016-05-15

    Cholestasis leads to acute hepatic injury, fibrosis/cirrhosis, inflammation, and duct proliferation. We investigated whether blocking receptor of advanced glycation end-products (RAGE) with polyclonal anti-RAGE antibodies (anti-RAGE) could regulate acute liver injury and fibrosis in a rat bile duct ligation (BDL) model. Male Wister rats received 0.5mg/kg rabbit anti-RAGE or an equal amount of rabbit IgG by subcutaneous injection twice a week after BDL. Samples of liver tissue and peripheral blood were collected at 14 days after BDL. Serum biochemistry and histology were used to analyze the degree of liver injury. Quantitative real-time PCR (qPCR) and immunohistochemical staining were used to further analyze liver injury. Anti-RAGE improved the gross appearance of the liver and the rat survival rate. Liver tissue histology and relevant serum biochemistry indicated that anti-RAGE attenuated liver necrosis, inflammation, liver fibrosis, and duct proliferation in the BDL model. qPCR and western blotting showed significant reductions in interleukin-1β expression levels in the liver by treatment with anti-RAGE. Anti-RAGE also significantly reduced the mRNA levels of α1(1) collagen (Col1α1) and cholesterol 7α-hydroxylase, and the ratio of tissue inhibitor of matrix metalloproteinase-1 to matrix metalloproteinases (MMPs) in the liver. In addition, anti-RAGE regulated the transcriptional level of Col1α1 and MMP-9 in transforming growth factor-β-induced activated LX-2 cells in vitro. Anti-RAGE was found to inhibit hepatic stellate cell proliferation in vivo and in vitro. Therefore, anti-RAGE can protect the liver from injury induced by BDL in rats. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

    DEFF Research Database (Denmark)

    Prentø, Jannick Cornelius; Bukh, Jens

    2011-01-01

    , flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar...... physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development....

  3. From affine Hecke algebras to boundary symmetries

    International Nuclear Information System (INIS)

    Doikou, Anastasia

    2005-01-01

    Motivated by earlier works we employ appropriate realizations of the affine Hecke algebra and we recover previously known non-diagonal solutions of the reflection equation for the U q (gl n -bar ) case. The corresponding N site spin chain with open boundary conditions is then constructed and boundary non-local charges associated to the non-diagonal solutions of the reflection equation are derived, as coproduct realizations of the reflection algebra. With the help of linear intertwining relations involving the aforementioned solutions of the reflection equation, the symmetry of the open spin chain with the corresponding boundary conditions is exhibited, being essentially a remnant of the U q (gl n -bar ) algebra. More specifically, we show that representations of certain boundary non-local charges commute with the generators of the affine Hecke algebra and with the local Hamiltonian of the open spin chain for a particular choice of boundary conditions. Furthermore, we are able to show that the transfer matrix of the open spin chain commutes with a certain number of boundary non-local charges, depending on the choice of boundary conditions

  4. Actualization of the Tandem-E N Accelerator of the Nuclear Centre of Mexico

    International Nuclear Information System (INIS)

    Villasenor S, P.; Aguilera R, E.; Aspiazu F, J.; Fernandez A, J.; Fernandez B, M.; Garcia R, B.; Lopez M, J.; Martinez Q, E.; Mendez G, B.; Moreno B, E.; Murillo O, G.; Policroniades R, R.; Ramirez T, J.; Reynoso V, R.; Varela G, A.; Vega C, J.

    2004-01-01

    In this work, the activities are described carried out to change the tubes accelerators and original resistances of the accelerator Tandem-E N of the Nuclear Center, for tubes DOWLISH and resistances again design, both donated ones for ORNL. This way same, the problem is described that imply this changes, and like it was solved by the personnel of the laboratory, without having to appeal to external services, what there is redounded in a considerable increment in the costs. In form preliminary the improvements are described observed after the rehabilitation of the Accelerator. (Author)

  5. Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors

    NARCIS (Netherlands)

    Aizpurua-Olaizola, Oier; Sastre Torano, Javier; Pukin, Aliaksei; Fu, Ou; Boons, Geert Jan; de Jong, Gerhardus J; Pieters, Roland J

    2018-01-01

    Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE)

  6. Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats.

    Science.gov (United States)

    Olsen, Ingrid; Balasingham, Seetha V; Davidsen, Tonje; Debebe, Ephrem; Rødland, Einar A; van Soolingen, Dick; Kremer, Kristin; Alseth, Ingrun; Tønjum, Tone

    2009-07-01

    The ability to repair DNA damage is likely to play an important role in the survival of facultative intracellular parasites because they are exposed to high levels of reactive oxygen species and nitrogen intermediates inside phagocytes. Correcting oxidative damage in purines and pyrimidines is the primary function of the enzymes formamidopyrimidine (faPy)-DNA glycosylase (Fpg) and endonuclease VIII (Nei) of the base excision repair pathway, respectively. Four gene homologs, belonging to the fpg/nei family, have been identified in Mycobacterium tuberculosis H37Rv. The recombinant protein encoded by M. tuberculosis Rv2924c, termed Mtb-Fpg1, was overexpressed, purified and biochemically characterized. The enzyme removed faPy and 5-hydroxycytosine lesions, as well as 8-oxo-7,8-dihydroguanine (8oxoG) opposite to C, T and G. Mtb-Fpg1 thus exhibited substrate specificities typical for Fpg enzymes. Although Mtb-fpg1 showed nearly complete nucleotide sequence conservation in 32 M. tuberculosis isolates, the region upstream of Mtb-fpg1 in these strains contained tandem repeat motifs of variable length. A relationship between repeat length and Mtb-fpg1 expression level was demonstrated in M. tuberculosis strains, indicating that an increased length of the tandem repeats positively influenced the expression levels of Mtb-fpg1. This is the first example of such a tandem repeat region of variable length being linked to the expression level of a bacterial gene.

  7. Proteomic analysis of protein purified derivative of Mycobacterium bovis.

    Science.gov (United States)

    Roperto, Sante; Varano, Mariaconcetta; Russo, Valeria; Lucà, Roberta; Cagiola, Monica; Gaspari, Marco; Ceccarelli, Dora Maria; Cuda, Giovanni; Roperto, Franco

    2017-04-03

    Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these

  8. Hypolipidemic activities of xanthorrhizol purified from centrifugal TLC.

    Science.gov (United States)

    Oon, Seok Fang; Nallappan, Meenakshii; Kassim, Nur Kartinee; Shohaimi, Shamarina; Sa'ariwijaya, Mohd Shazrul Fazry; Tee, Thiam Tsui; Cheah, Yew Hoong

    2016-09-23

    Hyperlipidemia is defined as the presence of either hypertriglyceridemia or hypercholesterolemia, which could cause atherosclerosis. Although hyperlipidemia can be treated by hypolipidemic drugs, they are limited due to lack of effectiveness and safety. Previous studies demonstrated that xanthorrhizol (XNT) isolated from Curcuma xanthorrhizza Roxb. reduced the levels of free fatty acid and triglyceride in vivo. However, its ability to inhibit cholesterol uptake in HT29 colon cells and adipogenesis in 3T3-L1 cells are yet to be reported. In this study, XNT purified from centrifugal TLC demonstrated 98.3% purity, indicating it could be an alternative purification method. The IC50 values of XNT were 30.81 ± 0.78 μg/mL in HT29 cells and 35.07 ± 0.24 μg/mL in 3T3-L1 adipocytes, respectively. Cholesterol uptake inhibition study using HT29 colon cells showed that XNT (15 μg/mL) significantly inhibited the fluorescent cholesterol analogue NBD uptake by up to 27 ± 3.1% relative to control. On the other hand, higher concentration of XNT (50 μg/mL) significantly suppressed the growth of 3T3-L1 adipocytes (5.9 ± 0.58%) compared to 3T3-L1 preadipocytes (81.31 ± 0.55%). XNT was found to impede adipogenesis of 3T3-L1 adipocytes in a dose-dependent manner from 3.125 to 12.5 μg/mL, where 12.5 μg/mL significantly suppressed 36.13 ± 2.1% of lipid accumulation. We postulate that inhibition of cholesterol uptake, adipogenesis, preadipocyte and adipocyte number may be utilized as treatment modalities to reduce the prevalence of lipidemia. To conclude, XNT could be a potential hypolipidemic agent to improve cardiovascular health in the future. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Optical properties and ensemble characteristics of size purified Silicon nanocrystals

    Science.gov (United States)

    Miller, Joseph Bradley

    Nanotechnology is at the forefront of current scientific research and nanocrystals are being hailed as the 'artificial' atoms of the 21st century. Semiconducting silicon nanocrystals (SiNCs) are prime candidates for potential commercial applications because of silicon's already ubiquitous presence in the semiconductor industry, nontoxicity and abundance in nature. For realization of these potential applications, the properties and behavior of SiNCs need to be understood and enhanced. In this report, some of the main SiNC synthesis schemes are discussed, including those we are currently experimenting with to create our own SiNCs and the one utilized to create the SiNCs used in this study. The underlying physics that governs the unique behavior of SiNCs is then presented. The properties of the as-produced SiNCs are determined to depend strongly on surface passivation and environment. Size purification, an important aspect of nanomaterial utilization, was successfully performed on our SiNCs though density gradient ultracentrifugation. We demonstrate that the size-purified fractions exhibit an enhanced ability for colloidal self-assembly, with better aligned nanocrystal energy levels which promotes greater photostability in close-packed films and produces a slight increase in photoluminescence (PL) quantum yield. The qualities displayed by the fractions are exploited to form SiNC clusters that exhibit photostable PL. An analysis of SiNC cluster (from individual nanocrystals to collections of more than one thousand) blinking and PL shows an improvement in their PL emitting 'on' times. Pure SiNC films and SiNC-polymer nanocomposites are created and the dependence of their PL on temperature is measured. For such nanocomposites, the coupling between the 'coffee-ring' effect and liquid-liquid phase separation is also examined for ternary mixtures of solvent, polymer and semiconducting nanocrystal. We discover that with the right SiNC-polymer concentration and polymer

  10. Brief-stimulus presentations on multiform tandem schedules

    OpenAIRE

    Reed, Phil

    1994-01-01

    Three experiments examined the influence of a brief stimulus (a light) on the behavior of food-deprived rats whose lever pressing on tandem schedules comprising components of different schedule types resulted in food presentation. In Experiment 1, either a tandem variable-ratio variable-interval or a tandem variable-interval variable-ratio schedule was used. The variable-interval requirement in the tandem variable-ratio variable-interval schedule was yoked to the time taken to complete the va...

  11. Structure of a High-Affinity

    Energy Technology Data Exchange (ETDEWEB)

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  12. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    Science.gov (United States)

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). Copyright © 2014 Elsevier B.V. All rights reserved.

  13. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  14. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  15. Method of fabricating bifacial tandem solar cells

    Science.gov (United States)

    Wojtczuk, Steven J; Chiu, Philip T; Zhang, Xuebing; Gagnon, Edward; Timmons, Michael

    2014-10-07

    A method of fabricating on a semiconductor substrate bifacial tandem solar cells with semiconductor subcells having a lower bandgap than the substrate bandgap on one side of the substrate and with subcells having a higher bandgap than the substrate on the other including, first, growing a lower bandgap subcell on one substrate side that uses only the same periodic table group V material in the dislocation-reducing grading layers and bottom subcells as is present in the substrate and after the initial growth is complete and then flipping the substrate and growing the higher bandgap subcells on the opposite substrate side which can be of different group V material.

  16. Plasma confinement in the TMX tandem mirror

    International Nuclear Information System (INIS)

    Hooper, E.B. Jr.; Allen, S.L.; Casper, T.A.

    1981-01-01

    Plasma confinement in the Tandem Mirror Experiment (TMX) is described. Axially confining potentials are shown to exist throughout the central 20-cm core of TMX. Axial electron-confinement time is up to 100 times that of single-cell mirror machines. Radial transport of ions is smaller than axial transport near the axis. It has two parts at large radii: nonambipolar, in rough agreement with predictions from resonant-neoclassical transport theory, and ambipolar, observed near the plasma edge under certain conditions, accompanied by a low-frequency, m = 1 instability or strong turbulence

  17. The capacity of digital links in tandem

    Science.gov (United States)

    Posner, E. C.; Rubin, A. L.

    1984-01-01

    The present investigation is concerned with the error control strategy used in connection with digital data networks, taking into account the capacity of the end-to-end channel. It is assumed that the only source of noise is Gaussian and that bandwidth limitation on capacity can be ignored. Attention is given to L links in tandem with binary repeaters, the asymptotic capacity with a large number of links, some alternative repeater strategies, and approaches for lowering the bit error probability. Numerical results are presented, taking into consideration capacity versus link error probability for various numbers of links.

  18. Tandem mirror and tokamak reactor maintainability comparison

    International Nuclear Information System (INIS)

    Zahn, H.S.

    1981-01-01

    The analysis proceeds through estimates of downtime and resources required for selected maintenance actions and optimization of the replacement fraction, availability and cost of electricity. Scheduled downtime estimates and availability goals provide a basis for determining allowable forced outage downtimes. These analyses have been conducted with the assumption of redundancy wherever feasible but without the impact of maintenance equipment outages. Annual maintenance cost estimates and availabilities for both reactors are found to be approximately equal. However, the tandem mirror reactor capital costs are higher. Reduction of these costs appears feasible with the trend of current design studies toward smaller and more accessible machines

  19. NSF tandem stack support structure deflection characteristics

    International Nuclear Information System (INIS)

    Cook, J.

    1979-12-01

    Results are reported of load tests carried out on the glass legs of the insulating stack of the 30 MV tandem Van de Graaff accelerator now under construction at Daresbury Laboratory. The tests to investigate the vulnerability of the legs when subjected to tensile stresses were designed to; establish the angle of rotation of the pads from which the stresses in the glass legs may be calculated, proof-test the structure and at the same time reveal any asymmetry in pad rotations or deflections, and to confirm the validity of the computer design analysis. (UK)

  20. A tandem queue with delayed server release

    OpenAIRE

    Nawijn, W.M.

    1997-01-01

    We consider a tandem queue with two stations. The rst station is an s-server queue with Poisson arrivals and exponential service times. After terminating his service in the rst station, a customer enters the second station to require service at an exponential single server, while in the meantime he is blocking his server in station 1 until he completes service in station 2, whereupon the server in station 1 is released. An analysis of the generating function of the simultaneous probability di...

  1. Parametric systems analysis for tandem mirror hybrids

    International Nuclear Information System (INIS)

    Lee, J.D.; Chapin, D.L.; Chi, J.W.H.

    1980-09-01

    Fusion fission systems, consisting of fissile producing fusion hybrids combining a tandem mirror fusion driver with various blanket types and net fissile consuming LWR's, have been modeled and analyzed parametrically. Analysis to date indicates that hybrids can be competitive with mined uranium when U 3 O 8 cost is about 100 $/lb., adding less than 25% to present day cost of power from LWR's. Of the three blanket types considered, uranium fast fission (UFF), thorium fast fission (ThFF), and thorium fission supressed (ThFS), the ThFS blanket has a modest economic advantage under most conditions but has higher support ratios and potential safety advantages under all conditions

  2. Electron cyclotron resonance heating of tandem mirrors

    Energy Technology Data Exchange (ETDEWEB)

    Porkolab, M.

    1978-12-19

    Heating electrons by microwave power near either the electron cyclotron frequency or its harmonic is analyzed in some detail. The purpose of this study is to determine whether electrons in current or future mirror devices could be heated by electromagnetic waves in this frequency range. Absorption lengths, accessibility conditions, and appropriate polarization are among the concerns studied. It is found that the Tandem Mirror Experiment (TMX) may efficiently be heated by the extraordinary mode at omega = omega either in the solenoid-to-plug transition region or near the center of the plug at densities napprox. = 10/sup 13/cm/sup -3/.

  3. Data Stream Clustering With Affinity Propagation

    KAUST Repository

    Zhang, Xiangliang

    2014-07-09

    Data stream clustering provides insights into the underlying patterns of data flows. This paper focuses on selecting the best representatives from clusters of streaming data. There are two main challenges: how to cluster with the best representatives and how to handle the evolving patterns that are important characteristics of streaming data with dynamic distributions. We employ the Affinity Propagation (AP) algorithm presented in 2007 by Frey and Dueck for the first challenge, as it offers good guarantees of clustering optimality for selecting exemplars. The second challenging problem is solved by change detection. The presented StrAP algorithm combines AP with a statistical change point detection test; the clustering model is rebuilt whenever the test detects a change in the underlying data distribution. Besides the validation on two benchmark data sets, the presented algorithm is validated on a real-world application, monitoring the data flow of jobs submitted to the EGEE grid.

  4. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    Energy Technology Data Exchange (ETDEWEB)

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. (Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier (France))

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  5. Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel.

    Science.gov (United States)

    Beyaztaş, Serap; Arslan, Oktay

    2015-06-01

    A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150 kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60 °C and incubated for 60 min. These results indicated that the enzyme was heat stable.

  6. Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

    Directory of Open Access Journals (Sweden)

    Yangchao Dong

    2015-09-01

    Full Text Available Efficient isolation of endogenously assembled viral RNA-protein complexes is essential for understanding virus replication mechanisms. We have developed an affinity purification strategy based on an RNA affinity tag that allows large-scale preparation of native viral RNA-binding proteins (RBPs. The streptavidin-binding aptamer S1 sequence was inserted into the 3′ end of dengue virus (DENV 5′–3′ UTR RNA, and the DENV RNA UTR fused to the S1 RNA aptamer was expressed in living mammalian cells. This allowed endogenous viral ribonucleoprotein (RNP assembly and isolation of RNPs from whole cell extract, through binding the S1 aptamer to streptavidin magnetic beads. Several novel host DENV RBPs were subsequently identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS, including RPS8, which we further implicate in DENV replication. We proposed efficient S1 aptamer-based isolation of viral assembled RNPs from living mammalian cells will be generally applicable to the purification of high- and low-affinity RBPs and RNPs under endogenous conditions.

  7. Antibody-based affinity cryo-EM grid.

    Science.gov (United States)

    Yu, Guimei; Li, Kunpeng; Jiang, Wen

    2016-05-01

    The Affinity Grid technique combines sample purification and cryo-Electron Microscopy (cryo-EM) grid preparation into a single step. Several types of affinity surfaces, including functionalized lipids monolayers, streptavidin 2D crystals, and covalently functionalized carbon surfaces have been reported. More recently, we presented a new affinity cryo-EM approach, cryo-SPIEM, which applies the traditional Solid Phase Immune Electron Microscopy (SPIEM) technique to cryo-EM. This approach significantly simplifies the preparation of affinity grids and directly works with native macromolecular complexes without need of target modifications. With wide availability of high affinity and high specificity antibodies, the antibody-based affinity grid would enable cryo-EM studies of the native samples directly from cell cultures, targets of low abundance, and unstable or short-lived intermediate states. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs.

    Science.gov (United States)

    Nübel, Gabriele; Sorgenfrei, Frieda A; Jäschke, Andres

    2017-03-15

    RNA modifications are widely distributed in Nature, and their thorough analysis helps answering fundamental biological questions. Nowadays, mass spectrometry or deep-sequencing methods are often used for the analysis. With the raising number of newly discovered RNA modifications, such as the 5'-NAD cap in Escherichia coli, there is an important need for new, less complex and fast analytical tools to analyze the occurrence, amount, and distribution of modified RNAs in cells. To accomplish this task, we have revisited the previously developed affinity gel electrophoresis principles and copolymerized acryloylaminophenyl boronic acid (APB) in standard denaturing polyacrylamide gels to retard the NAD- or FAD-modified RNAs compared to the unmodified RNAs in the gels. The boronyl groups inside the gel form relatively stable complexes with 1,2-cis diols, occurring naturally at the 3'-end of RNA, and also in the nicotinamide riboside of NAD-modified RNA at the 5'-end. The transient formation of diesters between the immobilized boronic acid and the diols causes lower mobility of the modified RNAs, compared to unmodified RNAs, resulting in two distinct bands for one RNA sequence. We used APB affinity gel electrophoresis to preparatively purify in vitro transcribed NAD-RNA from triphosphorylated RNA, to study the enzyme kinetics of the NAD-RNA decapping enzyme NudC, and to determine the NAD modification ratios of various cellular sRNAs. In summary, APB affinity gels can be used to study cofactor-modified RNAs with low amounts of material, and to rapidly screen for their occurrence in total RNA while avoiding complex sample treatments. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. High blood oxygen affinity in the air-breathing swamp eel Monopterus albus.

    Science.gov (United States)

    Damsgaard, Christian; Findorf, Inge; Helbo, Signe; Kocagoz, Yigit; Buchanan, Rasmus; Huong, Do Thi Thanh; Weber, Roy E; Fago, Angela; Bayley, Mark; Wang, Tobias

    2014-12-01

    The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O₂ transport system to secure adequate O₂ delivery to the respiring tissues. We therefore investigated O₂ binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O₂ equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O₂ binding properties. The blood of M. albus has a high O₂ carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O₂ with an unusually high affinity (P₅₀=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O₂ affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue⁻¹, respectively). We suggest that the high Hct and high blood O₂ affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O₂ flux from the blood to the muscles. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

    Science.gov (United States)

    Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

    2012-11-01

    Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

  11. Method and device for feeding purified water to a pressure vessel

    International Nuclear Information System (INIS)

    Hirato, Miharu.

    1982-01-01

    Purpose: To prevent thermal wear at the junction of feedwater pipes and purified water pipes, as well as maintain the function of the purified water feeding system by stopping the introduction of purified water to the heated water feeding system and introducing purified water to the recycling water system upon transient operation or start-up. Constitution: Since a feedwater heater does not function well during transient operation or upon start-up, the temperature of heated water flowing through the feedwater pipe is reduced to produce a temperature difference relative to the set temperature for the purified water feeding system. The temperature difference is detected by a temperature sensor and, when it arrives at a predetermined difference, an operation valve is switched to interrupt the feed of the purified water to the heated water feeding system and it is sent to a water recycling system. Then, the purified water is sent from the water recycling system by way of the discharge portion to the inside of a pressure vessel. Thus, since only the heated water flows to the junction between the cleaned water pipes and the heated water pipes, neither shocks are generated nor the performance of the purified water feeding system is impaired. (Moriyama, K.)

  12. Characterization of crude and purified pumpkin seed oil.

    Directory of Open Access Journals (Sweden)

    Tsaknis, John

    1997-10-01

    Full Text Available Oil from hulled pumpkin seeds (Cucurbita pepo and Cucurbita Maxima was extracted with hot petroleum ether, and then it was degummed, neutralized and bleached, consecutively Physical and chemical characteristics of crude and purified oils were determined. Density, refractive index, viscosity and peroxide value were not affected by purification, while decreases in acidity, colour, unsaponifiable, E1%1cm 232, and oxidative stability, and increases in smoke point and E1%1cm 270 were observed. Purification did not affect the fatty acid and sterol profiles. GLC analysis for the fatty acid composition of the seed oil showed that the predominant unsaturates were linoleic (42% and oleic (38%, while the major saturates were palmitic (12,7% and stearic (6%. Only α-tocopherol was detected at a level of 126 mg/kg, which reduced to 78 mg/kg after purification. The main sterols of pumpkin seed oil unsaponifiable were Δ7.22,25 -stigmastatrien-3β-ol, α-spinasterol, Δ7,25_stigmastadienol and Δ7-avenasterol, followed by stigmasterol, 24-methylcholest-7-enol and Δ7-stigmastenol, and also trace to minor amounts of cholesterol, brassicasterol, campesterol, sitostanol, Δ5-avenasterol, erythrodiol and uvaol were found.

    Aceite de semillas de calabaza descascarada (Cucurbita pepo YCucurbita maxima fue extraído con éter de petróleo caliente, y luego desgomado, neutralizado y decolorado consecutivamente. Las características físicas y químicas de aceites crudo y purificado fueron determinadas. La densidad, el índice de refracción, la viscosidad y el índice de peróxido no se afectaron por la purificación, mientras que se observó una disminución en la acidez, color, insaponificable, E1%1cm 232, y estabilidad oxidativa, y un aumento en el punto de humo y de E1%1cm270. La purificaci

  13. Remote maintenance of tandem mirror hybrid coils

    International Nuclear Information System (INIS)

    Dietz, L.P.

    1983-01-01

    Hybrid Coils (superconducting coils with normal conducting inserts) are being employed with increasing frequency on Tandem Mirror Devices to obtain high field strengths. The normal conducting copper inserts are short lived in comparison to their encircling superconductors. It becomes desirable, therefore, to devise design features and maintenance procedures to replace the inner normal conducting coils without simultaneously replacing the longer lived (and significantly more costly) superconducting coils. The high neutron wall loadings require that the task be accomplished by remote control. The approach is to permanently mount the coil assemblies on track mounted carriages which serve, during machine operation merely as structural supports, but during maintenance procedures as moveable transport devices. The carriages incorporate all necessary provisions to facilitate remote maintenance operations and to adjust and align the coil assemblies with respect to adjacent machine components. The vacuum vessel is severed on both sides of the hybrid coil by means of a remote cutting machine. The entire coil is transported horizontally, normal to the machine axis to a nearby repair station. Prepositioned carriage mounted repair equipment at the repair station withdraws the damaged normal coil as a single entity and inserts a preassembled spare unit. The repaired hybrid coil is reassembled to the reactor. A cost and risk effective procedure has been evolved to maintain one of the more critical components of a Tandem Mirror Machine

  14. Progress in the tandem mirror program

    International Nuclear Information System (INIS)

    Fowler, T.K.; Borchers, R.R.

    1981-01-01

    Experimental results in TMX have confirmed the basic principles of the tandem-mirror concept. A center-cell particle confinement parameter eta tau approx. 10 11 cm -3 s has been obtained at ion temperatures around 100 eV, which is a hundred-fold improvement over single mirrors at the same temperatures. For TMX these results have been obtained at peak beta values in the center cell in the range 10 to 40%, not yet limited by MHD activity; and ion-cyclotron resonant heating (ICRH) in the Phaedrus tandem-mirror experiment has produced beta values approx. 25%, which is several times the ideal MHD limit for that device. In addition, it has been demonstrated that the end fan chambers of TMX simultaneously isolate the hot electrons from the end walls, provide adequate pumping and conveniently dispose of the exhaust plasma energy either by thermal deposition on the end wall or by direct conversion to electricity (at 48% efficiency in agreement with calculations). Also, evidence was obtained for inherent divertor action in TMX, presumably in part responsible for the observed low impurity level

  15. Cell-mediated immune responses to Plasmodium falciparum purified soluble antigens in sickle-cell trait subjects.

    Science.gov (United States)

    Bayoumi, R A; Abu-Zeid, Y A; Abdulhadi, N H; Saeed, B O; Theander, T G; Hviid, L; Ghalib, H W; Nugud, A H; Jepsen, S; Jensen, J B

    1990-08-01

    To determine the possible differences in the immune response to Plasmodium falciparum between sickle-cell trait (Hb AS) and normal haemoglobin (Hb AA) individuals, we examined 35 Hb AS and 24 Hb AA subjects matched for age and microenvironment. Their age was 2-55 years and all lived in a malaria endemic area 300 km south of Khartoum. Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) and to circumsporozoite (CS) protein (anti-NANP40) indicated equal exposure to falciparum malaria. Peripheral blood mononuclear cells (BMNCs) from 20/35 (57%) Hb AS subjects compared with 10/24 (42%) Hb AA subjects, responded to affinity-purified P. falciparum soluble antigens (SPAg). Of those responding to SPAg, 9 (26%) Hb AS subjects and only two (8%) Hb AA subjects had high responses. The mean proliferative response to SPAg of BMNCs from Hb AS individuals was significantly higher than in Hb AA individuals (P less than 0.025). Responses of BMNCs to PPD and PHA were also higher among Hb AS individuals and correlated positively with responses to SPAg. These findings support the hypotheses that the sickle-cell trait protects individuals from P. falciparum infections, at least in part, by modulating the immune response.

  16. Carbohydrate specificity of lectin, purified from the fruiting bodies of Mycena pura /Fr./ Kumm. and its use in histochemical investigation

    Directory of Open Access Journals (Sweden)

    Ambarova N. O.

    2009-12-01

    Full Text Available Aim. The purpose of this investigation was to research carbohydrate specificity of a new lectin from fruiting body of Mycena pura and possibilities of its application in histochemical studies. Methods. The lectin has been purified by affinity chromatography on «îvomucine». The lectin carbohydrate specificity has been determined by a reaction of inhibiting haemagglutination by haptens. Histological materials were fixed in 4 % neutral formalin solution. Alkaline phosphatase was revealed in the cryostat unfixed microscopical sections. Results. The lectin yield from fresh fruit bodies of raw material was 9 mg/kg. Mol. mass of the lectin is 40 kDa. The lectin poorly interacted with D-glucose and D-mannose in contrast to lectins from Pisum sativum and Leucojum vernum. The peculiarity of this lectin is its strong interaction with alkaline phosphatase, the highest among twenty tested lectins. However, the receptors for Mycena lectin binding in mammalian tissues are not limited by this enzyme being presented also by glycoconjugates of another structure, as it was shown for fetus calf small intestine and kidney of rat. Conclusions. An important role in the lectin interaction with glycoproteins probably belongs to the disaccharide links of GlcNAcb(1-2Mana(1-6 or GlcNAcb(1- 2Mana(1-2, which not necessarily are terminal

  17. Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: the performance of PEGylated protein A.

    Science.gov (United States)

    González-Valdez, José; Yoshikawa, Alex; Weinberg, Justin; Benavides, Jorge; Rito-Palomares, Marco; Przybycien, Todd M

    2014-01-01

    Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or "PEGylation" can potentially improve selectivity by sterically suppressing non-specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media-associated non-specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono-PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media-associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two- to three-fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass-transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands. © 2014 American Institute of Chemical Engineers.

  18. Status report on the folded tandem ion accelerator at BARC

    Indian Academy of Sciences (India)

    Folded tandem ion accelerator; charged particle beams; voltage stability; Rutherford backscattering; ion optics; beam lines. Abstract. The folded tandem ion accelerator (FOTIA) facility set up at BARC has become operational. At present, it is used for elemental analysis studies using the Rutherford backscattering technique.

  19. High-sensitivity mass spectrometry with a tandem accelerator

    International Nuclear Information System (INIS)

    Henning, W.

    1984-01-01

    The characteristic features of accelerator mass spectrometry are discussed. A short overview is given of the current status of mass spectrometry with high-energy (MeV/nucleon) heavy-ion accelerators. Emphasis is placed on studies with tandem accelerators and on future mass spectrometry of heavier isotopes with the new generation of higher-voltage tandems

  20. 14 CFR 23.302 - Canard or tandem wing configurations.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Canard or tandem wing configurations. 23.302 Section 23.302 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Structure General § 23.302 Canard or tandem wing...

  1. A solution process for inverted tandem solar cells

    DEFF Research Database (Denmark)

    Larsen-Olsen, Thue Trofod; Bundgaard, Eva; Sylvester-Hvid, Kristian O.

    2011-01-01

    Tandem solar cells with normal and inverted device geometries were prepared by a solution process. Both device types were based on the use of zinc(II)oxide as the electron transporting layer (ETL). The hole transporting layer (HTL) was either PEDOT:PSS for normal geometry tandem solar cells...... or vanadium(V)oxide in the case of inverted tandem cells. It was found that the inverted tandem solar cells performed comparable or better than the normal geometry devices, showing that the connection structure of vanadium(V)oxide, Ag nanoparticles and zinc(II)oxide functions both as a good recombination...... layer, ensuring serial connection, and as a solvent barrier, protecting the first photoactive layer from processing of the second layer. This successfully demonstrates a tandem solar cell fabrication process fully compatible with state-of-the-art solution based automated production procedures....

  2. The Bucharest FN Tandem Accelerator: Modernization and Development

    Science.gov (United States)

    Dobrescu, S.; Mosu, D. V.; Moisa, D.; Papureanu, S.

    2009-03-01

    The Bucharest FN tandem accelerator, installed in 1973 and upgraded in 1983 to 9 MV, has been used for atomic and nuclear physics studies as well as for different applications using accelerated ion beams. In the last three years a program of modernization of the tandem accelerator including the replacement of the old accelerator equipment by new ones, installation of a pelletron system for the Van de Graaff generator and installation of new negative ion injectors was undertaken. In parallel a development of the tandem accelerator was started. In 2009, a beam pulsing system in the nanosecond range is scheduled to be installed. All these works aimed to transform the tandem accelerator in a reliable and efficient tool for research and applications are presented. The main lines of the research program at the Bucharest tandem accelerator are shortly presented too.

  3. Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

    Science.gov (United States)

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J; Serwer, Philip; Thompson, David H; Jiang, Wen

    2014-07-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Cambrian trilobites with Siberian affinities, southwestern Alaska

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, A.R.; Egbert, R.M.; Sullivan, R.; Knoth, J.S.

    1985-02-01

    Cambrian trilobites occur in two levels (about 7 m apart) in the core of a large, complex anticlinal structure in the area between the Taylor Mountains and the Hoholitna River in southwestern Alaska. The lower collection contains Erbia, Macannaia (a species close to Soviet forms described as Pagetia ferox Lermontova), two species of Kootenia (including one perhaps cospecific with forms from the central Brooks range), and several species of ptychoparioid trilobites. It is clear that biogeographic affinities are with the transitional facies of the eastern Siberian platform and the south Siberian foldbelt. In Soviet terms, the age of the collection falls in a disputed interval called latest Early Cambrian (Tojonian) by some authors, and earliest Middle Cambrian (Amgan) by others. In North American terms, Macannaia is known only from early Middle Cambrian beds. The younger collection contains abundant agnostids, a variety of conocoryphids, Paradoxides, and several species of ptychoparioid trilobites. This is an assemblage of undoubted late Middle Cambrian age, comparable to faunas described from the Maya State of the Siberian platform and the Paradoxides paradoxissimus Stage of the Baltic region. Both faunas are from ocean-facing or outer shelf environments. None of the key non-agnostid or non-pagetiid elements have been seen previously in deposits of Cambrian North America.

  5. Set-Membership Proportionate Affine Projection Algorithms

    Directory of Open Access Journals (Sweden)

    Stefan Werner

    2007-01-01

    Full Text Available Proportionate adaptive filters can improve the convergence speed for the identification of sparse systems as compared to their conventional counterparts. In this paper, the idea of proportionate adaptation is combined with the framework of set-membership filtering (SMF in an attempt to derive novel computationally efficient algorithms. The resulting algorithms attain an attractive faster converge for both situations of sparse and dispersive channels while decreasing the average computational complexity due to the data discerning feature of the SMF approach. In addition, we propose a rule that allows us to automatically adjust the number of past data pairs employed in the update. This leads to a set-membership proportionate affine projection algorithm (SM-PAPA having a variable data-reuse factor allowing a significant reduction in the overall complexity when compared with a fixed data-reuse factor. Reduced-complexity implementations of the proposed algorithms are also considered that reduce the dimensions of the matrix inversions involved in the update. Simulations show good results in terms of reduced number of updates, speed of convergence, and final mean-squared error.

  6. Daily rhythmicity of high affinity copper transport.

    Science.gov (United States)

    Perea-García, Ana; Sanz, Amparo; Moreno, Joaquín; Andrés-Bordería, Amparo; de Andrés, Sonia Mayo; Davis, Amanda M; Huijser, Peter; Davis, Seth J; Peñarrubia, Lola

    2016-01-01

    A differential demand for copper (Cu) of essential cupro-proteins that act within the mitochondrial and chloroplastal electronic transport chains occurs along the daily light/dark cycles. This requires a fine-tuned spatiotemporal regulation of Cu delivery, becoming especially relevant under non-optimal growth conditions. When scarce, Cu is imported through plasma membrane-bound high affinity Cu transporters (COPTs) whose coding genes are transcriptionally induced by the SPL7 transcription factor. Temporal homeostatic mechanisms are evidenced by the presence of multiple light- and clock-responsive regulatory cis elements in the promoters of both SPL7 and its COPT targets. A model is presented here for such temporal regulation that is based on the synchrony between the basal oscillatory pattern of SPL7 and its targets, such as COPT2. Conversely, Cu feeds back to coordinate intracellular Cu availability on the SPL7-dependent regulation of further Cu acquisition. This occurs via regulation at COPT transporters. Moreover, exogenous Cu affects several circadian-clock components, such as the timing of GIGANTEA transcript abundance. Together we propose that there is a dynamic response to Cu that is integrated over diurnal time to maximize metabolic efficiency under challenging conditions.

  7. The electron affinity of CF3

    Science.gov (United States)

    Miller, Deanna M.

    State-of-the-art ab initio electronic structure methods have been employed to pinpoint the electron affinity of the trifluoromethyl radical, EA(CF3). The theoretical techniques entailed restricted and unrestricted Hartree-Fock reference wavefunctions, contemporary density functional schemes, second-order Moller-Plesset perturbation theory, the configuration interaction and coupled-cluster singles and doubles approaches, and the latter augmented perturbatively for connected triple excitations. The one-particle basis sets for carbon and fluorine ranged in quality from (9s5p1d/4s2p1d) to (11s7p2d1f/6s5p2d1f). In the derivation of EA(CF3), both lower and upper bounds were first inferred by invoking selected methods in direct computational routes; a primary evaluation was then achieved by applying systematically increasing levels of theory to the isogyric (CF3-,3CF2) electron-transfer process; and confirmatory results were extracted from a thermochemical cycle involving the gas-phase acidity of CHF3. The collective analysis engenders a final proposal of EA(CF3) = 1ṡ78 ± 0ṡ10 eV, which clearly discounts a spectrum of early observations, poses a compromise between the best values given by competing experimental approaches, and significantly reduces the uncertainty in this quantity.

  8. Generalized Warburg impedance on realistic self-affine fractals ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals.

  9. Generalized Warburg impedance on realistic self-affine fractals

    Indian Academy of Sciences (India)

    We analyse the problem of impedance for a diffusion controlled charge transfer process across an irregular interface. These interfacial irregularities are characterized as two class of random fractals: (i) a statistically isotropic self-affine fractals and (ii) a statistically corrugated self-affine fractals. The information about the ...

  10. Affine Toda equations and solutions in the homogeneous grading

    Czech Academy of Sciences Publication Activity Database

    Zuevsky, Alexander

    2018-01-01

    Roč. 542, April 1 (2018), s. 149-161 ISSN 0024-3795 Institutional support: RVO:67985840 Keywords : affine Lie gebras * affine Toda modes * solitons Subject RIV: BA - General Mathematics OBOR OECD: Pure mathematics Impact factor: 0.973, year: 2016 https://www.sciencedirect.com/science/article/pii/S0024379517302100

  11. Isolation of bovine serum albumin from whey using affinity chromatography

    NARCIS (Netherlands)

    Besselink, T.; Janssen, A.E.M.; Boom, R.M.

    2015-01-01

    The adsorption of bovine serum albumin (BSA) to a chromatography resin with immobilised llama antibody fragments as affinity ligands was investigated. The maximum adsorption capacity of the affinity resin was 21.6 mg mL-1 with a Langmuir equilibrium constant of 20.4 mg mg-1. Using packed bed

  12. Calcium, Copper Protein And Oxygen Affinity In Haemocyanins Of ...

    African Journals Online (AJOL)

    ... to buffer the decrease in extracellular pH during aestivation is likely responsible for the high oxygen affinity of haemocyanin (43.0% increase) from aestivating snails through co-operative oxygen binding. Key Words: Aestivation, snail, Achatina achatina, inorganic ions, haemocyanin, absorption spectra, oxygen affinity.

  13. Self-affine roughness influence on redox reaction charge admittance

    NARCIS (Netherlands)

    Palasantzas, G

    2005-01-01

    In this work we investigate the influence of self-affine electrode roughness on the admittance of redox reactions during facile charge transfer kinetics. The self-affine roughness is characterized by the rms roughness amplitude w, the correlation length xi and the roughness exponent H (0

  14. Polynomial Primal-Dual Cone Affine Scaling for Semidefinite Programming

    NARCIS (Netherlands)

    A.B. Berkelaar (Arjan); J.F. Sturm; S. Zhang (Shuzhong)

    1996-01-01

    textabstractIn this paper we generalize the primal--dual cone affine scaling algorithm of Sturm and Zhang to semidefinite programming. We show in this paper that the underlying ideas of the cone affine scaling algorithm can be naturely applied to semidefinite programming, resulting in a new

  15. Fermionic construction of vertex operators for twisted affine algebras

    International Nuclear Information System (INIS)

    Frappat, L.; Sorba, P.; Sciarrino, A.

    1988-03-01

    We construct vertex operator representations of the twisted affine algebras in terms of fermionic (or parafermionic in some cases) elementary fields. The folding method applied to the extended Dynkin diagrams of the affine algebras allows us to determine explicitly these fermionic fields as vertex operators

  16. Affine Toda equations and solutions in the homogeneous grading

    Czech Academy of Sciences Publication Activity Database

    Zuevsky, Alexander

    2018-01-01

    Roč. 542, April 1 (2018), s. 149-161 ISSN 0024-3795 Institutional support: RVO:67985840 Keywords : affine Lie gebras * affine Toda modes * solitons Subject RIV: BA - General Mathematics OBOR OECD: Pure mathematics Impact factor: 0.973, year: 2016 https:// www .sciencedirect.com/science/article/pii/S0024379517302100

  17. Algorithm-Architecture Affinity - Parallelism Changes the Picture

    DEFF Research Database (Denmark)

    Abildgren, Rasmus; Šaramentovas, Aleksandras; Ruzgys, Paulius

    the hardware-software partitioning step. This extension builds upon and complement our own work with metrics in the Design Trotter project, and is combined with the affinity metric approach. We show that the proposed extension improves the original affinity metric in terms of parallelism detection, and thus...

  18. Pseudo-affinity chromatography of rumen microbial cellulase on ...

    African Journals Online (AJOL)

    Pseudo-affinity chromatography of rumen microbial cellulase on Sepharose- Cibacron Blue F3GA. ... African Journal of Biotechnology ... Pseudo affinity adsorption of bioproducts on Sepharose-cibacron blue F3-GA was subjected to rumen microbial enzyme evaluation through batch binding and column chromatography of ...

  19. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic acids...

  20. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a

  1. Congruence of genomic and ethnolinguistic affinities among five ...

    Indian Academy of Sciences (India)

    The central Indian state of Madhya Pradesh is home to a large number of tribal populations of diverse linguistic and ethnic backgrounds. With a view to examining how well genomic affinities among tribal populations of this state correspond with their ethnic and linguistic affinities, we analysed DNA samples of individuals ...

  2. Online identification of continuous bimodal and trimodal piecewise affine systems

    NARCIS (Netherlands)

    Le, Q.T.; van den Boom, A.J.J.; Baldi, S.; Rantzer, Anders; Bagterp Jørgensen, John; Stoustrup, Jakob

    2016-01-01

    This paper investigates the identification of continuous piecewise affine systems in state space form with jointly unknown partition and subsystem matrices. The partition of the system is generated by the so-called centers. By representing continuous piecewise affine systems in the max-form and

  3. A genomic view of short tandem repeats.

    Science.gov (United States)

    Gymrek, Melissa

    2017-06-01

    Short tandem repeats (STRs) are some of the fastest mutating loci in the genome. Tools for accurately profiling STRs from high-throughput sequencing data have enabled genome-wide interrogation of more than a million STRs across hundreds of individuals. These catalogs have revealed that STRs are highly multiallelic and may contribute more de novo mutations than any other variant class. Recent studies have leveraged these catalogs to show that STRs play a widespread role in regulating gene expression and other molecular phenotypes. These analyses suggest that STRs are an underappreciated but rich reservoir of variation that likely make significant contributions to Mendelian diseases, complex traits, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Protein Sequencing with Tandem Mass Spectrometry

    Science.gov (United States)

    Ziady, Assem G.; Kinter, Michael

    The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

  5. Tandem mirror reactor power balance studies

    International Nuclear Information System (INIS)

    Gorker, G.E.; Perkins, L.J.

    1985-01-01

    A tandem mirror reactor (TMR) power plant balance model has been developed and is now being used as a computer aid for performing parametric studies. End-cell power injection into the plasma and the physics thermal Q are used to determine the fusion power. About 80% of the fusion power is transferred by high-energy neutrons to the blanket modules and structures. The other 20% of the fusion power in the high-energy alpha particles is used to heat the deuterium-tritium (D-T) plasma. Most of the plasma-ionized particles transfer their energy to the halo dumps and direct converters. The plant efficiency is calculated for three different system cycles: (1) the pressurized water/saturated steam cycle; (2) the superheated steam cycle; and (3) the more complex superheat/reheat cycle. There is a signficiant improvement in plant efficiency as the electrical power multiplication factor and steam cycle efficiency increases

  6. Vacuum system for the Tandem Mirror Experiment

    International Nuclear Information System (INIS)

    Richardson, M.J.; Atkinson, D.P.; Calderon, M.O.

    1979-01-01

    This paper is a sequel to the one prepared by Atkinson, et al., in which the authors described the vacuum system of Lawrence Livermore Laboratory's Tandem Mirror Experiment (TMX). We discuss here the final configuration, liquid nitrogen (LN 2 ) supply, and operation of the complete TMX vacuum system. The assembled vacuum system consists of two plug tanks with a volume of approximately 60 m 3 each and a center cell tank with a volume of approximately 10 m 3 . In each plug tank there are 145 m 2 of titanium-gettered, LN 2 -filled panels, which allow a pumping speed calculated to be 5 x 10 7 l/s for a period of 50 ms. The system maintains an operating pressure in the plasma chamber on the order of 10 -6 Torr while 24 neutral-beam injectors are introducing 700 Torr l/s of hydrogen into the vacuum chamber

  7. Status of tandem-mirror confinement

    International Nuclear Information System (INIS)

    Baldwin, D.E.

    1983-01-01

    Recent end-stopping experiments in TMX-Upgrade show strong plugging of the central cell by lower-density plugs, requiring both electron-cyclotron heating (ECRH) and 47 0 neutral-beam injection, consistent with the thermal-barrier concept. These experiments have low density (n 12 cm -3 ) due to inefficient ECRH power coupling. Hot-ion and hot-electron buildup are consistent with Fokker-Planck calculations. No ion-cyclotron activity is observed in the plugs; occasional electron-cyclotron activity is observed. With plugging, axial lifetimes (tau/sub parallel/ > 40 ms) are larger than radial (tau/sub perpendicular/ = 5 to 10 ms) due to observed non-ambipolar ion transport. Recent tandem-mirror theoretical activities are also surveyed

  8. Tandem come tirocinio: la riflessione nel corso dell'azione - in un tandem in presenza italiano-portoghese

    Directory of Open Access Journals (Sweden)

    Maria Luisa Vassallo

    2006-04-01

    Full Text Available II presente studio è dedicato al Tandem, un metodo di studio delle lingue straniere autonomo e collaborativo. Esso illustra alcune potenzialità di questa forma didattica per la formazione degli insegnanti, utilizzando il concetto di riflessione nel corso dell'azione elaborato dal filosofo statunitense Donald Schön (1983. Il capitolo 1 introduce il metodo Tandem. Il capitolo 2 analizza il Tandem alla luce della teoria di Schön. Il capitolo 3 descrive l'aspetto riflessivo in un Tandem in presenza, svolto nel 2004 in Brasile. II capitolo 4 propone alcuni motivi per considerare il metodo Tandem un contesto complementare utile per la formazione alla pratica docente attraverso il tirocinio.

  9. Microbial Transglutaminase Separation by pH-Responsive Affinity Precipitation with Crocein Orange G as the Ligand.

    Science.gov (United States)

    Ding, Zhaoyang; Li, Sipeng; Cao, Xuejun

    2015-09-01

    A microbial transglutaminase (MTG) was efficiently purified by using pH-responsive affinity precipitation with Crocein orange G (COG) as the affinity ligand. The docking method was used to identify the appropriate ligand. The molecular simulation results were compared with the label-free detection data analyzed by ForteBio's Octet. A pH-responsive polymer, PMMDN, was polymerized and subsequently coupled with COG as the ligand. The isoelectric point (pI) and recovery of PMMDN and PMMDN-COG were 4.51, 99.8,% and 4.78, 98.1 %, respectively. The optimal adsorption conditions were found to be a ligand density of 60.0 μmol/g, pH 7.0, and 0.2 mol/L NaCl. The adsorption isotherm showed that the maximum adsorption capacity was 91.32 mg/g polymer and the dissociation constant was 0.021 mg/mL. Interaction information between PMMDN-COG and MTG in the whole process of affinity precipitation were obtained by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). PB (0.02 mol/L pH 7.0) in 20.0 % glycol was used to elute the binding MTG from PMMDN-COG. Under these conditions, electrophoretically pure MTG was obtained in only one step with elution recoveries of 98.96 % (protein) and 95.09 % (activity).

  10. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    Directory of Open Access Journals (Sweden)

    Shangyong Li

    2016-12-01

    Full Text Available Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

  11. Exploring Girls' Science Affinities Through an Informal Science Education Program

    Science.gov (United States)

    Todd, Brandy; Zvoch, Keith

    2017-10-01

    This study examines science interests, efficacy, attitudes, and identity—referred to as affinities, in the context of an informal science outreach program for girls. A mixed methods design was used to explore girls' science affinities before, during, and after participation in a cohort-based summer science camp. Multivariate analysis of survey data revealed that girls' science affinities varied as a function of the joint relationship between family background and number of years in the program, with girls from more affluent families predicted to increase affinities over time and girls from lower income families to experience initial gains in affinities that diminish over time. Qualitative examination of girls' perspectives on gender and science efficacy, attitudes toward science, and elements of science identities revealed a complex interplay of gendered stereotypes of science and girls' personal desires to prove themselves knowledgeable and competent scientists. Implications for the best practice in fostering science engagement and identities in middle school-aged girls are discussed.

  12. Convulsant bicuculline modifies CNS muscarinic receptor affinity

    Directory of Open Access Journals (Sweden)

    Rodríguez de Lores Arnaiz Georgina

    2006-04-01

    Full Text Available Abstract Background Previous work from this laboratory has shown that the administration of the convulsant drug 3-mercaptopropionic acid (MP, a GAD inhibitor, modifies not only GABA synthesis but also binding of the antagonist [3H]-quinuclidinyl benzilate ([3H]-QNB to central muscarinic receptors, an effect due to an increase in affinity without modifications in binding site number. The cholinergic system has been implicated in several experimental epilepsy models and the ability of acetylcholine to regulate neuronal excitability in the neocortex is well known. To study the potential relationship between GABAergic and cholinergic systems with seizure activity, we analyzed the muscarinic receptor after inducing seizure by bicuculline (BIC, known to antagonize the GABA-A postsynaptic receptor subtype. Results We analyzed binding of muscarinic antagonist [3H]-QNB to rat CNS membranes after i.p. administration of BIC at subconvulsant (1.0 mg/kg and convulsant (7.5 mg/kg doses. Subconvulsant BIC dose failed to develop seizures but produced binding alteration in the cerebellum and hippocampus with roughly 40% increase and 10% decrease, respectively. After convulsant BIC dose, which invariably led to generalized tonic-clonic seizures, binding increased 36% and 15% to cerebellar and striatal membranes respectively, but decreased 12% to hippocampal membranes. Kd value was accordingly modified: with the subconvulsant dose it decreased 27% in cerebellum whereas it increased 61% in hippocampus; with the convulsant dose, Kd value decreased 33% in cerebellum but increased 85% in hippocampus. No change in receptor number site was found, and Hill number was invariably close to unity. Conclusion Results indicate dissimilar central nervous system area susceptibility of muscarinic receptor to BIC. Ligand binding was modified not only by a convulsant BIC dose but also by a subconvulsant dose, indicating that changes are not attributable to the seizure process

  13. Electron affinities of atoms, molecules, and radicals

    International Nuclear Information System (INIS)

    Christodoulides, A.A.; McCorkle, D.L.; Christophorou, L.G.

    1982-01-01

    We review briefly but comprehensively the theoretical, semiempirical and experimental methods employed to determine electron affinities (EAs) of atoms, molecules and radicals, and summarize the EA data obtained by these methods. The detailed processes underlying the principles of the experimental methods are discussed very briefly. It is, nonetheless, instructive to recapitulate the definition of EA and those of the related quantities, namely, the vertical detachment energy, VDE, and the vertical attachment energy, VAE. The EA of an atom is defined as the difference in total energy between the ground state of the neutral atom (plus the electron at rest at infinity) and its negative ion. The EA of a molecule is defined as the difference in energy between the neutral molecule plus an electron at rest at infinity and the molecular negative ion when both, the neutral molecules and the negative ion, are in their ground electronic, vibrational and rotational states. The VDE is defined as the minimum energy required to eject the electron from the negative ion (in its ground electronic and nuclear state) without changing the internuclear separation; since the vertical transition may leave the neutral molecule in an excited vibrational/rotational state, the VDE, although the same as the EA for atoms is, in general, different (larger than), from the EA for molecules. Similarly, the VAE is defined as the difference in energy between the neutral molecule in its ground electronic, vibrational and rotational states plus an electron at rest at infinity and the molecular negative ion formed by addition of an electron to the neutral molecule without allowing a change in the intermolecular separation of the constituent nuclei; it is a quantity appropriate to those cases where the lowest negative ion state lies above the ground states of the neutral species and is less or equal to EA

  14. Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

    Science.gov (United States)

    Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

    2017-06-01

    Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Simultaneous purifying of Hg0, SO2, and NOx from flue gas by Fe3+/H2O2: the performance and purifying mechanism.

    Science.gov (United States)

    Xing, Yi; Li, Liuliu; Lu, Pei; Cui, Jiansheng; Li, Qianli; Yan, Bojun; Jiang, Bo; Wang, Mengsi

    2018-03-01

    Hg 0 , SO 2 , and NOx result in heavily global environmental pollution and serious health hazards. Up to now, how to efficiently remove mercury with SO 2 and NOx from flue gas is still a tough task. In this study, series of high oxidizing Fenton systems were employed to purify the pollutants. The experimental results showed that Fe 3+ /H 2 O 2 was more suitable to purify Hg 0 than Fe 2+ /H 2 O 2 and Cu 2+ /H 2 O 2. The optimal condition includes Fe 3+ concentration of 0.008 mol/L, Hg 0 inlet concentration of 40 μg/m 3 , solution temperature of 50 °C, pH of 3, H 2 O 2 concentration of 0.7 mol/L, and O 2 percentage of 6%. When SO 2 and NOx were taken into account under the optimal condition, Hg 0 removal efficiency could be enhanced to 91.11% while the removal efficiency of both NOx and SO 2 was slightly declined, which was consistent to the analysis of purifying mechanism. The removal efficiency of Hg 0 was stimulated by accelerating the conversion of Fe 2+ to Fe 3+ , which resulted from the existence of SO 2 and NOx. The results of this study suggested that simultaneously purifying Hg 0 , SO 2 , and NOx from flue gas is feasible.

  16. In vitro determination of 6PGD enzyme activity purified from Lake Van fish (Chalcalburnus tarichii Pallas, 1811) liver exposed to pesticides.

    Science.gov (United States)

    Guler, Muhammet; Kivanc, M Riza; Turkoglu, Vedat; Basi, Zehra; Kivrak, Hilal

    2013-11-01

    In the present study, the effect of methidathion, cypermethrin, and deltamethrin pesticides on Lake Van fish (Chalcalburnus tarichii Pallas, 1811) liver 6-phosphogluconate dehydrogenase enzyme activity was investigated due to the fact that these pesticides are extensively used to improve agricultural productivity in the Van region. 2',5'-ADP Sepharose 4B affinity chromatography was used to purify 6-phosphogluconate dehydrogenase enzyme from fish liver and SDS-PAGE technique was used to control the purity of this enzyme. The in vitro effect of methidathion, cypermethrin, and deltamethrin pesticides on the enzyme activity was investigated. The enzyme was purified 1,050-fold with specific activity of 27.04 EU/mg protein. Moreover, Ki constants of methidathion, cypermethrin, and deltamethrin were to be 3.294 ± 0.215, 0.718 ± 0.095, and 0.084 ± 0.009 mM respectively. The IC50 value were estimated as 9.95 × 10(-5) ± 0.1844 × 10(-5) mM for methidathion, 1.01 × 10(-4) ± 0.01413 × 10(-4) mM for cypermethrin, and 4.43 × 10(-6) ± 0.05653 × 10(-6) mM for deltamethrin. In conclusion, deltamethrin inhibits the enzyme activity more than methidathion and cypermethrin.

  17. A biochemical correlate of dimorphism in a zygomycete Benjaminiella poitrasii: characterization of purified NAD-dependent glutamate dehydrogenase, a target for antifungal agents.

    Science.gov (United States)

    Joshi, C V; Pathan, E K; Punekar, N S; Tupe, S G; Kapadnis, B P; Deshpande, M V

    2013-07-01

    The fungal organisms, especially pathogens, change their vegetative (Y, unicellular yeast and H, hypha) morphology reversibly for survival and proliferation in the host environment. NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from a non-pathogenic dimorphic zygomycete Benjaminiella poitrasii was previously reported to be an important biochemical correlate of the transition process. The enzyme was purified to homogeneity and characterized. It is a 371 kDa native molecular weight protein made up of four identical subunits. Kinetic studies showed that unlike other NAD-GDHs, it may act as an anabolic enzyme and has more affinity towards 2-oxoglutarate than L-glutamate. Chemical modifications revealed the involvement of single histidine and lysine residues in the catalytic activity of the enzyme. The phosphorylation and dephosphorylation study showed that the NAD-GDH is present in active phosphorylated form in hyphal cells of B. poitrasii. Two of the 1,2,3 triazole linked β-lactam-bile acid conjugates synthesized in the laboratory (B18, B20) were found to be potent inhibitors of purified NAD-GDH which also significantly affected Y-H transition in B. poitrasii. Furthermore, the compound B20 inhibited germ tube formation during Y-H transition in Candida albicans strains and Yarrowia lipolytica. The possible use of NAD-GDH as a target for antifungal agents is discussed.

  18. Induction of protective immunity against Schistosoma mansoni infection by antigens purified from PIII, a fraction of adult worm, associated to the downregulation of granuloma formation

    Directory of Open Access Journals (Sweden)

    Gustavson Shauma

    1998-01-01

    Full Text Available This study was performed in order to define Schistosoma mansoni antigens able to function as modulator agents in BALB/c mice granulomatous hypersensitivity to parasite egg. The antigens P-24, P-35 and P-97 were purified by affinity chromatography from a fraction of S. mansoni adult worm antigenic preparation, denominated PIII, involved in the inhibition of granulomatous response to eggs. Immunization of mice with these antigens, in the presence of Corynebacterium parvum and Al(OH3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system. In vitro blastogenesis assays revealed that purified antigens were able to induce significant proliferation of spleen cells from S. mansoni-infected mice. This protection was correlated to significant decrease in granuloma size induced by PIII. From these results, we concluded that PIII preparation contains antigens capable of mediating protective anti-parasite immunity and down-regulating granulomatous hypersensitivity to S. mansoni eggs.

  19. Comparative study of two purified inulinases from thermophile Thielavia Terrestris NRRL 8126 and mesophile Aspergillus Foetidus NRRL 337 grown on Cichorium Intybus l

    Directory of Open Access Journals (Sweden)

    Eman Mohamed Fawzi

    2011-06-01

    Full Text Available Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml and higher maximum initial velocity (115 µmol/min/mg of protein for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  20. Identification and characterization of a chitin-binding protein purified from coelomic fluid of the lugworm Arenicola marina defining a novel protein sequence family

    DEFF Research Database (Denmark)

    Vitashenkova, Nina; Moeller, Jesper Bonnet; Leth-Larsen, Rikke

    2012-01-01

    We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm Arenicola marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass......, respectively, were isolated from a coelomocyte cDNA library. The two clones designated AML-1a and AML-1b were 92% identical at the protein level, and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N......-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a reduced lacked a 27-kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed, that AML-1 is expressed...

  1. Composition and potency characterization of Mycobacterium avium subsp. paratuberculosis purified protein derivatives

    Science.gov (United States)

    Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain ATCC 19698. Traditional production consists of floating culture incubation at 37oC, organism inactivation by autoclaving, coarse filtrat...

  2. On the technological development of cotton primary processing, using a new drying-purifying unit

    Science.gov (United States)

    Agzamov, M. M.; Yunusov, S. Z.; Gafurov, J. K.

    2017-10-01

    The article reflects feasibility study of conducting research on technological development of cotton primary processing with the modified parameters of drying and cleaning process for small litter. As a result of theoretical and experimental research, drying and purifying unit is designed, in which in the existing processes a heat source, exhaust fans, a dryer drum, a peg-drum cleaner of cotton and the vehicle transmitting raw cotton from the dryer to the purifier will be excluded. The experience has shown that when a drying-purifying unit is installed (with eight wheels) purifying effect on the small litter of 34%, ie cleaning effect is higher than of that currently in operation 1XK drum cleaner. According to the research patent of RU UZ FAP 00674 “Apparatus for drying and cleaning fibrous material” is received.

  3. 77 FR 14733 - Purified Carboxymethylcellulose From Finland and the Netherlands: Extension of Time Limit for...

    Science.gov (United States)

    2012-03-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE International Trade Administration Purified Carboxymethylcellulose From Finland and the Netherlands: Extension of Time Limit for Preliminary Results of Antidumping Duty Administrative Reviews AGENCY: Import Administration, International Trade...

  4. 76 FR 18156 - Purified Carboxymethylcellulose From the Netherlands: Extension of Time Limit for Preliminary...

    Science.gov (United States)

    2011-04-01

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF COMMERCE International Trade Administration Purified Carboxymethylcellulose From the Netherlands: Extension of Time Limit for Preliminary Results of Antidumping Duty Administrative Review AGENCY: Import Administration, International Trade Administration,...

  5. Integrated Microchannel Reformer/Hydrogen Purifier for Fuel Cell Power Systems, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Makel Engineering, Inc. (MEI) supported by Lockheed Martin and the Colorado School of Mines (CSM) propose to develop an integrated hydrogen generator and purifier...

  6. Can a photocatalytic air purifier be used to improve the perceived air quality indoors?

    DEFF Research Database (Denmark)

    Kolarik, Jakub; Wargocki, Pawel

    2010-01-01

    The effect of a photocatalytic air purifier on perceived air quality(PAQ) was examined in rooms polluted by typical sources of indoor pollution.The rooms were ventilated at three different outdoor air supply rates. The air quality was assessed by a sensory panel when the purifier was in operation...... as well as when it was off. Operation of the purifier significantly improved PAQ in the rooms polluted by building materials (used carpet, old linoleum, and old chip-board), and a used ventilation filter as well as a mixture of building materials, used ventilation filter and cathode-ray tube computer...... monitors. The effect cor-responded to approximately doubling the outdoor air supply rate. Operation of the purifier significantly worsened the PAQ in rooms with human bioeffluents, probably due to incomplete oxidation of alcohols which are one of the main pollutants emitted by humans. Present results show...

  7. Simple and Efficient Method to Purify DNA–Protein Conjugates and Its Sensing Applications

    Science.gov (United States)

    2015-01-01

    DNA–protein conjugates are very useful in analytical chemistry for target recognition and signal amplification. While a number of methods for conjugating DNA with proteins are known, methods for purification of DNA–protein conjugates from reaction mixture containing unreacted proteins are much less investigated. In this work, a simple and efficient approach to purify DNA–invertase conjugates from reaction mixture via a biotin displacement strategy to release desthiobiotinylated DNA–invertase conjugates from streptavidin-coated magnetic beads was developed. The conjugates purified by this approach were utilized for quantitative detection of cocaine and DNA using a personal glucose meter through structure-switching DNA aptamer sensors and competitive DNA hybridization assays, respectively. In both cases, the purified DNA–invertase conjugates showed better performance compared to the same assays using unpurified conjugates. The approach demonstrated here can be further expanded to other DNA and proteins to generate purified DNA–protein conjugates for analytical and other applications. PMID:24605905

  8. Endurance Pump Tests With Fresh and Purified MIL-PRF-83282 Hydraulic Fluid

    National Research Council Canada - National Science Library

    Sharma, Shashi

    1999-01-01

    .... Two endurance pump tests were conducted with F-16 aircraft hydraulic pumps, using both fresh and purified MIL-PRF-83282 hydraulic fluid, to determine if fluid purification had any adverse effect on pump life...

  9. Effect of streamer plasma air purifier on sbs symptoms and performance of office work

    DEFF Research Database (Denmark)

    Zhang, X.J.; Fang, Lei; Wargocki, Pawel

    2011-01-01

    level of air pollution. Intensity of SBS symptoms were indicated using visual-analogue scales. Subjects’ performance was evaluated with several computer tasks. The results show that operation of the air purifiers improved perceived air quality and reduced the odor intensity of indoor air. Eye dryness......Subjective experiments were conducted to evaluate the effect of a streamer plasma air purifier on perceived air quality, SBS symptoms and performance of office work during 5-hour exposure of 32 recruited subjects in field laboratory in which real materials were used to establishing a realistic...... symptom was found significantly improved when the air purifiers were used but no other SBS symptoms or performance of office work were improved when the air purifiers were in operation compared to the condition when they were off....

  10. Integrated Microchannel Reformer/Hydrogen Purifier for Fuel Cell Power Systems, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Makel Engineering, Inc. (MEI) and Colorado School of Mines (CSM) propose to develop an integrated hydrogen generator and purifier system for conversion of in-situ...

  11. Investigating the characteristic strength of flocs formed from crude and purified Hibiscus extracts in water treatment.

    Science.gov (United States)

    Jones, Alfred Ndahi; Bridgeman, John

    2016-10-15

    The growth, breakage and re-growth of flocs formed using crude and purified seed extracts of Okra (OK), Sabdariffa (SB) and Kenaf (KE) as coagulants and coagulant aids was assessed. The results showed floc size increased from 300 μm when aluminium sulphate (AS) was used as a coagulant to between 696 μm and 722 μm with the addition of 50 mg/l of OK, KE and SB crude samples as coagulant aids. Similarly, an increase in floc size was observed when each of the purified proteins was used as coagulant aid at doses of between 0.123 and 0.74 mg/l. The largest floc sizes of 741 μm, 460 μm and 571 μm were obtained with a 0.123 mg/l dose of purified Okra protein (POP), purified Sabdariffa (PSP) and purified Kenaf (PKP) respectively. Further coagulant aid addition from 0.123 to 0.74 mg/l resulted in a decrease in floc size and strength in POP and PSP. However, an increase in floc strength and reduced d50 size was observed in PKP at a dose of 0.74 mg/l. Flocs produced when using purified and crude extract samples as coagulant aids exhibited high recovery factors and strength. However, flocs exhibited greater recovery post-breakage when the extracts were used as a primary coagulant. It was observed that the combination of purified proteins and AS improved floc size, strength and recovery factors. Therefore, the applications of Hibiscus seeds in either crude or purified form increases floc growth, strength, recoverability and can also reduce the cost associated with the import of AS in developing countries. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  12. An Experiment with Air Purifiers in Delhi during Winter 2015-2016.

    Science.gov (United States)

    Vyas, Sangita; Srivastav, Nikhil; Spears, Dean

    2016-01-01

    Particulate pollution has important consequences for human health, and is an issue of global concern. Outdoor air pollution has become a cause for alarm in India in particular because recent data suggest that ambient pollution levels in Indian cities are some of the highest in the world. We study the number of particles between 0.5μm and 2.5μm indoors while using affordable air purifiers in the highly polluted city of Delhi. Though substantial reductions in indoor number concentrations are observed during air purifier use, indoor air quality while using an air purifier is frequently worse than in cities with moderate pollution, and often worse than levels observed even in polluted cities. When outdoor pollution levels are higher, on average, indoor pollution levels while using an air purifier are also higher. Moreover, the ratio of indoor air quality during air purifier use to two comparison measures of air quality without an air purifier are also positively correlated with outdoor pollution levels, suggesting that as ambient air quality worsens there are diminishing returns to improvements in indoor air quality during air purifier use. The findings of this study indicate that although the most affordable air purifiers currently available are associated with significant improvements in the indoor environment, they are not a replacement for public action in regions like Delhi. Although private solutions may serve as a stopgap, reducing ambient air pollution must be a public health and policy priority in any region where air pollution is as high as Delhi's during the winter.

  13. [Diurnal variations in purifying-tanks when use Pontederia cordata treating the Malodorous River water].

    Science.gov (United States)

    Chen, Jian-jun; Lu, Xiao-ming; Lu, Shao-yong; Jin, Xiang-can; Huang, Min-sheng; Zhang, Yong; Zhao, Feng

    2009-12-01

    Aquatic plants (Ponsederie cordaza) were waked in two purifying-tanks to investigate the effects of illumination intensity and aeration on diurnal variations of Chla, SP, POD of Ponsederia cordaza and pH, DO, COD, NH4+ -N, TP of water from purifying-tanks when treating the malodorous river water at seven different times, another blank purifying-tank was set as a control. Comparative studies and correlation analysis of these different indicators were carried out to improve the plants working efficiency and provide scientific basis for optimal operation of plant purifying-tanks. Results showed that all indicators affected by changes of light, TP shows best correlation coefficient Cr = 0.93, p < 0.01) of physicochemical indicators and SP behaves best correlation coefficient Cr = 0.91 , p < 0.01) of plant physiology indicators in non-aeration purifying-tank;aeration is necessary as diurnal average of DO shows an increase of 0.13 mg/L by treatment of plant meanwhile 1.8 mg/L by plant with aeration,purifying-tanks with aeration got 7.1%, 6.3% higher removing rates of COD, NH4+ -N and 38% less TP removing rate than non-aeration plant purifying-tanks (p < 0.01); with aeration treatment, significant reduction of Chla, SP content (p < 0.05) and increase of POD activity (p < 0.05) observed in plants; the changes of illumination intensity and aeration can significantly affect physiological characteristics of plants and should be considered carefully and need further study when treating malodorous river water by plant purifying-tanks.

  14. Effect of Purified Mushroom Tyrosinase on Melanin Content and Melanogenic Protein Expression

    OpenAIRE

    Zaidi, Kamal Uddin; Ali, Sharique A.; Ali, Ayesha S.

    2016-01-01

    In mammalian melanocytes, melanosome is a highly specialized organelle where melanin is synthesized. Melanin synthesis is controlled by tyrosinase, the vital enzyme in melanogenic pathway. The present investigation is based on an effect of purified mushroom tyrosinase of Agaricus bisporus on B16F10 melanocytes for the melanin production via blocking pigment cell machinery. Using B16F10 melanocytes showed that the stimulation of melanogenesis by purified tyrosinase is due to increased tyrosina...

  15. A Comparison of the Inflammatory Response Produced by Commercial Eugenol and Purified Eugenol,

    Science.gov (United States)

    1980-05-22

    irritant. However, commercial or "stock" eugenol contains a number of impurities; 20,21 e.g. acetyl eugenol , furfurai, methyl -n-amyl-ketone, and others...block nmber) Eugenol is one of the major therapeutic agents In dentistry. The inflammatory.0 response to purified and commercial eugenol are compared...and the Inflammatory C response to zinc oxide/ eugenol prepared with either purified’or commercial eugenol are also compared. The results indicate

  16. Genetic Algorithm-based Affine Parameter Estimation for Shape Recognition

    Directory of Open Access Journals (Sweden)

    Yuxing Mao

    2014-06-01

    Full Text Available Shape recognition is a classically difficult problem because of the affine transformation between two shapes. The current study proposes an affine parameter estimation method for shape recognition based on a genetic algorithm (GA. The contributions of this study are focused on the extraction of affine-invariant features, the individual encoding scheme, and the fitness function construction policy for a GA. First, the affine-invariant characteristics of the centroid distance ratios (CDRs of any two opposite contour points to the barycentre are analysed. Using different intervals along the azimuth angle, the different numbers of CDRs of two candidate shapes are computed as representations of the shapes, respectively. Then, the CDRs are selected based on predesigned affine parameters to construct the fitness function. After that, a GA is used to search for the affine parameters with optimal matching between candidate shapes, which serve as actual descriptions of the affine transformation between the shapes. Finally, the CDRs are resampled based on the estimated parameters to evaluate the similarity of the shapes for classification. The experimental results demonstrate the robust performance of the proposed method in shape recognition with translation, scaling, rotation and distortion.

  17. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    Science.gov (United States)

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

  18. Determination of ractopamine and salbutamol in pig hair by liquid chromatography tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Kai-Chun Chang

    2018-04-01

    Full Text Available A liquid chromatography tandem mass spectrometric method was developed for the determination of two β-agonists (ractopamine and salbutamol in pig hair samples. An isotope of ractopamine-d5 or salbutamol-d6 as an internal standard was used to carry out quantitative analysis. Concentrated sodium hydroxide was used to pretreat hair samples and then purified by the solid phase extraction (SPE procedure. The extracted solution was evaporated and reconstituted for injection in the instrument with electrospray ionization (ESI operating in a positive multiple-reaction-monitoring (MRM mode. Ractopamine and salbutamol separation were performed on C18 analytical column under gradient condition. The internal standard calibration curve was linear in the range of concentration from 0.5 to 100 ng mL−1 (R2 > 0.995. Recoveries of this method estimated at three spiked concentrations of 100, 250 and 500 ng mL−1 in pig hair samples, were 79–82% for ractopamine and 77–96% for salbutamol. The corresponding inter-day and intra-day precisions expressed as relative standard deviation (RSD % were 3.8–6.4% and 3.8–8.6%, respectively. The analytical time for one sample was 8 min. The detection limit of this method was 0.6 and 8.3 ng mL−1 for ractopamine and salbutamol, respectively. This developed method can be applied for monitoring the use of the β-agonists salbutamol and ractopamine in swine feed incurred pig hair. Keywords: Salbutamol, Ractopamine, Hair, Tandem mass

  19. Purification, Biochemical Characterization, and Bioactive Properties of a Lectin Purified from the Seeds of White Tepary Bean (Phaseolus Acutifolius Variety Latifolius

    Directory of Open Access Journals (Sweden)

    Marco A. Becerril-Flores

    2011-03-01

    Full Text Available The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90–100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL; J20 (DI50 275 µg/mL, and N5 (DI50 72.5 µg/mL.

  20. Production and Purification of a Polyclonal Antibody Against Purified Mouse IgG2b in Rabbits Towards Designing Mouse Monoclonal Isotyping Kits

    Directory of Open Access Journals (Sweden)

    Sadeq Eivazi

    2015-03-01

    Full Text Available Purpose: Mouse IgG subclasses containing IgG1, IgG2a, IgG2b and IgG3 have been defined and described both physiochemically and immunologically. Methods: Sepharose beads conjugated with protein A affinity chromatography was used for purification of mouse IgG2b. Sodium citrate buffer (0.1 M, pH: 3.5 was used for separation of mouse IgG2b. Verification of the purified fractions was monitored by SDS-PAGE (polyacrylamide gel electrophoresis in reducing condition. Immunized rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. After dialysis against tris-phosphate buffer (pH: 8.1 ion exchange chromatography column was used for purification of rabbit anti-mouse IgG2b. The periodate method was performed for conjugation with some variations. After conjugation, direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against mouse IgG2b. Results: The titer of rabbit anti-mouse IgG2b that determined by ELISA was 32000. The purity of rabbit anti-mouse IgG2b was about 95%. The optimum dilution of prepared HRP conjugated IgG was 1:10000. This study showed that ion-exchange chromatography and affinity chromatography could be appropriate techniques for purification of mouse IgG and IgG subclasses respectively. Conclusion: This study showed that affinity chromatography could be an appropriate method for purification of IgG2b antibodies.

  1. An improved affine projection algorithm for active noise cancellation

    Science.gov (United States)

    Zhang, Congyan; Wang, Mingjiang; Han, Yufei; Sun, Yunzhuo

    2017-08-01

    Affine projection algorithm is a signal reuse algorithm, and it has a good convergence rate compared to other traditional adaptive filtering algorithm. There are two factors that affect the performance of the algorithm, which are step factor and the projection length. In the paper, we propose a new variable step size affine projection algorithm (VSS-APA). It dynamically changes the step size according to certain rules, so that it can get smaller steady-state error and faster convergence speed. Simulation results can prove that its performance is superior to the traditional affine projection algorithm and in the active noise control (ANC) applications, the new algorithm can get very good results.

  2. Molecular modeling of protein A affinity chromatography.

    Science.gov (United States)

    Salvalaglio, Matteo; Zamolo, Laura; Busini, Valentina; Moscatelli, Davide; Cavallotti, Carlo

    2009-12-11

    The properties of the complex between fragment B of Protein A and the Fc domain of IgG were investigated adopting molecular dynamics with the intent of providing useful insight that might be exploited to design mimetic ligands with properties similar to those of Protein A. Simulations were performed both for the complex in solution and supported on an agarose surface, which was modeled as an entangled structure constituted by two agarose double chains. The energetic analysis was performed by means of the molecular mechanics Poisson Boltzmann surface area (MM/PBSA), molecular mechanics generalized Born surface area (MM/GBSA), and the linear interaction energy (LIE) approaches. An alanine scan was performed to determine the relative contribution of Protein A key amino acids to the complex interaction energy. It was found that three amino acids play a dominant role: Gln 129, Phe 132 and Lys 154, though also four other residues, Tyr 133, Leu 136, Glu 143 and Gln 151 contribute significantly to the overall binding energy. A successive molecular dynamics analysis of Protein A re-organization performed when it is not in complex with IgG has however shown that Phe 132 and Tyr 133 interact among themselves establishing a significant pi-pi interaction, which is disrupted upon formation of the complex with IgG and thus reduces consistently their contribution to the protein-antibody bond. The effect that adsorbing fragment B of Protein A on an agarose support has on the stability of the protein-antibody bond was investigated using a minimal molecular model and compared to a similar study performed for a synthetic ligand. It was found that the interaction with the surface does not hinder significantly the capability of Protein A to interact with IgG, while it is crucial for the synthetic ligand. These results indicate that ligand-surface interactions should be considered in the design of new synthetic affinity ligands in order to achieve results comparable to those of Protein A

  3. Screening of L-histidine-based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform.

    Science.gov (United States)

    Amorim, Lúcia F A; Sousa, Fani; Queiroz, João A; Cruz, Carla; Sousa, Ângela

    2015-06-01

    The growing demand of pharmaceutical-grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l-histidine and its derivatives, 1-benzyl-L-histidine and 1-methyl-L-histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l-histidine and its derivatives was high (KD  > 10(-8)  M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K(D)  = 1.1 × 10(-10)  M and KD  = 3.34 × 10(-10)  M for open circular and linear plasmid isoforms, respectively). L-Histidine and 1-benzyl-L-histidine were immobilized on monolithic matrices. Chromatographic studies of L-histidine and 1-benzyl-L-histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1-benzyl-L-histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l-histidine monolith was 29-fold higher than that obtained with conventional L-histidine agarose. Overall, the combination of either L-histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Design of tandem mirror reactors with thermal barriers

    International Nuclear Information System (INIS)

    Carlson, G.A.

    1980-01-01

    End-plug technologies for tandem mirror reactors include high-field superconducting magnets, neutral beam injectors, and gyrotrons for electron cyclotron resonant heating (ECRH). In addition to their normal use for sustenance of the end-plug plasmas, neutral beam injectors are used for ''pumping'' trapped ions from the thermal barrier regions by charge exchange. An extra function of the axially directed pump beams is the removal of thermalized alpha particles from the reactor. The principles of tandem mirror operation with thermal barriers will be demonstrated in the upgrade of the Tandem Mirror Experiment (TMX-U) in 1981 and the tandem configuration of the Mirror fusion Test Facility (MFTF-B) in 1984

  5. Green oxidations: Titanium dioxide induced tandem oxidation coupling reactions

    OpenAIRE

    Jeena, Vineet; Robinson, Ross S

    2009-01-01

    Summary The application of titanium dioxide as an oxidant in tandem oxidation type processes is described. Under microwave irradiation, quinoxalines have been synthesized in good yields from the corresponding ?-hydroxyketones.

  6. Solar-to-Chemical Energy Conversion with Photoelectrochemical Tandem Cells.

    Science.gov (United States)

    Sivula, Kevin

    2013-01-01

    Efficiently and inexpensively converting solar energy into chemical fuels is an important goal towards a sustainable energy economy. An integrated tandem cell approach could reasonably convert over 20% of the sun's energy directly into chemical fuels like H2 via water splitting. Many different systems have been investigated using various combinations of photovoltaic cells and photoelectrodes, but in order to be economically competitive with the production of H2 from fossil fuels, a practical water splitting tandem cell must optimize cost, longevity and performance. In this short review, the practical aspects of solar fuel production are considered from the perspective of a semiconductor-based tandem cell and the latest advances with a very promising technology - metal oxide photoelectrochemical tandem cells - are presented.

  7. Monolithic Perovskite Silicon Tandem Solar Cells with Advanced Optics

    Energy Technology Data Exchange (ETDEWEB)

    Goldschmidt, Jan C.; Bett, Alexander J.; Bivour, Martin; Blasi, Benedikt; Eisenlohr, Johannes; Kohlstadt, Markus; Lee, Seunghun; Mastroianni, Simone; Mundt, Laura; Mundus, Markus; Ndione, Paul; Reichel, Christian; Schubert, Martin; Schulze, Patricia S.; Tucher, Nico; Veit, Clemens; Veurman, Welmoed; Wienands, Karl; Winkler, Kristina; Wurfel, Uli; Glunz, Stefan W.; Hermle, Martin

    2016-11-14

    For high efficiency monolithic perovskite silicon tandem solar cells, we develop low-temperature processes for the perovskite top cell, rear-side light trapping, optimized perovskite growth, transparent contacts and adapted characterization methods.

  8. Tandem Wittig-ene reaction approach to kainic acid

    Digital Repository Service at National Institute of Oceanography (India)

    Majik, M.S.; Parameswaran, P; Tilve, S.G.

    The first example of a tandem Wittig-intramolecular ene reaction approach and its application toward the synthesis of kainic acid is reported. The synthetic pathway involves conversion of prenyl bromide into phosphorane 3, followed by one-pot Wittig...

  9. 42 CFR 84.1143 - Dust, fume, and mist air-purifying filter tests; performance requirements; general.

    Science.gov (United States)

    2010-10-01

    ... RESPIRATORY PROTECTIVE DEVICES Dust, Fume, and Mist; Pesticide; Paint Spray; Powered Air-Purifying High Efficiency Respirators and Combination Gas Masks § 84.1143 Dust, fume, and mist air-purifying filter tests... 42 Public Health 1 2010-10-01 2010-10-01 false Dust, fume, and mist air-purifying filter tests...

  10. Solution-Processed Nanocrystal Quantum Dot Tandem Solar Cells

    KAUST Repository

    Choi, Joshua J.

    2011-06-03

    Solution-processed tandem solar cells created from nanocrystal quantum dots with size-tuned energy levels are demonstrated. Prototype devices featuring interconnected quantum dot layers of cascaded energy gaps exhibit IR sensitivity and an open circuit voltage, V oc, approaching 1 V. The tandem solar cell performance depends critically on the optical and electrical properties of the interlayer. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Zinc Mediated Tandem Fragmentation-Allylation of Methyl 5-Iodopentofuranosides

    DEFF Research Database (Denmark)

    Hyldtoft, Lene; Madsen, Robert

    1999-01-01

    In the presence of zinc and allyl bromide methyl 5-iodopentofuranosides undergo a tandem fragmentation alkylation to give functionalized dienes. These can undergo ring-closing olefin metathesis to produce cyclohexenes which on dihydroxylation give quercitols.......In the presence of zinc and allyl bromide methyl 5-iodopentofuranosides undergo a tandem fragmentation alkylation to give functionalized dienes. These can undergo ring-closing olefin metathesis to produce cyclohexenes which on dihydroxylation give quercitols....

  12. Engineering problems of tandem-mirror reactors

    International Nuclear Information System (INIS)

    Moir, R.W.; Barr, W.L.; Boghosian, B.M.

    1981-01-01

    We have completed a comparative evaluation of several end plug configurations for tandem mirror fusion reactors with thermal barriers. The axi-cell configuration has been selected for further study and will be the basis for a detailed conceptual design study to be carried out over the next two years. The axi-cell end plug has a simple mirror cell produced by two circular coils followed by a transition coil and a yin-yang pair, which provides for MHD stability. This paper discusses some of the many engineering problems facing the designer. We estimated the direct cost to be 2$/W/sub e/. Assuming total (direct and indirect) costs to be twice this number, we need to reduce total costs by factors between 1.7 and 2.3 to compete with future LWRs levelized cost of electricity. These reductions may be possible by designing magnets producing over 20T made possible by use of combinations of superconducting and normal conducting coils as well as improvements in performance and cost of neutral beam and microwave power systems. Scientific and technological understanding and innovation are needed in the area of thermal barrier pumping - a process by which unwanted particles are removed (pumped) from certain regions of velocity and real space in the end plug. Removal of exhaust fuel ions, fusion ash and impurities by action of a halo plasma and plasma dump in the mirror end region is another challenging engineering problem discussed in this paper

  13. Results from the Tara tandem mirror experiment

    International Nuclear Information System (INIS)

    Lane, B.G.

    1987-09-01

    A summary of the experimental results from the Tara tandem mirror experiment is presented. Optimization of the fueling configuration, slow wave ion cyclotron heating from a magnetic ''plateau'' using an aperture antenna design, and enhanced stabilization from a magnetic divertor have allowed the attainment of a stable start up plasma (T/sub i,perpendicular/ = 800 - 1500 eV, n/sub e/ = 4 - 5 x 10 12 cm -3 , T/sub e/ = 70 - 80 eV). Plugging experiments using radiofrequency waves near the plug midplane ion cyclotron frequency have proved successful in reducing endloss, while simultaneously leading to an increase in central cell density. The plugging potentials have been limited to approximately the ion parallel temperature. This limitation is due to low frequency instabilities localized in the plug. Axial plugging experiments using electron cyclotron (ECH) resonant microwave radiation in the plug cells have had ambiguous results. Endloss reductions up to 100% have been achieved without build-up of central cell densities or the appearance of the reflected particles at the other end of the machine. We conjecture that rapid radial losses accompany the use of ECH, although the mechanisms for this loss remain unidentified. 9 refs., 9 figs

  14. Software dependability in the Tandem GUARDIAN system

    Science.gov (United States)

    Lee, Inhwan; Iyer, Ravishankar K.

    1995-01-01

    Based on extensive field failure data for Tandem's GUARDIAN operating system this paper discusses evaluation of the dependability of operational software. Software faults considered are major defects that result in processor failures and invoke backup processes to take over. The paper categorizes the underlying causes of software failures and evaluates the effectiveness of the process pair technique in tolerating software faults. A model to describe the impact of software faults on the reliability of an overall system is proposed. The model is used to evaluate the significance of key factors that determine software dependability and to identify areas for improvement. An analysis of the data shows that about 77% of processor failures that are initially considered due to software are confirmed as software problems. The analysis shows that the use of process pairs to provide checkpointing and restart (originally intended for tolerating hardware faults) allows the system to tolerate about 75% of reported software faults that result in processor failures. The loose coupling between processors, which results in the backup execution (the processor state and the sequence of events) being different from the original execution, is a major reason for the measured software fault tolerance. Over two-thirds (72%) of measured software failures are recurrences of previously reported faults. Modeling, based on the data, shows that, in addition to reducing the number of software faults, software dependability can be enhanced by reducing the recurrence rate.

  15. Atmospheric pressure ionization-tandem mass spectrometry of the phenicol drug family.

    Science.gov (United States)

    Alechaga, Élida; Moyano, Encarnación; Galceran, M Teresa

    2013-11-01

    In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool.

    Science.gov (United States)

    Jackson, Thomas R; Springall, Jeremy S; Rogalle, Damien; Masumoto, Naoko; Ching Li, Hung; D'Hooge, François; Perera, Semali P; Jenkins, A Toby A; James, Tony D; Fossey, John S; van den Elsen, Jean M H

    2008-11-01

    The incorporation of specialised carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for fluorophore-assisted carbohydrate electrophoresis greatly improved the effective separation of saccharides that show similar mobilities in standard electrophoresis. Polyacrylamide gel electrophoresis using methacrylamido phenylboronic acid in low loading (typically 0.5-1% dry weight) was unequivocally shown to alter retention of labelled saccharides depending on their boronate affinity. While conventional fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone labelled glucose oligomers showed an inverted parabolic migration, an undesired trait of small oligosaccharides labelled with this neutral fluorophore, boron affinity saccharide electrophoresis separation of these carbohydrates completely restored their predicted running order, based on their charge/mass ratio, and resulted in improved separation of the analyte saccharides. These results exemplify boron affinity saccharide electrophoresis as an important new technique for analysing carbohydrates and sugar-containing molecules.

  17. Methods for quantifying T cell receptor binding affinities and thermodynamics

    Science.gov (United States)

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  18. ASIFT: An Algorithm for Fully Affine Invariant Comparison

    Directory of Open Access Journals (Sweden)

    Guoshen Yu

    2011-02-01

    Full Text Available If a physical object has a smooth or piecewise smooth boundary, its images obtained by cameras in varying positions undergo smooth apparent deformations. These deformations are locally well approximated by affine transforms of the image plane. In consequence the solid object recognition problem has often been led back to the computation of affine invariant image local features. The similarity invariance (invariance to translation, rotation, and zoom is dealt with rigorously by the SIFT method The method illustrated and demonstrated in this work, Affine-SIFT (ASIFT, simulates a set of sample views of the initial images, obtainable by varying the two camera axis orientation parameters, namely the latitude and the longitude angles, which are not treated by the SIFT method. Then it applies the SIFT method itself to all images thus generated. Thus, ASIFT covers effectively all six parameters of the affine transform.

  19. Affinity between information retrieval system and search topic

    International Nuclear Information System (INIS)

    Ebinuma, Yukio

    1979-01-01

    Ten search profiles are tested on the INIS system at the Japan Atomic Energy Research Institute. The results are plotted on recall-precision chart ranging from 100% recall to 100% precision. The curves are not purely systems-dependent nor search-dependent, and are determined substantially by the ''affinity'' between the system and the search topic. The curves are named ''Affinity curves of search topics with information retrieval systems'', and hence retrieval affinity factors are derived. They are obtained not only for individual search topics but also for averages in the system. By such a quantitative examination, the difference of affinity among search topics in a given system, that of the same search topic among various systems, and that of systems to the same group of search topics can be compared reasonably. (author)

  20. Antibody Fragments and Their Purification by Protein L Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Gustav Rodrigo

    2015-09-01

    Full Text Available Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing.

  1. Elective affinities and economic thought: 1870-1914

    OpenAIRE

    João Antônio de Paula; Pedro Rocha de Oliveira

    2006-01-01

    This article seeks to demonstrate that the concept of "elective affinities" can be applied to the relations between economic thought, literature, and philosophy. Emphasis is given to Institutionalist thought, the German historical school, and neoclassical thought.

  2. Ab initio studies on proton affinities of substituted furans

    International Nuclear Information System (INIS)

    Lee, Gab Yong; Lee, Hyun Mee

    1998-01-01

    The geometry of furan, relevant to the binding of bis-furan lexitropsin that contains this ring to the base pair of minor groove of DNA, is optimized by semiempirical (MNDO) and ab initio (Hartree-Fock) methods. The proton affinity and electronic structure are evaluated at the 6-31G and 6-31G level of theory for the optimized geometry. The proton affinities are also studied for various substituted furans with the electron donating and -withdrawing groups to estimate the substituent effect on the proton affinity of furans. It has been found that the electron-donating substituents increase the proton affinity of of furan, whereas the electron-withdrawing substituents decrease it. This result can be explained with atomic charge and electron density at oxygen of substituted furans

  3. STAR: an algorithm to Search for Tandem Approximate Repeats.

    Science.gov (United States)

    Delgrange, Olivier; Rivals, Eric

    2004-11-01

    Tandem repeats consist in approximate and adjacent repetitions of a DNA motif. Such repeats account for large portions of eukaryotic genomes and have also been found in other life kingdoms. Owing to their polymorphism, tandem repeats have proven useful in genome cartography, forensic and population studies, etc. Nevertheless, they are not systematically detected nor annotated in genome projects. Partially because of this lack of data, their evolution is still poorly understood. In this work, we design an exact algorithm to locate approximate tandem repeats (ATR) of a motif in a DNA sequence. Given a motif and a DNA sequence, our method named STAR, identifies all segments of the sequence that correspond to significant approximate tandem repetitions of the motif. In our model, an Exact Tandem Repeat (ETR) comes from the tandem duplication of the motif and an ATR derives from an ETR by a series of point mutations. An ATR can then be encoded as a number of duplications of the motif together with a list of mutations. Consequently, any sequence that is not an ATR cannot be encoded efficiently by this description, while a true ATR can. Our method uses the minimum description length criterion to identify which sequence segments are ATR. Our optimization procedure guarantees that STAR finds a combination of ATR that minimizes this criterion. for use at http://atgc.lirmm.fr/star

  4. Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria.

    Directory of Open Access Journals (Sweden)

    Ingo L Brand

    Full Text Available Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins may underestimate their activity.

  5. [Determination of congo red in beef by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry].

    Science.gov (United States)

    Lin, Hui; Xu, Chunxiang; Yan, Chunrong; Zhang, Zheng; Wang, Suilou

    2013-09-01

    A method was developed for the determination of congo red in beef. The analyte was identified by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (LC-QTOF MS) and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the congo red in the beef sample was separated on an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD UPLC column (50 mm x 2.1 mm, 1.8 microm) HPLC , using 95% (volume percentage) methanol as the mobile phase at 0.2 mL/min. The detection was performed on an AB 4000 + triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The results showed that the linear range of congo red mass concentration was 0.03 - 1 mg/L with the correlation coefficient of 0.999 8. The method had a good precision with the RSDs lower than 5% and the recoveries ranging from 88% to 91%. The limit of detection (LOD) of congo red was 0.01 mg/L. With good reproducibility, the method is simple, fast and effective for the determination of the illegally added congo red in beef and other meat products.

  6. Color and Contrast Enhancement by Controlled Piecewise Affine Histogram Equalization

    Directory of Open Access Journals (Sweden)

    Jose-Luis Lisani

    2012-10-01

    Full Text Available This paper presents a simple contrast enhancement algorithm based on histogram equalization (HE. The proposed algorithm performs a piecewise affine transform of the intensity levels of a digital image such that the new cumulative distribution function will be approximately uniform (as with HE, but where the stretching of the range is locally controlled to avoid brutal noise enhancement. We call this algorithm Piecewise Affine Equalization (PAE. Several experiments show that, in general, the new algorithm improves HE results.

  7. New analogues of agmatine with higher affinity to imidazoline receptors.

    Science.gov (United States)

    Treder, Adam P; Andruszkiewicz, Ryszard; Zgoda, Włodzimierz; Ford, Celeste; Hudson, Alan L

    2009-02-01

    Compilation of agmatine structure and imidazoline ring leads to a new family of imidazoline/alpha(2)-adrenoceptor ligands, 4(5)-(2-aminoethyl)imidazoline derivatives. Constraining of the guanidine moiety into heterocyclic ring improved the affinities of the resultant fusion compounds in comparison to agmatine itself. In this work, the synthetic approach and results for I(1), I(2), and alpha(2)-adrenoceptors affinities are reported.

  8. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    International Nuclear Information System (INIS)

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    An overview of the methods used for high-throughput cloning and protein-expression screening of SSGCID hexahistidine recombinant proteins is provided. It is demonstrated that screening for recombinant proteins that are highly recoverable from immobilized metal-affinity chromatography improves the likelihood that a protein will produce a structure. The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure

  9. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  10. Quantification of endogenous brassinosteroids in sub-gram plant tissues by in-line matrix solid-phase dispersion-tandem solid phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Wang, Lu; Duan, Chunfeng; Wu, Dapeng; Guan, Yafeng

    2014-09-12

    A matrix solid-phase dispersion (MSPD)-tandem mixed mode anion exchange (MAX)-mixed mode cation exchange (MCX) solid phase extraction-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of six endogenous brassinosteroids (BRs) (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, dolichosterone, teasterone and typhasterol) in rice plant tissues. Non-polar interferences were removed effectively by C8 dispersant used in MSPD, while the following tandem MAX-MCX process facilitated the elimination of polar and ionizable compounds. The weak reversed-phase retention feature of MAX-MCX leaded to good compatibility of the elution solvents in the in-line coupled MSPD-MAX-MCX system. This system was optimized for extraction and purification of BRs in plant samples. The effects of the type of solid phase, the elution solvent, the extraction temperature and the clean-up material were studied. Before HPLC separation, BRs purified were derivatized by m-aminophenylboronic acid to enhance the sensitivity of MS/MS to BRs. Compared with traditional liquid-liquid extraction and solid phase extraction (LLE-SPE), the proposed MSPD-MAX-MCX method showed higher extraction efficiency, lower matrix effect, and advantages of easy manipulation and time-saving. The in-line MSPD-MAX-MCX coupled with HPLC-MS/MS method provided a linear response over two orders of magnitude of BRs concentration with correlation coefficients above 0.9982, limits of detection between 0.008 and 0.04ngmL(-1), relative standard deviations (RSDs) below 29.4%, and recoveries above 77.8%. The proposed method has been successfully applied to analysis of endogenous BRs in rice plant at booting stage and maturity stage. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Process for the winning of a concentrate containing uranium and purified phosphoric acid, as well as the concentrate containing uranium and purified phosphoric acid obtained by this process

    International Nuclear Information System (INIS)

    1980-01-01

    The uranium containing concentrate and purified phosphoric acid are obtained by treating wet phosphoric acid with an inorganic fluorine compound (ammonium fluoride) and an aliphatic ketone (acetone) in the presence of a reducing agent (finely divided iron). The ketone is added first and the formed uranium precipitate is separated from the solution. If the fluorine compound is added first, the yield is lowered by a factor of 2. (Th.P.)

  12. Surfactant-free Colloidal Particles with Specific Binding Affinity.

    Science.gov (United States)

    van der Wel, Casper; Bossert, Nelli; Mank, Quinten J; Winter, Marcel G T; Heinrich, Doris; Kraft, Daniela J

    2017-09-26

    Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles.

  13. Actualization of the Tandem-E N Accelerator of the Nuclear Centre of Mexico; Actualizacion del Acelerador Tandem-EN del Centro Nuclear

    Energy Technology Data Exchange (ETDEWEB)

    Villasenor S, P.; Aguilera R, E.; Aspiazu F, J.; Fernandez A, J.; Fernandez B, M.; Garcia R, B.; Lopez M, J.; Martinez Q, E.; Mendez G, B.; Moreno B, E.; Murillo O, G.; Policroniades R, R.; Ramirez T, J.; Reynoso V, R.; Varela G, A.; Vega C, J. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    2004-07-01

    In this work, the activities are described carried out to change the tubes accelerators and original resistances of the accelerator Tandem-E N of the Nuclear Center, for tubes DOWLISH and resistances again design, both donated ones for ORNL. This way same, the problem is described that imply this changes, and like it was solved by the personnel of the laboratory, without having to appeal to external services, what there is redounded in a considerable increment in the costs. In form preliminary the improvements are described observed after the rehabilitation of the Accelerator. (Author)

  14. Mechanical performance of HMA-2 modified with purified and unpurified carbon nanotubes and nanofibers

    Directory of Open Access Journals (Sweden)

    Mario Rodrigo Rubio

    2017-05-01

    Full Text Available The present study evaluates the mechanical performance of a Hot Mix Asphalt – Type II (HMA-2 modified with carbon nanotubes and carbon nanofibers (CNTF. CNTF were made by means the Catalytic Vapor Deposition (CVD technique at 700° C using a Nickel, Copper and Aluminum (NiCuAl catalyst with a Cu/Ni molar relation of 0,33. In order to properly assess HMA-2 performance, three different mixtures were analyzed: 1 HMA-2 modified with purified CNTF; 2 HMA-2 modified with non-purified CNTF and, 3 a Conventional HMA-2 (control. Samples manufactured in accordance with the Marshall Mix Design were tested in the laboratory to study rutting, resilient modulus (Mr and fatigue. In addition to the aforementioned dynamic characterization, the effect of CNTF purification on the asphalt mixture’s mechanical properties was analyzed. In short, a comparative study was designed to determine whether or not CNTF should be purified before introduction into the HMA-2. This investigation responds to the growing demand for economical materials capable of withstanding traffic loads while simultaneously enhancing pavement durability and mechanical properties. Although purified CNTF increased HMA-2 stiffness and elastic modulus, non-purified CNTF increased the asphalt mixture’s elastic modulus without considerable increases in stiffness. Thus, the latter modification is deemed to help address fatiguerelated issues and improve the long-term durability of flexible pavements.

  15. Evaluating purifying selection in the mitochondrial DNA of various mammalian species.

    Directory of Open Access Journals (Sweden)

    Pedro Soares

    Full Text Available Mitochondrial DNA (mtDNA, the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.

  16. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    Science.gov (United States)

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  17. [3H] 5,7-dichlorokynurenic acid, a high affinity ligand for the NMDA receptor glycine regulatory site

    International Nuclear Information System (INIS)

    Hurt, S.D.; Baron, B.M.

    1991-01-01

    The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. The authors have prepared [3H] 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield [3H] 5,7-DCKA with a specific activity of 17.6 Ci/mmol. [3H] 5,7-DCKA bound to rat brain synaptosomes with a Kd of 69 ± 23 nM and Bmax = 14.5 ± 3.2 pmoles/mg protein. Binding was 65-70% specific at 10 nM [3H] 5,7-DCKA. This ligand is thus more selective and has higher affinity than [3H] glycine, in addition to being an antagonist

  18. Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

    Science.gov (United States)

    Wu, Ming-Chang; Lin, Guan-Hui; Wang, Yuh-Tai; Jiang, Chii-Ming; Chang, Hung-Min

    2005-10-05

    Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

  19. Purified humanism

    DEFF Research Database (Denmark)

    Nickelsen, Niels Christian Mossfeldt

    2016-01-01

    Abstract. The aim of the Leicester Conference is to help managers by way of experiential learning to acquire the prerequisites to influence effectively organizational change. For some time there has been an ongoing debate on the innovative potential of social psychological experiments...... and culturally specific attitudes in relation to leadership and the question of authority among participants. Keywords: The Leicester Conference, experiential learning, authority, socio-materiality, social techniques...

  20. Engineering GCaMP affinity and kinetics for improved tracking of neuronal activity

    Science.gov (United States)

    Sun, Xiaonan Richard

    Fluorescent calcium indicator proteins (FCIPs) are powerful tools for monitoring neural activity. However, they still have significant performance limitations compared with synthetic indicators based on the small-molecule chelator BAPTA. Because of high cooperativity originating from a calmodulin-based recombinant calcium sensor, a given GECI is only sensitive to a small part of a neuron's likely calcium concentration range, which can span a range of 0.1-10 microM. GECIs also have up to 100-fold slower reponse kinetics than BAPTA-based indicators. Overcoming limitations in range and kinetics is a key step toward monitoring spike times and firing rates in cell-type-specific brain circuits. We are engaged in structure-based design to vary the affinity and accelerate the response kinetics of a widely used GECI, GCaMP3. We have designed more than 50 novel variants by targeted mutation of GCaMP3's calmodulin (CaM) domain and its intraprobe peptide partner, RS20. In our cuvet characterizations of purified protein, we have attained a nearly 40-fold (0.16-6 microM) range of KD without impairing per-molecule brightness. In stopped-flow biochemical measurements, off-responses to sharp decreases in calcium are more than 10 times faster than any other published GECI. Most of the gap in off-response speed between G-CaMP3 and BAPTA-based indicators could be closed without perturbing KD. In Drosophila antennal nerve axons, sensory stimulation-evoked fluorescence responses were significantly enhanced in speed and amplitude in two novel GECIs. With our biophysical measurements, we discovered that the N-lobe of the bilobular CaM domain is required for the high-fluorescence state and the C-lobe contributes to high affinity Ca2+ binding. To account for our observations, we propose a molecular dynamics model of GCaMP3 with two kinetic pathways leading to a high-fluorescence state. First, small amounts of Ca 2+ activate a slow "C-like" pathway through sequential binding to the C

  1. Fabrication of Simple Indoor Air Haze Purifier using Domestic Discarded Substances and Its Haze Removal Performance

    Science.gov (United States)

    Wang, Zhou; Cao, Haoshu; Zhao, Shuang

    2018-01-01

    Based on the concept of circular economy, discarded plastic bottles stuffed with discarded cotton, clothing and sofa cushion were used as pre-filter to remove big particles (dust and coal dust) in air and 4 L tap water in discarded plastic bottle was worked as an absorbing medium to dissolve the water soluble ions in air (SO4 2-, NO3-, NH4+, Cl- and Ca2+). Moreover, the internet control design was used in this homemade indoor air haze purifier to achieve the performance of remote control and intelligent management. The experimental results showed that this indoor air haze purifier can effectively reduce the level of indoor air haze and the air quality after 20 minutes treatment is higher than that of two commercial well-known air haze purifier

  2. Oxidative Stability of Dispersions Prepared from Purified Marine Phospholipid and the Role of α-Tocopherol

    DEFF Research Database (Denmark)

    Lu, Henna Fung Sieng; Nielsen, Nina Skall; Baron, Caroline P.

    2012-01-01

    , respectively, during 32 days of storage at 2 °C. Nonenzymatic browning was investigated through measurement of Strecker aldehydes, color changes, and pyrrole content. Dispersions containing α-tocopherol or higher levels of purified marine PL showed a lower increment of volatiles after 32 days storage......The objective of this study was to investigate the oxidative stability of dispersions prepared from different levels of purified marine phospholipid (PL) obtained by acetone precipitation, with particular focus on the interaction between α-tocopherol and PL in dispersions. This also included...... the investigation of nonenzymatic browning in purified marine PL dispersions. Dispersions were prepared by high-pressure homogenizer. The oxidative and hydrolytic stabilities of dispersions were investigated by determination of hydroperoxides, secondary volatile oxidation products, and free fatty acids...

  3. Inference of purifying and positive selection in three subspecies of chimpanzees (Pan troglodytes) from exome sequencing

    DEFF Research Database (Denmark)

    Bataillon, Thomas; Duan, Jinjie; Hvilsom, Christina

    2015-01-01

    of recent gene flow from Western into Eastern chimpanzees. The striking contrast in X-linked vs. autosomal polymorphism and divergence previously reported in Central chimpanzees is also found in Eastern and Western chimpanzees. We show that the direction of selection (DoS) statistic exhibits a strong non......-monotonic relationship with the strength of purifying selection S, making it inappropriate for estimating S. We instead use counts in synonymous vs. non-synonymous frequency classes to infer the distribution of S coefficients acting on non-synonymous mutations in each subspecies. The strength of purifying selection we...... infer is congruent with the differences in effective sizes of each subspecies: Central chimpanzees are undergoing the strongest purifying selection followed by Eastern and Western chimpanzees. Coding indels show stronger selection against indels changing the reading frame than observed in human...

  4. Experimental studies on removal of airborne haloanisoles by non-thermal plasma air purifiers

    DEFF Research Database (Denmark)

    Fang, Lei; Hallam, David; Bermúdez, Raúl

    2016-01-01

    was collected by Tenax tubes and the concentration of TCA and TBA were analyzed by thermal desorption GC–MS. The results showed that the plasma air purifier was effective on removing TCA and TBA with a single pass efficiency of better than 82%. The effect was further validated in a wine cellar under a realistic......A laboratory study was conducted to test the performance of non-thermal plasma air purifiers on its removal effectiveness of two haloanisoles – 2,4,6-trichloroanisole (TCA) and 2,4,6-Tribromoanisole (TBA). TCA and TBA are the two major compounds found in wine cellars that can contaminate wine...... condition. The concentrations of TCA and TBA in the wine cellar decreased 94% and 50% respectively after running two plasma air purifiers for 5 days. The non-thermal plasma air purification technology may be used in wine cellar to remove the two airborne contaminants and prevent the wine from being...

  5. Structural transformation of the tandem ubiquitin-interacting motifs in ataxin-3 and their cooperative interactions with ubiquitin chains.

    Directory of Open Access Journals (Sweden)

    Ai-Xin Song

    Full Text Available The ubiquitin-interacting motif (UIM is a short peptide with dual function of binding ubiquitin (Ub and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12 both in free and Ub-bound forms. The solution structure of free AT3-UIM12 consists of two α-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates.

  6. Improved detection of a staphylococcal infection by monomeric and protein A-purified polyclonal human immunoglobulin

    International Nuclear Information System (INIS)

    Calame, W.

    1993-01-01

    The present study was undertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99m Tc-labelled monomeric human immunoglobulin (m-Ig), 99m Tc-labelled, protein A-purified, human immunoglobulin (A-IG) and 99m Tc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99m Tc-labelled Igs bound to bacteria in vitro: The percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-to-non-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purified Ig yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99m Tc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99m Tc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein A-purified monomeric Ig was the highest of all tracer agents tested. (orig.)

  7. Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, Stefan [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Seiler, Cornelia; Gerecke, Andreas C. [Swiss Federal Laboratories for Material Science and Technology (EMPA), CH-8600 Dübendorf (Switzerland); Hächler, Herbert [University of Zürich, Institute for Food Safety and Hygiene, National Centre for Enteropathogenic Bacteria and Listeria (NENT), CH-8057 Zürich (Switzerland); Hilbi, Hubert [Ludwig-Maximilians-Universität München Max von Pettenkofer-Institut, D-80336 München (Germany); Frey, Joachim [University of Bern, Institute for Veterinary Bacteriology, CH-3001 Bern (Switzerland); Weidmann, Simon; Meier, Lukas; Berchtold, Christian [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Zenobi, Renato, E-mail: zenobi@org.chem.ethz.ch [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland)

    2013-07-15

    Highlights: • Degradation of environmental toxins, a protein, and bioparticles were studied. • A commercial air purifier based on a cold plasma was used. • Passage through the device reduced the concentration of the compounds/particles. • Deposition inside the plasma air purifier was the main removal process. -- Abstract: Degradation of non-volatile organic compounds–environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)–in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0 m s{sup −1} (3200 L min{sup −1}), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10 L min{sup −1}. Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10 Hz or 50 Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative “degradation” efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

  8. [Rapid determination of 8 urinary carbamate pesticides by liquid chromatography-tandem mass spectrometry].

    Science.gov (United States)

    Liu, Hualiang; Wang, Yuan; Zhu, Baoli

    2015-11-01

    To establish a method for simultaneously determining the urinary concentrations of 8 carbamate pesticides. After being purified by acetonitrile precipitation, urine samples were transferred to a liquid chromatography-tandem mass spectrometry system, and the concentrations of 8 carbamate pesticides were determined by external standard method. A C18 column was used for ultra-high-performance liquid chromatography; methanol/ammonium acetate solution was used as the mobile phase for gradient elution; the mass spectrometer was operated in a multi-reaction monitoring mode. The calibration curves were linear when the urinary concentrations of these carbamate pesticides were 20~800 µg/L, and the recovery rates were 61.0%~121% at spiked levels of 20, 200 and 800 µg/L, with a relative standard deviation of 1.7%~5.5%. This determination method meets the Guide for establishing occupational health standards-part 5: Determination methods of chemicals in biological materials, and can be used for simultaneous determination of 8 carbamate pesticides in the urine of poisoning patients.

  9. Tandem SUMO fusion vectors for improving soluble protein expression and purification.

    Science.gov (United States)

    Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

    2015-12-01

    Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Tarkowski, Petr, E-mail: petr.tarkowski@upol.cz [Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Vaclavikova, Katerina, E-mail: katka.vaclavik@seznam.cz [Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Novak, Ondrej, E-mail: ondrej.novak@upol.cz [Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Pertry, Ine, E-mail: ine.pertry@ugent.BE [Department of Plant Biotechnology and Genetics, Ghent University, K.L.Ledeganckstraat 35, B-9000 Gent (Belgium); Hanus, Jan, E-mail: helehan@seznam.cz [Isotope Laboratory, Institute of Experimental Botany ASCR, Videnska 1083, 142 20 Prague (Czech Republic); Whenham, Robert [Apex Organics, Devon, England (United Kingdom); Vereecke, Danny, E-mail: danny.vereecke@hogent.BE [Department of Plant Production, University College Ghent, Ghent University, Schoonmeersstraat 52, B-9000 Gent (Belgium); Sebela, Marek, E-mail: marek.sebela@upol.cz [Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Strnad, Miroslav, E-mail: miroslav.strnad@upol.cz [Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany ASCR, Slechtitelu 11, 783 71 Olomouc (Czech Republic)

    2010-11-08

    A sensitive and reliable high-performance liquid chromatographic method with tandem mass spectrometric detection has been developed and used for the determination of 2-methylthio-cytokinin derivatives produced by the phytopathogenic actinomycete Rhodococcus fascians. The cultivation medium containing secreted cytokinins was concentrated and subjected to a solid-phase extraction (C18 and ion-exchange). The purified samples were further separated and analyzed by HPLC-ESI-MS/MS. This allowed to achieve chromatographic resolution of six highly hydrophobic cytokinin species including 2-methylthio-isopentenyladenine, 2-methylthio-isopentenyladenosine, 2-methylthio-trans-zeatin and 2-methylthio-trans-zeatin riboside and their cis-isomers when a reversed-phase chromatographic column (C4) and a mobile phase consisting of acetonitrile and 20 mM ammonium formate, pH 5, were used. Quantification was performed by a standard isotope dilution method using a multiple-reaction monitoring (MRM) mode. In the MRM mode, limits of detection reached 20-30 fmol and linear ranges spanned four orders of magnitude. Recovery values were between 35% and 65% and the analytical accuracy between 95% and 149%. The proposed bioanalytical method, which takes advantage of effective chromatographic separation of six 2-methyltio-derivatives (including isomers of zeatin-type cytokinins) and sensitive mass spectrometric detection, may become useful for plant biologists studying the significance of these substances in plant-microbe interactions.

  11. Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry

    International Nuclear Information System (INIS)

    Tarkowski, Petr; Vaclavikova, Katerina; Novak, Ondrej; Pertry, Ine; Hanus, Jan; Whenham, Robert; Vereecke, Danny; Sebela, Marek; Strnad, Miroslav

    2010-01-01

    A sensitive and reliable high-performance liquid chromatographic method with tandem mass spectrometric detection has been developed and used for the determination of 2-methylthio-cytokinin derivatives produced by the phytopathogenic actinomycete Rhodococcus fascians. The cultivation medium containing secreted cytokinins was concentrated and subjected to a solid-phase extraction (C18 and ion-exchange). The purified samples were further separated and analyzed by HPLC-ESI-MS/MS. This allowed to achieve chromatographic resolution of six highly hydrophobic cytokinin species including 2-methylthio-isopentenyladenine, 2-methylthio-isopentenyladenosine, 2-methylthio-trans-zeatin and 2-methylthio-trans-zeatin riboside and their cis-isomers when a reversed-phase chromatographic column (C4) and a mobile phase consisting of acetonitrile and 20 mM ammonium formate, pH 5, were used. Quantification was performed by a standard isotope dilution method using a multiple-reaction monitoring (MRM) mode. In the MRM mode, limits of detection reached 20-30 fmol and linear ranges spanned four orders of magnitude. Recovery values were between 35% and 65% and the analytical accuracy between 95% and 149%. The proposed bioanalytical method, which takes advantage of effective chromatographic separation of six 2-methyltio-derivatives (including isomers of zeatin-type cytokinins) and sensitive mass spectrometric detection, may become useful for plant biologists studying the significance of these substances in plant-microbe interactions.

  12. 78 FR 9884 - Purified Carboxymethylcellulose From the Netherlands: Final Results of Antidumping Duty...

    Science.gov (United States)

    2013-02-12

    ...On August 2, 2012, the Department of Commerce (the Department) published its preliminary results of the 2010-2011 administrative review of the antidumping duty order on purified carboxymethylcellulose (purified CMC) from the Netherlands.\\1\\ This review covers two respondents, Akzo Nobel Functional Chemicals B.V. (Akzo Nobel) and CP Kelco B.V. (CP Kelco). The period of review (POR) is July 1, 2010, through June 30, 2011. The final weighted-average dumping margin is listed below in the ``Final Results of Review'' section of this notice. ---------------------------------------------------------------------------

  13. 78 FR 48649 - Purified Carboxymethylcellulose From the Netherlands: Preliminary Results of Antidumping Duty...

    Science.gov (United States)

    2013-08-09

    ...The Department of Commerce (the Department) is conducting an administrative review of the antidumping duty order on purified carboxymethylcellulose (purified CMC) from the Netherlands. The period of review (POR) is July 1, 2011, through June 30, 2012. The review covers two producers/exporters of the subject merchandise, Akzo Nobel Functional Chemicals, B.V. (Akzo Nobel) and CP Kelco, B.V. (CP Kelco). We preliminarily determine that sales of subject merchandise by Akzo Nobel were made at less than normal value and CP Kelco had no shipments of subject merchandise during the POR.

  14. The impact of genome triplication on tandem gene evolution in Brassica rapa

    Directory of Open Access Journals (Sweden)

    Lu eFang

    2012-11-01

    Full Text Available Whole genome duplication (WGD and tandem duplication (TD are both important modes of gene expansion. However, how whole genome duplication influences tandemly duplicated genes is not well studied. We used Brassica rapa, which has undergone an additional genome triplication (WGT and shares a common ancestor with Arabidopsis thaliana, Arabidopsis lyrata and Thellungiella parvula, to investigate the impact of genome triplication on tandem gene evolution. We identified 2,137, 1,569, 1,751 and 1,135 tandem gene arrays in B. rapa, A. thaliana, A. lyrata and T. parvula respectively. Among them, 414 conserved tandem arrays are shared by the 3 species without WGT, which were also considered as existing in the diploid ancestor of B. rapa. Thus, after genome triplication, B. rapa should have 1,242 tandem arrays according to the 414 conserved tandems. Here, we found 400 out of the 414 tandems had at least one syntenic ortholog in the genome of B. rapa. Furthermore, 294 out of the 400 shared syntenic orthologs maintain tandem arrays (more than one gene for each syntenic hit in B. rapa. For the 294 tandem arrays, we obtained 426 copies of syntenic paralogous tandems in the triplicated genome of B. rapa. In this study, we demonstrated that tandem arrays in B. rapa were dramatically fractionated after WGT when compared either to non-tandem genes in the B. rapa genome or to the tandem arrays in closely related species that have not experienced a recent whole-genome polyploidization event.

  15. Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

    Science.gov (United States)

    Peled, Michael; Tocheva, Anna S; Sandigursky, Sabina; Nayak, Shruti; Philips, Elliot A; Nichols, Kim E; Strazza, Marianne; Azoulay-Alfaguter, Inbar; Askenazi, Manor; Neel, Benjamin G; Pelzek, Adam J; Ueberheide, Beatrix; Mor, Adam

    2018-01-16

    Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

  16. Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin.

    Science.gov (United States)

    Narahari, Akkaladevi; Swamy, Musti J

    2010-04-21

    The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% alpha-helix, 35.8% beta-sheet, 22.5% beta-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4-70 degrees C, but a sharp decrease was seen between 75 and 85 degrees C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 degrees C, indicating that it is a relatively stable protein.

  17. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    Directory of Open Access Journals (Sweden)

    Frank eSainsbury

    2016-02-01

    Full Text Available The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to rapidly purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

  18. RECOVERY OF CYCLODEXTRIN GLUCANOTRANSFERASE (CGTase USING IMMOBILIZED METAL CHELATING AFFINITY CHROMATOGRAPHY

    Directory of Open Access Journals (Sweden)

    M. Sivapragasam

    2015-03-01

    Full Text Available Abstract Immobilized metal affinity chromatography (IMAC was chosen as a method of purification for the recovery of CGTase from E. coli homogenate. E. coli harbouring the Bacillus sp. G1 gene expressed extracellularly secreted CGTase into ampicillin supplied LB broth. Culture was pre-purified using SnakeSkin dialysis tubing (3.5 MWCO with an enzyme activity of 147.80 U/mL. Three strategies (A, B and C were employed for the purification of CGTase using column adsorption chromatography with Ni2+-Sepharose resin. Strategy A employed an elution buffer of 50 mM EDTA, pH 7, Strategy B used 0.1 M imidazole, pH 7 and Strategy C employed 45 mM imidazole pH 7 as the elution buffer. Strategy C was found to be most suitable yielding a total CGTase recovery of 87.04% from an initial activity of 147.80 U/mL.

  19. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success.

    Science.gov (United States)

    Choi, Ryan; Kelley, Angela; Leibly, David; Hewitt, Stephen Nakazawa; Napuli, Alberto; Van Voorhis, Wesley

    2011-09-01

    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.

  20. Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

    Science.gov (United States)

    Inoguchi, Noriko; Oshlo, Jake R.; Natarajan, Chandrasekhar; Weber, Roy E.; Fago, Angela; Storz, Jay F.; Moriyama, Hideaki

    2013-01-01

    The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128. PMID:23545644