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Affinity chromatography

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https://www.readbyqxmd.com/read/28549117/engineering-of-recombinant-wisteria-floribunda-agglutinin-specifically-binding-to-galnac%C3%AE-1-4glcnac-lacdinac
#1
Takashi Sato, Hiroaki Tateno, Hiroyuki Kaji, Yasunori Chiba, Tomomi Kubota, Jun Hirabayashi, Hisashi Narimatsu
Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA was expressed in Escherichia coli and purified. The natural cDNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation...
May 26, 2017: Glycobiology
https://www.readbyqxmd.com/read/28547925/blockade-of-the-dissociation-of-nmmhc-iia-from-tnfr2-by-d39-a-saponin-from-liriope-muscari-suppresses-tissue-factor-expression-and-venous-thrombosis
#2
Ke-Feng Zhai, Jin-Rong Zheng, You-Mei Tang, Fang Li, Yan-Ni Lv, Yuan-Yuan Zhang, Zhen Gao, Jin Qi, Bo-Yang Yu, Jun-Ping Kou
BACKGROUND AND PURPOSE: Non-muscular myosin heavy chain IIA (NMMHC IIA) has been demonstrated to play a key role in tissue factor (TF) expression and venous thrombosis. However, whether natural products could inhibit thrombosis via NMMHC IIA remains unclear. In this study, we identified that a representative natural saponin D39 from Liriope muscari showed anti-thrombotic activity by targeting NMMHC IIA. EXPERIMENTAL APPROACH: TF expression and activity in endothelial cells were analysed in vitro by western blot and simplified chromogenic assays...
May 26, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28546025/molecular-and-functional-characterization-of-a-glycosylated-galactose-binding-lectin-from-mytilus-californianus
#3
Efrén García-Maldonado, Patricia Cano-Sánchez, Alejandra Hernández-Santoyo
Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular weight of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose...
May 22, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28545467/expression-of-deleted-atoxic-atypical-recombinant-beta2-toxin-in-a-baculovirus-system-and-production-of-polyclonal-and-monoclonal-antibodies
#4
Anna Serroni, Chiara Francesca Magistrali, Giovanni Pezzotti, Luca Bano, Martina Pellegrini, Giulio Severi, Chiara Di Pancrazio, Mirella Luciani, Manuela Tittarelli, Silvia Tofani, Antonio De Giuseppe
BACKGROUND: Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen...
May 25, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28544560/fatal-intoxication-by-5f-adb-and-diphenidine-detection-quantification-and-investigation-of-their-main-metabolic-pathways-in-human-by-lc-ms-ms-and-lc-q-tofms
#5
Maiko Kusano, Kei Zaitsua, Kentaro Taki, Kazuaki Hisatsune, Jun'ichi Nakajima, Takako Moriyasu, Tomomi Asano, Yumi Hayashi, Hitoshi Tsuchihashi, Akira Ishii
Despite the implementation of the new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F-ADB (methyl 2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS) in the information dependent acquisition (IDA) mode only detected diphenidine. Further urinary screening using an in-house database containing new NPS and metabolites detected not only diphenidine but also possible 5F-ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of very low level of unchanged 5F-ADB in postmortem heart blood...
May 21, 2017: Drug Testing and Analysis
https://www.readbyqxmd.com/read/28539047/identifying-the-protein-interactions-of-the-cytosolic-iron-sulfur-cluster-targeting-complex-essential-for-its-assembly-and-recognition-of-apo-targets
#6
Amanda Thuy Van Vo, Nicholas M Fleischman, Mary J Froehlich, Claudia Y Lee, Jessica A Cosman, Calina A Glynn, Zanub O Hassan, Deborah L Perlstein
The cytosolic iron sulfur cluster assembly (CIA) system assembles iron sulfur (FeS) cluster cofactors and inserts them into >20 apo-protein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex...
May 25, 2017: Biochemistry
https://www.readbyqxmd.com/read/28533032/design-and-synthesis-of-an-immobilized-metal-affinity-chromatography-and-metal-oxide-affinity-chromatography-hybrid-material-for-improved-phosphopeptide-enrichment
#7
Da-Song Yang, Xi-Ying Ding, Hong-Ping Min, Bo Li, Meng-Xiang Su, Miao-Miao Niu, Bin Di, Fang Yan
Reversible phosphorylation of proteins is one of the most important post-translational modifications, while the detection of phosphopeptides is difficult due to their low abundance and the signal suppression of nonphosphorylated peptides. Therefore, selective enrichment of phosphopeptides from highly complicated mixtures is vital for MS-based phosphoproteome analysis. Despite various strategies have been developed, there is no single method that is capable of providing full coverage of the whole phosphoproteome...
May 10, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28532594/the-endocrine-disrupting-alkylphenols-and-4-4-ddt-interfere-with-estrogen-conversion-and-clearance-by-mouse-liver-cytosol
#8
Talal El-Hefnawy, Claudia Hernandez, Laura P Stabile
Endocrine disrupting chemicals (EDCs) are ubiquitous compounds known for negative impacts on reproductive functions and for increasing cancer risk. EDCs are believed to cause the harmful effects in part through their inappropriate low-affinity binding to steroid receptors and other possible non-receptor mediated paradigms, however there is a need to further elucidate other mechanisms involving the direct and indirect impact of EDCs on reproductive functions. We examined the metabolism of 17β-estradiol (E2) and estrone (E1) by cell-free hepatic cytosol in the presence of alkylphenols (nonylphenol/NP and 4-tert-octylphenol/tOP), Dichlorodiphenyltrichloroethane (4,4'-DDT) and other EDCs...
May 19, 2017: Reproductive Biology
https://www.readbyqxmd.com/read/28532286/hb-bakersfield-hba1-c-151_152insggagcc-the-insertion-of-arg-his-between-codons-49-and-50-of-the-%C3%AE-1-globin-chain-leads-to-increased-oxygen-affinity
#9
Saskia Brunner-Agten, Thomas von Känel, Benno Röthlisberger, Charles Broquet, Andreas R Huber
We describe an insertion variant on the α1-globin gene (HBA1) identified in a 49-year-old woman of Jurassian ancestry presenting with macrocytosis and erythrocytosis. The variant resulted in a peak of 15.5% of the total hemoglobin (Hb) on high performance liquid chromatography (HPLC). Stability and oxygen affinity testing revealed that the variant was stable and had an increased oxygen affinity. Molecular genetic testing detected the heterozygous sequence variant Hb Bakersfield [α50(CE8)His→0; Arg-Ser-His- inserted between 49(CE7) and 51(CE9) of α1; HBA1: c...
January 2017: Hemoglobin
https://www.readbyqxmd.com/read/28530613/combination-of-solvent-extractants-for-dispersive-liquid-liquid-microextraction-of-fungicides-from-water-and-fruit-samples-by-liquid-chromatography-with-tandem-mass-spectrometry
#10
Marta Pastor-Belda, Isabel Garrido, Natalia Campillo, Pilar Viñas, Pilar Hellín, Pilar Flores, José Fenoll
A multiresidue method was developed to determine twenty-five fungicides belonging to three different chemical families, oxazoles, strobilurins and triazoles, in water and fruit samples, using dispersive liquid-liquid microextraction (DLLME) and liquid chromatography/tandem mass spectrometry (LC-MS(2)). Solid-liquid extraction with acetonitrile was used for the analysis in fruits, the extract being used as dispersant solvent in DLLME. Since some of the analytes showed high affinity for chloroform and the others were more efficiently extracted with undecanol, a mixture of both solvents was used as extractant in DLLME...
October 15, 2017: Food Chemistry
https://www.readbyqxmd.com/read/28529501/microbial-degradation-of-lobster-shells-to-extract-chitin-derivatives-for-plant-disease-management
#11
Gayathri Ilangumaran, Glenn Stratton, Sridhar Ravichandran, Pushp S Shukla, Philippe Potin, Samuel Asiedu, Balakrishnan Prithiviraj
Biodegradation of lobster shells by chitinolytic microorganisms are an environment safe approach to utilize lobster processing wastes for chitin derivation. In this study, we report degradation activities of two microbes, "S223" and "S224" isolated from soil samples that had the highest rate of deproteinization, demineralization and chitinolysis among ten microorganisms screened. Isolates S223 and S224 had 27.3 and 103.8 protease units mg(-1) protein and 12.3 and 11.2 μg ml(-1) of calcium in their samples, respectively, after 1 week of incubation with raw lobster shells...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28528790/characterization-of-a-novel-thermostable-gh45-endoglucanase-from-chaetomium-thermophilum-and-its-biodegradation-of-pectin
#12
Qinzheng Zhou, Peng Ji, Jianye Zhang, Xue Li, Chao Han
A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni(2+) affinity chromatography, and its apparent molecular mass was estimated to be 32 kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70°C and pH 4. CTendo45 was stable at 60°C for 1 h, and residual activities of 78...
May 19, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28528681/multilayer-adsorption-in-liquid-chromatography-the-surface-heterogeneity-below-an-adsorbed-multilayer
#13
Krisztián Horváth, Péter Vajda, Attila Felinger
A numerical method was introduced for the estimation of the surface heterogeneity below an adsorbed multilayer of the analyte. The calculation procedure is based on the raw adsorption isotherm data points obtained by frontal analysis experiments. To permit the mapping of the nature of the analyte-surface interaction, a numerical procedure was used to pre-estimate the adsorbate-adsorbate interactions occurring during the adsorption process. The surface heterogeneity estimation was carried out using the affinity-energy distribution calculations with assuming local BET isotherm...
May 10, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28522347/functional-analysis-of-tcctlp-5c2-a-chymotrypsin-like-serine-protease-needed-for-molting-in-tribolium-castaneum
#14
Daniel Albaum, Gunnar Broehan, Subbaratnam Muthukrishnan, Hans Merzendorfer
In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C2), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al., 2010; Insect Biochem. Mol. Biol 40, 274-83). Thus, the enzyme has distinct functions in different physiological environments...
May 15, 2017: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28520402/catch-and-release-engineered-allosterically-regulated-%C3%AE-roll-peptides-enable-on-off-biomolecular-recognition
#15
Beyza Bulutoglu, Kevin Dooley, Géza R Szilvay, Mark Blenner, Scott Banta
Alternative scaffolds for biomolecular recognition are being developed to overcome some of the limitations associated with immunoglobulin domains. The repeat-in-toxin (RTX) domain is a repeat protein sequence that reversibly adopts the β-roll secondary structure motif specifically upon calcium binding. This conformational change was exploited for controlled biomolecular recognition. Using ribosome display, an RTX peptide library was selected to identify binders to a model protein, lysozyme, exclusively in the folded state of the peptide...
May 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28520401/identification-of-galnac-conjugated-antisense-oligonucleotide-metabolites-using-an-untargeted-and-generic-approach-based-on-high-resolution-mass-spectrometry
#16
Christophe Husser, Andreas Brink, Manfred Zell, Martina B Müller, Erich Koller, Simone Schadt
Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl ga-lactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells...
May 18, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28516279/a-new-carbon-based-magnetic-material-for-the-dispersive-solid-phase-extraction-of-uv-filters-from-water-samples-before-liquid-chromatography-tandem-mass-spectrometry-analysis
#17
Susy Piovesana, Anna Laura Capriotti, Chiara Cavaliere, Giorgia La Barbera, Roberto Samperi, Riccardo Zenezini Chiozzi, Aldo Laganà
Magnetic solid-phase extraction is one of the most promising new extraction methods for liquid samples before ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Several types of materials, including carbonaceous ones, have been prepared for this purpose. In this paper, for the first time, the preparation, characterization, and sorption capability of Fe3O4-graphitized carbon black (mGCB) composite toward some compounds of environmental interest were investigated. The synthesized mGCB consisted of micrometric GCB particles with 55 m(2) g(-1) surface area bearing some carbonyl and hydroxyl functionalities and the surface partially decorated by Fe3O4 microparticles...
May 18, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28512205/phorbol-12-myristate-13-acetate-mediated-stabilization-of-leukemia-inhibitory-factor-lif-mrna-involvement-of-nucleolin-and-pcbp1
#18
Alina Chakraborty, Srimoyee Mukherjee, Sucharita Saha, Soumasree De, Sumita Sengupta Bandyopadhyay
LIF is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of Leukemia Inhibitory Factor in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell-line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region (3'-UTR) of human lif -mRNA revealed several conserved sequences with conventional cis -acting elements...
May 16, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28510356/purification-of-recombinant-a%C3%AE-1-42-and-pglu-a%C3%AE-3-42-using-preparative-sds-page
#19
Claudia Spahn, Michael Wermann, Rico Eichentopf, Gerd Hause, Dagmar Schlenzig, Stephan Schilling
Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation...
May 16, 2017: Electrophoresis
https://www.readbyqxmd.com/read/28510018/a-hilly-path-through-the-thermodynamics-and-statistical-mechanics-of-protein-solutions
#20
REVIEW
Peter R Wills
The opus of Don Winzor in the fields of physical and analytical biochemistry is a major component of that certain antipodean approach to this broad area of research that blossomed in the second half of the twentieth century. The need to formulate problems in terms of thermodynamic nonideality posed the challenge of describing a clear route from molecular interactions to the parameters that biochemists routinely measure. Mapping out this route required delving into the statistical mechanics of solutions of macromolecules, and at every turn mathematically complex, rigorous, general results that had previously been derived previously, often by Terrell Hill, came to the fore...
December 2016: Biophysical Reviews
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