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Sample records for 4b affinity column

  1. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

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

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    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.

  3. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  4. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. PMID:25748537

  5. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  6. [Preparation and evaluation of pepsin affinity organic polymer capillary monolithic column].

    PubMed

    Chi, Cuijie; Wang, Wei; Ji, Yibing

    2014-08-01

    The protein modified monolithic column in affinity capillary electrochromatography (CEC) has attracted considerable attention over the past decades because of its great enantioseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly (glycidyl methacrylate-co-ethyleneglycol dimethacrylate) (poly (GMA-co-EDMA)) was prepared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile (AIBN). The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions, including the proportion of monomer and porogens, reaction temperature etc, the column exhibited a uniform structure, sufficient permeability and excel- lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0%. A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly(GMA-co-EDMA) monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pepsin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam, amlodipine, citalopram and chlorpheniramine were resolved, and baseline separations of nefopam, amlodipine, citalopram were achieved. The influences of pH, operating voltage, temperature and sample quantity used on the chiral separation were studied. The chiral recognition mechanism of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the prepataion and application of protein affinity monolith in CEC.

  7. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  8. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  9. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  10. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  11. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  12. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  13. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  14. Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

    PubMed Central

    Ruprecht, Benjamin; Koch, Heiner; Medard, Guillaume; Mundt, Max; Kuster, Bernhard; Lemeer, Simone

    2015-01-01

    Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. PMID

  15. Evaluation of SDS depletion using an affinity spin column and IMS-MS detection

    SciTech Connect

    Hengel, Shawna M.; Floyd, Erica A.; Baker, Erin Shammel; Zhao, Rui; Wu, Si; Pasa-Tolic, Ljiljana

    2012-11-01

    While the use of detergents is necessary for a variety of protein isolation preparation protocols, often prior to mass spectral (MS) analysis, they are not compatible with MS analysis due to ion suppression and adduct formation. This manuscript describes optimization of detergent removal, using commercially available SDS depletion spin columns containing an affinity resin, providing for both increased protein recovery and thorough SDS removal. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) allowed for a concurrent analysis of both analyte and detergent. In the case of both proteins and peptides, higher detergent concentrations than previously reported provided an increase of sample recovery; however there was a limit as SDS was detected by IMS-MS at higher levels of SDS indicating incomplete detergent depletion. The results also suggest optimal conditions for SDS removal are dependent on the sample concentration. Overall, this study provides a useful guide for proteomic studies where SDS is required for efficient sample preparation.

  16. Synthesis and characterization of a cellular membrane affinity chromatography column containing histamine 1 and P2Y1 receptors: A multiple G-protein coupled receptor column

    PubMed Central

    Moaddel, Ruin; Musyimi, Harrison K.; Sanghvi, Mitesh; Bashore, Charlene; Frazier, Chester R.; Khadeer, Mohammad; Bhatia, Prateek; Wainer, Irving W.

    2015-01-01

    A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding. PMID:19608372

  17. One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

    PubMed

    Xiao, Yuan; Guo, Jialiang; Ran, Danni; Duan, Qianqian; Crommen, Jacques; Jiang, Zhengjin

    2015-06-26

    A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 μm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy.

  18. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 1: Theory

    PubMed Central

    2015-01-01

    We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants. PMID:24937679

  19. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    PubMed

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27091327

  20. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  1. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  2. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  3. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study

    PubMed Central

    2015-01-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the range of 10–50 μm s–1, and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10 000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  4. Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

    PubMed Central

    Muraro, P A; Vergelli, M; Kalbus, M; Banks, D E; Nagle, J W; Tranquill, L R; Nepom, G T; Biddison, W E; McFarland, H F; Martin, R

    1997-01-01

    The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans. PMID:9218510

  5. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  6. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    PubMed

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  7. Affinity column for purification of the human platelet thromboxane A/sub 2//prostaglandin H/sub 2/ (TXA/sub 2//PGH/sub 2/) receptor

    SciTech Connect

    Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

    1987-05-01

    The TXA/sub 2//PGH/sub 2/ receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific /sup 3/H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA/sub 2//PGH/sub 2/ receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor.

  8. Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry.

    PubMed

    Zhan, Xianquan; Desiderio, Dominic M

    2006-07-15

    The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein-protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein-protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, sphingosine-1-phosphate lyase 1, centaurin beta 1, proteasome subunit alpha type 2, interleukin 1 family member 6, and rhophilin 2) and three proteins (interleukin 1 receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.

  9. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

  10. Interference between eplerenone and digoxin in fluorescence polarization immunoassay, microparticle enzyme immunoassay, and affinity column-mediated immunoassay.

    PubMed

    Yamada, Tomoyuki; Suzuki, Kaoru; Iguchi, Ken; Kanada, Yasutaka; Kato, Ryuji; Ijiri, Yoshio; Nishihara, Masami; Murakami, Sumiko; Hayashi, Tetsuya; Tamai, Hiroshi; Tanaka, Kazuhiko

    2010-12-01

    Digitalis-like immunoreactive substances have crossreactivity with antidigoxin antibodies and the interference between digoxin and spironolactone/canrenone has been reported. The structure of eplerenone is similar to that of spironolactone/canrenone. Therefore, we hypothesized that eplerenone might also interfere with the measurement of digoxin by immunoassay. We performed three types of assays (fluorescence polarization immunoassay [FPIA], microparticle enzyme immunoassay [MEIA], and affinity column-mediated immunoassay [ACMIA]) to determine crossreactions between eplerenone and antidigoxin antibodies. Furthermore, we used FPIA, MEIA, and ACMIA to measure the apparent digoxin concentration in mixed solutions of eplerenone (1-100 μg/mL) and digoxin (1-3 ng/mL). In the crossreaction tests, eplerenone was detected as digoxin by FPIA and ACMIA. By FPIA, a known concentration of 1 μg/mL of eplerenone was measured as 0.33 ± 0.11 ng/mL of digoxin (crossreaction rate, 0.03%). By ACMIA, a known concentration of 10 μg/mL of eplerenone was measured as 0.13 ± 0.05 ng/mL of digoxin (crossreaction rate, 0.001%). No crossreaction between eplerenone and digoxin was determined by MEIA. In the interference of eplerenone coadministered with digoxin, the apparent concentration of digoxin was increased in FPIA, but decreased in MEIA and ACMIA. The results suggest that eplerenone crossreacts with antidigoxin antibodies in FPIA, MEIA, and ACMIA, but that the interference of eplerenone might be smaller than that of spironolactone/canrenone. PMID:20625353

  11. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  12. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent. PMID:27289464

  13. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  14. Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

    PubMed Central

    Marszałł, Michał P.; Moaddel, Ruin; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2008-01-01

    Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N-terminus to create the Hsp90α(NT)-column or C-terminus to create the Hsp90α(CT)-column. Binding to the exposed C-terminus on the Hsp90α(NT)-column was characterized using frontal chromatography and C-terminus ligands coumermycin A1(CA1) and novobiocin (NOVO). The calculated Kd values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Non-linear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex, 22.2 (±8.8) μM−1 sec−1 and 2.7 (±0.6) sec−1, respectively. Binding to the exposed N-terminus on the Hsp90α(CT)-column was characterized using frontal chromatography. The Kd values of N-terminus ligands geldanamycin (GM) (90 ± 50 nM) 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) (210 ± 50 nM) and radicicol (RAD) (20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC50 values for inhibition of ATPase activity on the Hsp90α(CT)-column. The IC50 for GM was 2.80 ± 0.18 μM and the relative IC50 values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition. PMID:18047824

  15. Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

    PubMed

    Senyuva, Hamide Z; Cimen, Dilek; Gilbert, John

    2009-01-01

    The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pişmaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes. PMID:19714981

  16. RECEPTOR AFFINITY AND PHOSPHODIESTERASES 4B AND 10A ACTIVITY OF OCTAHYDRO- AND 6,7-DIMETHOXY-3,4-DIHYDRO- ISOQUINOLIN-2(1H)-YL-ALKYL DERIVATIVES OF IMIDAZO- AND PYRIMIDINO[2,1-f]PURINES.

    PubMed

    Zagórska, Agnieszka; Gryzło, Beata; Satała, Grzegorz; Bojarski, Andrzej J; Głuch-Lutwin, Monika; Mordyl, Barbara; Kazek, Grzegorz; Pawłowski, Maciej

    2016-01-01

    A series of octahydro- and 6,7-dimethoxy-3,4-dihydro- isoquinolin-2(1H)-yl-alkyl derivatives of imidazo- and pyrimidino[2,1-f]purines were synthesized and biologically evaluated in in vitro competition binding experiments for serotonin 5-HT(1A), 5-HT(6), 5-HT(7), and dopamine D2 receptors and inhibitory potencies for phosphodiesterases - PDE4B1 and PDE10A. The structure-activity relationships allowed to determine the structural features responsible for receptor and enzyme activity. Compound 5 (8-(4-(6,7-dimethoxy-3,4-dihydroiso- quinolin-2(1H)butyl)1,3-dimethyl-H-imidazo[2,1-f]purine-2,4(3H,8H)-dione) could be regarded as promising structure for further modification and detailed mechanistic study for obtained hybrid ligands.

  17. Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-09-01

    More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

  18. Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-09-01

    More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

  19. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

    PubMed

    Ciesla, L; Okine, M; Rosenberg, A; Dossou, K S S; Toll, L; Wainer, I W; Moaddel, R

    2016-01-29

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  20. Facile fabrication and instant application of miniaturized antibody-decorated affinity columns for higher-order structure and functional characterization of TRIM21 epitope peptides.

    PubMed

    Al-Majdoub, M; Opuni, K F M; Koy, C; Glocker, M O

    2013-11-01

    Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an "L-E-Q-L" motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen. PMID:24094071

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

    PubMed Central

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

    2010-01-01

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

  2. The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

    PubMed

    Habicht, K-L; Frazier, C; Singh, N; Shimmo, R; Wainer, I W; Moaddel, R

    2013-01-01

    BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

  3. Boeing F4B-4

    NASA Technical Reports Server (NTRS)

    1932-01-01

    The Boeing F4B-4 was seen to differ from earlier F4Bs in having a vertical fin with slightly more area. The Boeing model 235 was not fitted with a NACA cowling, but rather the less efficient 'Townend' ring around the Pratt & Whitney Wasp radials cylinders. This aircraft was much used by both the Navy and the Army Air Corps in the late 1920's and early 1930's. The Army variation was known as the P-12E. The engine cowling was a British development known as the 'Townend' ring. It differed from the NACA cowling and was less effective in reducing the drag.

  4. Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with MS detection

    PubMed Central

    Temporini, C.; Pochetti, G.; Fracchiolla, G.; Piemontese, L.; Montanari, R.; Moaddel, R.; Laghezza, A.; Altieri, F.; Cervoni, L.; Ubiali, D.; Prada, E.; Loiodice, F.; Massolini, G.; Calleri, E.

    2013-01-01

    The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening towards PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of Frontal Affinity Chromatography coupled to Mass Spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments towards new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes. PMID:23466198

  5. Selective Phosphodiesterase 4B Inhibitors: A Review

    PubMed Central

    Azam, Mohammed Afzal; Tripuraneni, Naga Srinivas

    2014-01-01

    Abstract Phosphodiesterase 4B (PDE4B) is a member of the phosphodiesterase family of proteins that plays a critical role in regulating intracellular levels of cyclic adenosine monophosphate (cAMP) by controlling its rate of degradation. It has been demonstrated that this isoform is involved in the orchestra of events which includes inflammation, schizophrenia, cancers, chronic obstructive pulmonary disease, contractility of the myocardium, and psoriatic arthritis. Phosphodiesterase 4B has constituted an interesting target for drug development. In recent years, a number of PDE4B inhibitors have been developed for their use as therapeutic agents. In this review, an up-to-date status of the inhibitors investigated for the inhibition of PDE4B has been given so that this rich source of structural information of presently known PDE4B inhibitors could be helpful in generating a selective and potent inhibitor of PDE4B. PMID:25853062

  6. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  7. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes. PMID:25764651

  8. KINETIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY USING PEAK PROFILING AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: EXAMINATION OF MULTI-SITE INTERACTIONS OF DRUGS WITH HUMAN SERUM ALBUMIN COLUMNS

    PubMed Central

    Tong, Zenghan; Schiel, John E.; Papastavros, Efthimia; Ohnmacht, Corey M.; Smith, Quentin R.; Hage, David S.

    2010-01-01

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (± 0.2) s-1 and 0.67 (± 0.04) s-1 at pH 7.4 and 37 °C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins. PMID:21067755

  9. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    SciTech Connect

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  10. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  11. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  12. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  13. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  14. Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

    PubMed

    Larson, T J; Hirabayshi, T; Dowhan, W

    1976-03-01

    Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent... PMID:175832

  15. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  16. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  17. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  18. 49 CFR 173.4b - De minimis exceptions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... “Scientific research specimens, 49 CFR 173.4b applies.” (6) Documentation. (i) For transportation by highway... “Scientific research specimens, 49 CFR 173.4b applies” and the number of packages indicated. (iii) For... the statement “Scientific research specimens, 49 CFR 173.4b applies” and the number of...

  19. 49 CFR 173.4b - De minimis exceptions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... “Scientific research specimens, 49 CFR 173.4b applies.” (6) Documentation. (i) For transportation by highway... “Scientific research specimens, 49 CFR 173.4b applies” and the number of packages indicated. (iii) For... the statement “Scientific research specimens, 49 CFR 173.4b applies” and the number of...

  20. 49 CFR 173.4b - De minimis exceptions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... “Scientific research specimens, 49 CFR 173.4b applies.” (6) Documentation. (i) For transportation by highway... “Scientific research specimens, 49 CFR 173.4b applies” and the number of packages indicated. (iii) For... the statement “Scientific research specimens, 49 CFR 173.4b applies” and the number of...

  1. 49 CFR 173.4b - De minimis exceptions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... “Scientific research specimens, 49 CFR 173.4b applies.” (6) Documentation. (i) For transportation by highway... “Scientific research specimens, 49 CFR 173.4b applies” and the number of packages indicated. (iii) For... the statement “Scientific research specimens, 49 CFR 173.4b applies” and the number of...

  2. 49 CFR 173.4b - De minimis exceptions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false De minimis exceptions. 173.4b Section 173.4b Transportation Other Regulations Relating to Transportation PIPELINE AND HAZARDOUS MATERIALS SAFETY... SHIPMENTS AND PACKAGINGS General § 173.4b De minimis exceptions. (a) Packing Group II and III materials...

  3. Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

    PubMed Central

    Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

    2016-01-01

    ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication

  4. Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis.

    PubMed

    Qi, Xiao-Hua; Zhang, Li-Wei; Zhang, Xin-Xiang

    2008-08-01

    A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.

  5. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  6. Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

    PubMed Central

    Lim, Kue Peng; Chun, Nicole Ai Leng; Gan, Chai Phei; Teo, Soo-Hwang; Rahman, Zainal Ariff Abdul; Abraham, Mannil Thomas; Zain, Rosnah Binti; Ponniah, Sathibalan; Cheong, Sok Ching

    2014-01-01

    The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC. PMID:25483651

  7. The hypervariable region of K-Ras4B is responsible for its specific interactions with calmodulin.

    PubMed

    Abraham, Sherwin J; Nolet, Ryan P; Calvert, Richard J; Anderson, Lucy M; Gaponenko, Vadim

    2009-08-18

    K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival, and motility. The p21 Ras proteins, such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry, we demonstrate that the hypervariable region of K-Ras4B contributes in a major way to the interaction with calmodulin, while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca(2+)-loaded calmodulin with micromolar affinity, while the GTP-gamma-S-loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin.

  8. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    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)

  9. PULSE COLUMN

    DOEpatents

    Grimmett, E.S.

    1964-01-01

    This patent covers a continuous countercurrent liquidsolids contactor column having a number of contactor states each comprising a perforated plate, a layer of balls, and a downcomer tube; a liquid-pulsing piston; and a solids discharger formed of a conical section at the bottom of the column, and a tubular extension on the lowest downcomer terminating in the conical section. Between the conical section and the downcomer extension is formed a small annular opening, through which solids fall coming through the perforated plate of the lowest contactor stage. This annular opening is small enough that the pressure drop thereacross is greater than the pressure drop upward through the lowest contactor stage. (AEC)

  10. The hypervariable region of K-Ras4B is responsible for its specific interactions with Calmodulin

    PubMed Central

    Abraham, Sherwin J.; Nolet, Ryan P.; Calvert, Richard J.; Anderson, Lucy M.; Gaponenko, Vadim

    2009-01-01

    K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca2+-loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. PMID:19583261

  11. Continuous affinity-gradient nano-stationary phase served as a column for reversed-phase electrochromatography and matrix carrier in time-of-flight mass spectrometry for protein analysis.

    PubMed

    Wu, Jen-Kuei; Yang, Chung-Shi; Wu, Yi-Shiuan; Wang, Pen-Cheng; Tseng, Fan-Gang

    2015-08-19

    This study developed an affinity-gradient nano-stationary phase (AG-NSP) for protein analysis using nanofluidic capillary electrochromatography (nano-CEC) conjugated with matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). The AG-NSP can be used for protein pre-separation in nano-CEC and as a matrix carrier for protein analysis in MALDI-TOF-MS. A hydrophobicity gradient in AG-NSP was photochemically formed by grafting 4-azidoaniline hydrochloride on vertically arrayed multi-wall carbon nanotubes (MWCNTs) through gray-level exposure to UV light. The reversed-phase gradient stationary phase in AG-NSP was tailored according to the properties of the mobile phase gradient in capillary electrochromatography. As a result, the operation of the system is easily automated using a single buffer solution without the need for multiple solvents for elution. The use of nano-CEC with AG-NSP demonstrated excellent separation efficiency and high resolution for various types of DNA/protein/peptide. MALDI-TOF-MS analysis was then performed directly on the separated proteins and peptides on the chip. The proposed system was then used for the detection of three types of proteins with different molecular weights and PI values, including Cytochrome c (12,360, pI = 10), Lysozyme (14,300, pI = 11), and BSA (86,000, pI = 5)), and digested IgG fragments. The proposed system provided resolution of 1000 Da for the proteins in this study and the separation of digested IgG fragments at a low concentration of 1.2 pmol μL(-1).

  12. Column internals

    SciTech Connect

    Bravo, J.L.

    1998-02-01

    In the fields of distillation, absorption, stripping and extraction, theory and technology go hand in hand. The thermodynamic principles of phase equilibrium and the concepts of mass transfer and fluid flow are of primary importance in all of these operations. The engineer must understand these phenomena to select equipment effectively. This article discusses the latest in commercial technology in column internals for gas-liquid and liquid-liquid contacting. The principles of operation are explained vis-a-vis the characteristics of the applications in which they are used. The focus is on moderate-to-large columns for refining and chemical applications. Guidelines for selecting the most appropriate type of device are presented, and examples of typical applications are described.

  13. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  14. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  15. MCNP4B{sup {trademark}} verification and validation

    SciTech Connect

    Hendricks, J.S.; Court, J.D.

    1996-08-01

    Several new features and bug fixes have been incorporated into the new release of MCNP. As required by the MCNP Software Quality Assurance Plan, these changes to the code and the test set are documented here for user reference. This document summarizes the new MCNP4B features and corrections, separated into major and minor groupings. Also included are a code cleanup section and a section delineating problems identified in LA-12839 which have not been corrected. Finally, we document the MCNP4B test set modifications and explain how test set coverage has been improved.

  16. Pyrazolopyridines as potent PDE4B inhibitors: 5-Heterocycle SAR

    SciTech Connect

    Mitchell, Charlotte J.; Ballantine, Stuart P.; Coe, Diane M.; Cook, Caroline M.; Delves, Christopher J.; Dowle, Mike D.; Edlin, Chris D.; Hamblin, J. Nicole; Holman, Stuart; Johnson, Martin R.; Jones, Paul S.; Keeling, Sue E.; Kranz, Michael; Lindvall, Mika; Lucas, Fiona S.; Neu, Margarete; Solanke, Yemisi E.; Somers, Don O.; Trivedi, Naimisha A.; Wiseman, Joanne O.

    2012-05-03

    Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett.2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-{alpha} production from isolated human peripheral blood mononuclear cells.

  17. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  18. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  19. Affine projective Osserman structures

    NASA Astrophysics Data System (ADS)

    Gilkey, P.; Nikčević, S.

    2013-08-01

    By considering the projectivized spectrum of the Jacobi operator, we introduce the concept of projective Osserman manifold in both the affine and in the pseudo-Riemannian settings. If M is an affine projective Osserman manifold, then the deformed Riemannian extension metric on the cotangent bundle is both spacelike and timelike projective Osserman. Since any rank-1-symmetric space is affine projective Osserman, this provides additional information concerning the cotangent bundle of a rank-1 Riemannian symmetric space with the deformed Riemannian extension metric. We construct other examples of affine projective Osserman manifolds where the Ricci tensor is not symmetric and thus the connection in question is not the Levi-Civita connection of any metric. If the dimension is odd, we use methods of algebraic topology to show the Jacobi operator of an affine projective Osserman manifold has only one non-zero eigenvalue and that eigenvalue is real.

  20. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  1. PDE4B as a microglia target to reduce neuroinflammation.

    PubMed

    Pearse, Damien D; Hughes, Zoë A

    2016-10-01

    The importance of microglia in immune homeostasis within the brain is undisputed. Their role in a diversity of neurological and psychiatric diseases as well as CNS injury is the subject of much investigation. Cyclic adenosine monophosphate (AMP) is a critical regulator of microglia homeostasis; as the predominant negative modulator of cyclic AMP signaling within microglia, phosphodiesterase 4 (PDE4) represents a promising target for modulating immune function. PDE4 expression is regulated by inflammation, and in turn, PDE4 inhibition can alter microglia reactivity. As the prototypic PDE4 inhibitor, rolipram, was tested clinically in the 1980s, drug discovery and clinical development of PDE4 inhibitors have been severely hampered by tolerability issues involving nausea and emesis. The two PDE4 inhibitors approved for peripheral inflammatory disorders (roflumilast and apremilast) lack brain penetration and are dose-limited by side effects making them unsuitable for modulating microglial function. Subtype selective inhibitors targeting PDE4B are of high interest given the critical role PDE4B plays in immune function versus the association of PDE4D with nausea and emesis. The challenges and requirements for successful development of a novel brain-penetrant PDE4B inhibitor are discussed in the context of early clinical development strategies. Furthermore, the challenges of monitoring the state of microglia in vivo are highlighted, including a description of the currently available tools and their limitations. Continued drug discovery efforts to identify safe and well-tolerated, brain-penetrant PDE4 inhibitors are a reflection of the confidence in the rationale for modulation of this target to produce meaningful therapeutic benefit in a wide range of neurological conditions and injury. GLIA 2016;64:1698-1709. PMID:27038323

  2. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  3. Generation and characterization of a Cyp4b1 null mouse and the role of CYP4B1 in the activation and toxicity of Ipomeanol.

    PubMed

    Parkinson, Oliver T; Liggitt, H Denny; Rettie, Allan E; Kelly, Edward J

    2013-08-01

    4-Ipomeanol (IPO) is a prototypical pulmonary toxin that requires P450-mediated metabolic activation to reactive intermediates in order to elicit its toxic effects. CYP4B1 is a pulmonary enzyme that has been shown, in vitro, to have a high capacity for bioactivating IPO. In order to determine, unambiguously, the role of CYP4B1 in IPO bioactivation in vivo, we generated Cyp4b1 null mice following targeted disruption of the gene downstream of exon 1. Cyp4b1 (-/-) mice are viable and healthy, with no overt phenotype, and no evidence of compensatory upregulation of other P450 isoforms in any of the tissues examined. Pulmonary and renal microsomes prepared from male Cyp4b1 (-/-) mice exhibited no detectable expression of the protein and catalyzed the in vitro bioactivation of IPO at < 10% of the rates observed in tissue microsomes from Cyp4b1 (+/+) animals. Administration of IPO (20mg/kg) to Cyp4b1 (+/+) mice resulted in characteristic lesions in the lung, and to a lesser extent in the kidney, which were completely absent in Cyp4b1 (-/-) mice. We conclude that CYP4B1 is a critical enzyme for the bioactivation of IPO in vivo and that the Cyp4b1 (-/-) mouse is a useful model for studying CYP4B1-dependent metabolism and toxicity. PMID:23748241

  4. Generation and Characterization of a Cyp4b1 Null Mouse and the Role of CYP4B1 in the Activation and Toxicity of Ipomeanol

    PubMed Central

    Kelly, Edward J.

    2013-01-01

    4-Ipomeanol (IPO) is a prototypical pulmonary toxin that requires P450-mediated metabolic activation to reactive intermediates in order to elicit its toxic effects. CYP4B1 is a pulmonary enzyme that has been shown, in vitro, to have a high capacity for bioactivating IPO. In order to determine, unambiguously, the role of CYP4B1 in IPO bioactivation in vivo, we generated Cyp4b1 null mice following targeted disruption of the gene downstream of exon 1. Cyp4b1 −/− mice are viable and healthy, with no overt phenotype, and no evidence of compensatory upregulation of other P450 isoforms in any of the tissues examined. Pulmonary and renal microsomes prepared from male Cyp4b1 −/− mice exhibited no detectable expression of the protein and catalyzed the in vitro bioactivation of IPO at < 10% of the rates observed in tissue microsomes from Cyp4b1 +/+ animals. Administration of IPO (20mg/kg) to Cyp4b1 +/+ mice resulted in characteristic lesions in the lung, and to a lesser extent in the kidney, which were completely absent in Cyp4b1 −/− mice. We conclude that CYP4B1 is a critical enzyme for the bioactivation of IPO in vivo and that the Cyp4b1 −/− mouse is a useful model for studying CYP4B1-dependent metabolism and toxicity. PMID:23748241

  5. Secondary Eclipse Photometry of WASP-4b with Warm Spitzer

    NASA Astrophysics Data System (ADS)

    Beerer, Ingrid M.; Knutson, Heather A.; Burrows, Adam; Fortney, Jonathan J.; Agol, Eric; Charbonneau, David; Cowan, Nicolas B.; Deming, Drake; Desert, Jean-Michel; Langton, Jonathan; Laughlin, Gregory; Lewis, Nikole K.; Showman, Adam P.

    2011-01-01

    We present photometry of the giant extrasolar planet WASP-4b at 3.6 and 4.5 μm taken with the Infrared Array Camera on board the Spitzer Space Telescope as part of Spitzer's extended warm mission. We find secondary eclipse depths of 0.319% ± 0.031% and 0.343% ± 0.027% for the 3.6 and 4.5 μm bands, respectively, and show model emission spectra and pressure-temperature profiles for the planetary atmosphere. These eclipse depths are well fit by model emission spectra with water and other molecules in absorption, similar to those used for TrES-3 and HD 189733b. Depending on our choice of model, these results indicate that this planet has either a weak dayside temperature inversion or no inversion at all. The absence of a strong thermal inversion on this highly irradiated planet is contrary to the idea that highly irradiated planets are expected to have inversions, perhaps due the presence of an unknown absorber in the upper atmosphere. This result might be explained by the modestly enhanced activity level of WASP-4b's G7V host star, which could increase the amount of UV flux received by the planet, therefore reducing the abundance of the unknown stratospheric absorber in the planetary atmosphere as suggested in Knutson et al. We also find no evidence for an offset in the timing of the secondary eclipse and place a 2σ upper limit on |ecos ω| of 0.0024, which constrains the range of tidal heating models that could explain this planet's inflated radius.

  6. RAMONA-4B development for SBWR safety studies

    SciTech Connect

    Rohatgi, U.S.; Aronson, A.L.; Cheng, H.S.; Khan, H.J.; Mallen, A.N.

    1993-12-31

    The Simplified Boiling Water Reactor (SBWR) is a revolutionary design of a boiling-water reactor. The reactor is based on passive safety systems such as natural circulation, gravity flow, pressurized gas, and condensation. SBWR has no active systems, and the flow in the vessel is by natural circulation. There is a large chimney section above the core to provide a buoyancy head for natural circulation. The reactor can be shut down by either of four systems; namely, scram, Fine Motion Control Rod Drive (FMCRD), Alternate Rod Insertion (ARI), and Standby Liquid Control System (SLCS). The safety injection is by gravity drain from the Gravity Driven Cooling System (GDCS) and Suppression Pool (SP). The heat sink is through two types of heat exchangers submerged in the tank of water. These heat exchangers are the Isolation Condenser (IC) and the Passive Containment Cooling System (PCCS). The RAMONA-4B code has been developed to simulate the normal operation, reactivity transients, and to address the instability issues for SBWR. The code has a three-dimensional neutron kinetics coupled to multiple parallel-channel thermal-hydraulics. The two-phase thermal hydraulics is based on a nonhomogeneous nonequilibrium drift-flux formulation. It employs an explicit integration to solve all state equations (except for neutron kinetics) in order to predict the instability without numerical damping. The objective of this project is to develop a Sun SPARC and IBM RISC 6000 based RAMONA-4B code for applications to SBWR safety analyses, in particular for stability and ATWS studies.

  7. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  8. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  9. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  10. Aggregation Properties of the Chromonic Liquid Crystal Benzopurpurin 4B

    NASA Astrophysics Data System (ADS)

    McKitterick, Christopher; Collings, Peter

    2009-03-01

    Benzopurpurin 4B (BPP) is a textile dye very similar to the common indicator Congo Red. As is true for all chromonics, the absorption spectrum is concentration dependent at low concentrations. If this dependence is used to estimate a free energy change for aggregation, it is higher than has been determined for other systems. Unlike other recently investigated chromonic liquid crystals, BPP forms a liquid crystal phase at extremely low concentrations, about 0.5 wt%. Also unlike these other chromonic liquid crystals, the aggregation kinetics are exceedingly slow. X-ray diffraction and light scattering measurements indicate that the aggregates of BPP are much larger than for chromonic systems that form liquid crystals at higher concentrations. BPP aggregates can be imaged using confocal microscopy, revealing a length distribution centered at 3 μm for a solution forced through a 0.2 μm filter. Over days the aggregates lengthen to well over 10 μm. The diameter of the aggregate images is slightly greater than the diffraction limit of the microscope, placing an upper limit on the diameter of 0.14 μm. These dimensions are consistent with the light scattering results.

  11. Starch columns: Analog model for basalt columns

    NASA Astrophysics Data System (ADS)

    Müller, Gerhard

    1998-07-01

    Desiccation of starch-water mixtures produces tensile-crack patterns which appear to be interesting, but largely unknown study objects for fracture mechanics, structural geology, and volcanology. This paper concentrates on columnar jointing and on columns in starch. Starch columns have polygonal cross sections and are very similar to basalt columns. They are produced by lamp drying starch specimens with dimensions of several centimeters and have diameters in the millimeter range. The columns develop behind a crack front which propagates from the surface into the interior. The experiments, supported by X ray tomograms, show that polygonal regularity of the crack pattern is not present at the surface but develops during penetration. This transition is steered by a minimum-fracture-energy principle. The analogy between basalt cooling and starch desiccation is far reaching: water concentration in starch is analogous to temperature in basalt, both quantities obey diffusion equations, water loss is equivalent to heat loss, the resulting contraction stresses have similar dependences on depth and time, and in both cases the material strength is exceeded. The starch experiments show that column diameters are controlled by the depth gradient of water concentration at the crack front. High (low) gradients are connected with thin (thick) columns. By analogy, a similar relation with the temperature gradient exists for basalt columns. The (normalized) starch gradients are about 3 orders of magnitude larger than the (normalized) gradients in basalt. This explains why starch columns are much thinner than basalt columns. The gradients are so different, because the crack front speeds differ by a factor of about 10: after 3 days the speed is about 10 mm/d in starch but about 100 mm/d in basalt [Peck, 1978]. The speed difference, in turn, results from the difference of the diffusion constants: the hydraulic diffusivity of starch is 2 orders of magnitude lower than the thermal

  12. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Cognate transfer ribonucleic acid as a ligand.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The use of tRNA affinity columns for the purification of aminoacyl-tRNA synthetases was investigated. A purification method for valyl-tRNA synthetase from Bacillus stearothermophilus is described that uses two affinity columns, one containing the pure cognate tRNA, and the other containing all tRNA species except the cognate tRNA. A method for the rapid preparation of the two columns was developed, which does not require prior isolation of cognate tRNA but makes use of the ability of the target synthetase to select its cognate tRNA. The usefulness of tRNA columns is compared with that of affinity columns derived from the aminoalkyladenylate reported in the preceding paper [Clarke & Knowles (1977) Biochem J. 167, 405-417]. PMID:23108

  13. Generation and characterization of mouse monoclonal antibodies against NS4B protein of dengue virus.

    PubMed

    Xie, Xuping; Zou, Jing; Wang, Qing-Yin; Noble, Christian G; Lescar, Julien; Shi, Pei-Yong

    2014-02-01

    Dengue virus (DENV) non-structural protein 4B (NS4B) has been demonstrated to be an attractive antiviral target. Due to its nature as an integral membrane protein, NS4B remains poorly characterized. In this study, we generated and characterized two monoclonal antibodies (mAb) that selectively bind to DENV NS4B protein. One mAb, 10-3-7, is specific for DENV-2 NS4B, and its epitope was mapped to residues 5-15 of NS4B. The other mAb, 44-4-7, cross-reacts with all the four serotypes of DENV NS4B, and its epitope was mapped to residues 141-147 of NS4B. Using the mAbs, we probed the intracellular orientation of the epitopes of NS4B by an epitope accessibility assay. The results showed that the N-terminus of NS4B is located in the ER lumen, whereas amino acids 130-148 of NS4B are located in the cytosol. The study demonstrates that the two anti-NS4B mAbs will be useful for future structural and functional analyses of DENV NS4B. PMID:24503088

  14. Stringent Regulation of Complement Lectin Pathway C3/C5 Convertase By C4b-Binding Protein (C4bp)

    PubMed Central

    Rawal, Nenoo; Rajagopalan, Rema; Salvi, Veena P.

    2009-01-01

    The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (EMan) was examined. While the C4BP concentration for inhibiting 50% (IC50) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), ∼3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and EMan (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000-431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on EMan was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a ∼7 to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions. PMID:19660812

  15. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  16. Column Liquid Chromatography.

    ERIC Educational Resources Information Center

    Majors, Ronald E.; And Others

    1984-01-01

    Reviews literature covering developments of column liquid chromatography during 1982-83. Areas considered include: books and reviews; general theory; columns; instrumentation; detectors; automation and data handling; multidimensional chromatographic and column switching techniques; liquid-solid chromatography; normal bonded-phase, reversed-phase,…

  17. Affinity driven social networks

    NASA Astrophysics Data System (ADS)

    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.

  18. Magnitude and range of the RKKy interaction in SmRh4B4

    NASA Astrophysics Data System (ADS)

    Terris, B. D.; Gray, K. E.; Dunlap, B. D.

    1985-04-01

    The superconductive and magnetic transition temperatures taken together are shown to provide a unique probe which separately determines both the magnitude and range of the RKKY interaction in the RERh4B4 magnetic-superconductors (RE = Er, Sm). Experimentally, an unexpected peak is found in the antiferromagnetic ordering temperature of SmRh4B4 vs. electron mean free path, while for ErRh4B4 the ferromagnetic ordering temperature decreases monotonically. These qualitative features, as well as the quanitative differences between SmRh4B4 and ErRh4B4, are in excellent agreement with calculations using a mean free path dependent RKKY interaction.

  19. Discovery of Spiro[cyclohexane-dihydropyrano[3,4-b]indole]-amines as Potent NOP and Opioid Receptor Agonists.

    PubMed

    Schunk, Stefan; Linz, Klaus; Frormann, Sven; Hinze, Claudia; Oberbörsch, Stefan; Sundermann, Bernd; Zemolka, Saskia; Englberger, Werner; Germann, Tieno; Christoph, Thomas; Kögel, Babette-Y; Schröder, Wolfgang; Harlfinger, Stephanie; Saunders, Derek; Kless, Achim; Schick, Hans; Sonnenschein, Helmut

    2014-08-14

    We report the discovery of spiro[cyclohexane-pyrano[3,4-b]indole]-amines, as functional nociceptin/orphanin FQ peptide (NOP) and opioid receptor agonists with strong efficacy in preclinical models of acute and neuropathic pain. Utilizing 4-(dimethylamino)-4-phenylcyclo-hexanone 1 and tryptophol in an oxa-Pictet-Spengler reaction led to the formation of spiroether 2, representing a novel NOP and opioid peptide receptor agonistic chemotype. This finding initially stems from the systematic derivatization of 1, which resulted in alcohols 3-5, ethers 6 and 7, amines 8-10, 22-24, and 26-28, amides 11 and 25, and urea 12, many with low nanomolar binding affinities at the NOP and mu opioid peptide (MOP) receptors. PMID:25147602

  20. Inelastic column behavior

    NASA Technical Reports Server (NTRS)

    Duberg, John E; Wilder, Thomas W , III

    1952-01-01

    The significant findings of a theoretical study of column behavior in the plastic stress range are presented. When the behavior of a straight column is regarded as the limiting behavior of an imperfect column as the initial imperfection (lack of straightness) approaches zero, the departure from the straight configuration occurs at the tangent-modulus load. Without such a concept of the behavior of a straight column, one is led to the unrealistic conclusion that lateral deflection of the column can begin at any load between the tangent-modulus value and the Euler load, based on the original elastic modulus. A family of curves showing load against lateral deflection is presented for idealized h-section columns of various lengths and of various materials that have a systematic variation of their stress-strain curves.

  1. Analysis of biomolecular interactions using affinity microcolumns: a review.

    PubMed

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L; White, Christopher J; Carter, NaTasha; Hage, David S

    2014-10-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  2. Distillation Column Modeling Tools

    SciTech Connect

    2001-09-01

    Advanced Computational and Experimental Techniques will Optimize Distillation Column Operation. Distillation is a low thermal efficiency unit operation that currently consumes 4.8 quadrillion BTUs of energy...

  3. BPO4@B2O3 and (BPO4@B2O3):Eu3+: The novel single-emitting-component phosphors for near UV-white LEDs

    NASA Astrophysics Data System (ADS)

    Cao, Xiyu; Liu, Wei; Jiang, Yu; Cao, Lixin; Su, Ge; Gao, Rongjie

    2016-08-01

    Nowadays much effort has been devoted to exploring novel luminescent materials with low-cost, high stability and excellent luminescent properties. In this paper, a new kind of luminescent material BPO4@B2O3 was prepared by using a facile method. The as-obtained samples contain numerous BPO4 nanoparticles enclosed by amorphous and crystalline B2O3 homogeneously, which exhibits a broad emission band ranging from 380 to 700 nm under near-UV irradiation. More importantly, it is worth noting that the BPO4@B2O3 phosphor exhibits the excellent thermal quenching property, which endows it with a promising prospect as phosphors for high power white LEDs. To further promote its application as white light phosphors, Eu3+ ions were doped into the BPO4@B2O3 samples and prepared the (BPO4@B2O3):Eu3+ phosphors with chromaticity coordinates (0.3022, 0.3122). The corresponding packaging of LEDs indicates that both BPO4@B2O3 and (BPO4@B2O3):Eu3+ can be considered as the promising phosphors for WLEDs.

  4. Inflatable Column Structure

    NASA Technical Reports Server (NTRS)

    Hedgepeth, J. M.

    1985-01-01

    Lightweight structural member easy to store. Billowing between circumferential loops of fiber inflated column becomes series of cells. Each fiber subjected to same tension along entire length (though tension is different in different fibers). Member is called "isotensoid" column. Serves as jack for automobiles or structures during repairs. Also used as support for temporary bleachers or swimming pools.

  5. Glass-silicon column

    DOEpatents

    Yu, Conrad M.

    2003-12-30

    A glass-silicon column that can operate in temperature variations between room temperature and about 450.degree. C. The glass-silicon column includes large area glass, such as a thin Corning 7740 boron-silicate glass bonded to a silicon wafer, with an electrode embedded in or mounted on glass of the column, and with a self alignment silicon post/glass hole structure. The glass/silicon components are bonded, for example be anodic bonding. In one embodiment, the column includes two outer layers of silicon each bonded to an inner layer of glass, with an electrode imbedded between the layers of glass, and with at least one self alignment hole and post arrangement. The electrode functions as a column heater, and one glass/silicon component is provided with a number of flow channels adjacent the bonded surfaces.

  6. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  7. Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

    PubMed

    Qiu, Zongxing; Kuhn, Bernd; Aebi, Johannes; Lin, Xianfeng; Ding, Haiyuan; Zhou, Zheng; Xu, Zhiheng; Xu, Danqing; Han, Li; Liu, Cheng; Qiu, Hongxia; Zhang, Yuxia; Haap, Wolfgang; Riemer, Claus; Stahl, Martin; Qin, Ning; Shen, Hong C; Tang, Guozhi

    2016-08-11

    ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed. PMID:27563406

  8. Yeast eukaryotic initiation factor 4B (eIF4B) enhances complex assembly between eIF4A and eIF4G in vivo.

    PubMed

    Park, Eun-Hee; Walker, Sarah E; Zhou, Fujun; Lee, Joseph M; Rajagopal, Vaishnavi; Lorsch, Jon R; Hinnebusch, Alan G

    2013-01-25

    Translation initiation factor eIF4F (eukaryotic initiation factor 4F), composed of eIF4E, eIF4G, and eIF4A, binds to the m(7)G cap structure of mRNA and stimulates recruitment of the 43S preinitiation complex and subsequent scanning to the initiation codon. The HEAT domain of eIF4G stabilizes the active conformation of eIF4A required for its RNA helicase activity. Mammalian eIF4B also stimulates eIF4A activity, but this function appears to be lacking in yeast, making it unclear how yeast eIF4B (yeIF4B/Tif3) stimulates translation. We identified Ts(-) mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A·eIF4G complexes in vivo, and the rescue of specific mutant eIF4A·eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.

  9. Structural basis for the design of selective phosphodiesterase 4B inhibitors.

    PubMed

    Fox, David; Burgin, Alex B; Gurney, Mark E

    2014-03-01

    Phosphodiesterase-4B (PDE4B) regulates the pro-inflammatory Toll Receptor -Tumor Necrosis Factor α (TNFα) pathway in monocytes, macrophages and microglial cells. As such, it is an important, although under-exploited molecular target for anti-inflammatory drugs. This is due in part to the difficulty of developing selective PDE4B inhibitors as the amino acid sequence of the PDE4 active site is identical in all PDE4 subtypes (PDE4A-D). We show that highly selective PDE4B inhibitors can be designed by exploiting sequence differences outside the active site. Specifically, PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix, now termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP. PDE4B selectivity is driven by a single amino acid polymorphism in CR3 (Leu674 in PDE4B1 versus Gln594 in PDE4D). The reciprocal mutations in PDE4B and PDE4D cause a 70-80 fold shift in selectivity. Our structural studies show that CR3 is flexible and can adopt multiple orientations and multiple registries in the closed conformation. The new co-crystal structure with bound ligand provides a guide map for the design of PDE4B selective anti-inflammatory drugs. PMID:24361374

  10. ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis.

    PubMed

    Cabrera, Sandra; Fernández, Alvaro F; Mariño, Guillermo; Aguirre, Alina; Suárez, María F; Español, Yaiza; Vega, José A; Laurà, Rosaria; Fueyo, Antonio; Fernández-García, M Soledad; Freije, José M P; Kroemer, Guido; López-Otín, Carlos

    2013-08-01

    The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b (-/-) mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b (-/-) mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.

  11. DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation.

    PubMed

    Yi, Juan; Lu, Guang; Li, Li; Wang, Xiaozhen; Cao, Li; Lin, Ming; Zhang, Sha; Shao, Genze

    2015-05-19

    The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

  12. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. PMID:26616099

  13. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

  14. Regenerated bacterial cellulose microfluidic column for glycoproteins separation.

    PubMed

    Chen, Chuntao; Zhu, Chunlin; Huang, Yang; Nie, Ying; Yang, Jiazhi; Shen, Ruiqi; Sun, Dongping

    2016-02-10

    To analysis and separate glycoproteins, a simple strategy to prepare regenerated bacterial cellulose (RBC) column with concanavalin A (Con A) lectin immobilized in microfluidic system was applied. RBC was filled into microchannel to fabricate RBC microcolumn after bacterial cellulose dissolved in NaOH-sulfourea water solution. Lectin Con A was covalently connected onto RBC matrix surface via Schiff-base formation. Lysozyme (non-glycoprotein) and transferrin (glycoprotein) were successfully separated based on their different affinities toward the immobilized Con A. Overall, the RBC microfluidic system presents great potential application in affinity chromatography of glycoproteins analysis, and this research represents a significant step to prepare bacterial cellulose (BC) as column packing material in microfluidic system. What is more, troublesome operations for lectin affinity chromatography were simplified by integrating the microfluidic chip onto a HPLC (High Performance Liquid Chromatography) system.

  15. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    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.

  16. Distillation Column Flooding Predictor

    SciTech Connect

    George E. Dzyacky

    2010-11-23

    The Flooding Predictor™ is a patented advanced control technology proven in research at the Separations Research Program, University of Texas at Austin, to increase distillation column throughput by over 6%, while also increasing energy efficiency by 10%. The research was conducted under a U. S. Department of Energy Cooperative Agreement awarded to George Dzyacky of 2ndpoint, LLC. The Flooding Predictor™ works by detecting the incipient flood point and controlling the column closer to its actual hydraulic limit than historical practices have allowed. Further, the technology uses existing column instrumentation, meaning no additional refining infrastructure is required. Refiners often push distillation columns to maximize throughput, improve separation, or simply to achieve day-to-day optimization. Attempting to achieve such operating objectives is a tricky undertaking that can result in flooding. Operators and advanced control strategies alike rely on the conventional use of delta-pressure instrumentation to approximate the column’s approach to flood. But column delta-pressure is more an inference of the column’s approach to flood than it is an actual measurement of it. As a consequence, delta pressure limits are established conservatively in order to operate in a regime where the column is never expected to flood. As a result, there is much “left on the table” when operating in such a regime, i.e. the capacity difference between controlling the column to an upper delta-pressure limit and controlling it to the actual hydraulic limit. The Flooding Predictor™, an innovative pattern recognition technology, controls columns at their actual hydraulic limit, which research shows leads to a throughput increase of over 6%. Controlling closer to the hydraulic limit also permits operation in a sweet spot of increased energy-efficiency. In this region of increased column loading, the Flooding Predictor is able to exploit the benefits of higher liquid

  17. Nuclear reactor control column

    DOEpatents

    Bachovchin, Dennis M.

    1982-01-01

    The nuclear reactor control column comprises a column disposed within the nuclear reactor core having a variable cross-section hollow channel and containing balls whose vertical location is determined by the flow of the reactor coolant through the column. The control column is divided into three basic sections wherein each of the sections has a different cross-sectional area. The uppermost section of the control column has the greatest cross-sectional area, the intermediate section of the control column has the smallest cross-sectional area, and the lowermost section of the control column has the intermediate cross-sectional area. In this manner, the area of the uppermost section can be established such that when the reactor coolant is flowing under normal conditions therethrough, the absorber balls will be lifted and suspended in a fluidized bed manner in the upper section. However, when the reactor coolant flow falls below a predetermined value, the absorber balls will fall through the intermediate section and into the lowermost section, thereby reducing the reactivity of the reactor core and shutting down the reactor.

  18. Investigation of the heterogeneity of heterogalactan from the fruit bodies of Fomitopsis pinicola, by employing concanavalin A-Sepharose affinity chromatography.

    PubMed

    Usui, T; Hosokawa, S; Mizuno, T; Suzuki, T; Meguro, H

    1981-04-01

    A heterogalactan was isolated from the hot water extract of fruit bodies of Fomitopsis pinicola by a combination of fractionation procedures including precipitation with ethanol and with Cetavlon, and chromatography on columns of DEAE-cellulose and Sephadex G-100. Despite its apparent homogeneity on gel filtration, zone electrophoresis, sedimentation equilibration, and immunodiffusion analyses, the neutral component of heterogalactan was further fractionated into unbound, weakly bound, and strongly bound forms by affinity chromatography on a column of concanavalin A-Sepharose CL 4B. The former two polysaccharides fractions eluted with 0.1 M phosphate buffer (pH 7.0) were found to be a fucogalactan and a mannofucogalactan, respectively. A more tightly bound fraction (mannofucogalactan) was subsequently eluted with 0.1 M glucose in 1 M NaCl. The results of methylation, complete Smith degradation, and proton and 13C NMR spectroscopic analyses indicated that the three kinds of heterogalactans are all highly branched polysaccharides containing a framework of (1 leads to 6)-linked alpha-D-galactopyranosyl residues, the C-2 positions of which are substituted in different proportions with either single L-fucopyranosyl residues or disaccharide units of 3-O-alpha-D-mannopyranosyl-L-fucopyranose residues.

  19. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  20. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  1. Affinity chromatography with an immobilized RNA enzyme.

    PubMed Central

    Vioque, A; Altman, S

    1986-01-01

    M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid. Images PMID:3526344

  2. Microminiature gas chromatographic column

    NASA Technical Reports Server (NTRS)

    Donaldson, R. W., Jr.

    1972-01-01

    Techniques commonly used for fabrication of integrated circuits are utilized to produce long capillary tubes for microminiature chromatographs. Method involves bonding of flat silicon plate to top of spirally grooved silicon chip to close groove and form capillary column.

  3. Distillation Column Flooding Predictor

    SciTech Connect

    2002-02-01

    This factsheet describes a research project whose goal is to develop the flooding predictor, an advanced process control strategy, into a universally useable tool that will maximize the separation yield of a distillation column.

  4. TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis

    PubMed Central

    Grive, Kathryn J.; Gustafson, Eric A.; Seymour, Kimberly A.; Baddoo, Melody; Schorl, Christoph; Golnoski, Kayla; Rajkovic, Aleksandar; Brodsky, Alexander S.; Freiman, Richard N.

    2016-01-01

    TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women. PMID:27341508

  5. TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis.

    PubMed

    Grive, Kathryn J; Gustafson, Eric A; Seymour, Kimberly A; Baddoo, Melody; Schorl, Christoph; Golnoski, Kayla; Rajkovic, Aleksandar; Brodsky, Alexander S; Freiman, Richard N

    2016-06-01

    TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women.

  6. Histone demethylase KDM4B regulates otic vesicle invagination via epigenetic control of Dlx3 expression.

    PubMed

    Uribe, Rosa A; Buzzi, Ailín L; Bronner, Marianne E; Strobl-Mazzulla, Pablo H

    2015-11-23

    In vertebrates, the inner ear arises from the otic placode, a thickened swathe of ectoderm that invaginates to form the otic vesicle. We report that histone demethylase KDM4B is dynamically expressed during early stages of chick inner ear formation. A loss of KDM4B results in defective invagination and striking morphological changes in the otic epithelium, characterized by abnormal localization of adhesion and cytoskeletal molecules and reduced expression of several inner ear markers, including Dlx3. In vivo chromatin immunoprecipitation reveals direct and dynamic occupancy of KDM4B and its target, H3K9me3, at regulatory regions of the Dlx3 locus. Accordingly, coelectroporations of DLX3 or KDM4B encoding constructs, but not a catalytically dead mutant of KDM4B, rescue the ear invagination phenotype caused by KDM4B knockdown. Moreover, a loss of DLX3 phenocopies a loss of KDM4B. Collectively, our findings suggest that KDM4B play a critical role during inner ear invagination via modulating histone methylation of the direct target Dlx3. PMID:26598618

  7. Standard Neutron, Photon, and Electron Data Libraries for MCNP4B.

    1997-04-01

    Version 00 US DOE 10CFR810 Jurisdiction. DLC-189/MCNPXS is for use with Version 4B and later of the MCNP transport code. This data library provides a comprehensive set of cross sections for a wide range of radiation transport applications using the Monte Carlo code package CCC-660/MCNP4B.

  8. 75 FR 18509 - International Conference on Harmonisation; Guidance on Q4B Evaluation and Recommendation of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-12

    ... 21, 2008 (73 FR 9575), FDA made available a guidance on the Q4B process entitled ``Q4B Evaluation and.... In the Federal Register of August 14, 2009 (74 FR 41143), FDA published a notice announcing the... Evaluation and Recommendation of Pharmacopoeial Texts for Use in the International Conference...

  9. LAPTM4B-35 protein is a weak tumor-associated antigen candidate

    PubMed Central

    SHI, GUILAN; ZHOU, CHUNXIA; WANG, DONGMEI; MA, WENBO; ZHANG, SHUREN

    2014-01-01

    Lysosome-associated protein transmembrane 4β (LAPTM4B) is a gene that has been indicated to be involved in cancer. It is located at chromosome 8q22 and is composed of seven exons and six introns. LAPTM4B encodes two protein isoforms: LAPTM4B-35 and LAPTM4B-24. LAPTM4B-35 is markedly upregulated and LAPTM4B-24 is downregulated in several types of cancer. LAPTM4B-35 is 91 amino acids (N91) longer than LAPTM4B-24 at the N-terminus. In the present study, western blotting, enzyme-linked immunosorbent spot analysis and the B16F10-N91 tumor bearing-mice experiments were used to evaluate whether the overexpression of N91 indicates its potential as a candidate tumor-associated antigen. The results revealed that N91 was expressed in a wide range of normal mouse tissues and human peripheral blood mononuclear cells, with varying expression levels. The weak immunogenicity of N91 protein suggested it was a weak candidate antigen; however, the N91 protein was associated with cell proliferation. PMID:24396432

  10. Mutations in the classical swine fever virus NS4B protein affects virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    NS4B is one of the non-structural proteins of Classical Swine Fever Virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a Toll/Inte...

  11. Monoclonal antibodies against NS4B protein of japanese encephalitis virus.

    PubMed

    Ruan, Xindi; Huang, Shaomei; Shao, Lin; Ye, Jing; Chen, Zheng; Chen, Huanchun; Cao, Shengbo

    2013-12-01

    Japanese encephalitis (JE) is one of the most prevalent global viral encephalitis viruses. The functions of JEV (virus) NS4B protein are still under investigation. In our study, NS4B was expressed in Escherichia coli and purified by dialysis. Two clones of monoclonal antibodies (MAbs 1B1 and 1C3) against NS4B protein were generated and their characterizations were investigated. IFA, Western blot, and ELISA results showed that the MAbs were specific against JEV NS4B protein. The epitope of the MAbs was further identified using pairs of synthesized overlapping peptides. These MAbs may provide valuable tools for further exploration of the functions of NS4B and the pathogenesis of Japanese encephalitis virus. PMID:24328740

  12. Interferon regulatory factor 4b (IRF4b) in Japanese flounder, Paralichthys olivaceus: Sequencing, ubiquitous tissue distribution and inducible expression by poly(I:C) and DNA virus.

    PubMed

    Liu, Dahai; Chen, Jinjing; Zhang, Haiyan; Hu, Mengzhu; Lou, Huimin; Liu, Qiuming; Zhang, Shicui; Hu, Guobin

    2016-09-01

    Interferon regulatory factor 4 (IRF4) in mammals is known to be critical in regulation of development and functions of lymphomyeloid cell lineages. Recent studies have demonstrated its involvement in immune responses to bacterial and viral challenges in teleosts. In this study, an IRF4 gene was cloned from Japanese flounder (Paralichthys olivaceus) and its expression in response to polyinosinic:polycytidylic acid [poly(I:C)] and lymphocystis disease virus (LCDV) stimulations was studied in vivo. The cloned gene spans over 5.9 kb, comprises eight exons and seven introns and encodes a putative protein of 456 amino acids. The deduced amino acid sequence possesses a conserved DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS). Phylogenetic analysis clustered it into the teleost IRF4b clade and, thus, it was named Paralichthys olivaceus (Po)IRF4b. The constitutive expression of PoIRF4b transcripts was detectable in all examined organs, with highest levels found in lymphomyeloid-rich tissues. They were induced by both poly(I:C) and LCDV with a similar inducibility in immune or non-immune organs. Two waves of induced expression of PoIRF4b were observed with the two stimuli during a 7-day time course in the immune organs, with the early-phase induction being stronger. The maximum increases of PoIRF4b transcript levels ranged from 1.3 to 4.0-fold and appeared at day 1-5 post-injection depending on different organs and stimuli. In both stimulation cases, the strongest induction was detected in spleen and the weakest in muscle. These results indicate that PoIRF4b may participate in regulation of immune responses of flounders to both RNA and DNA virus infections. PMID:27084058

  13. Towards Atomic Column-by-Column Spectroscopy

    SciTech Connect

    Pennycook, S.J.; Rafferty, B.

    1998-09-06

    The optical arrangement of the scanning transmission electron microscope (STEM) is ideally suited for performing analysis of individual atomic columns in materials. Using the incoherent Z-contrast image as a reference, and arranging incoherent conditions also for the spectroscopy, a precise correspondence is ensured between features in the inelastic image and elastic signals. In this way the exact probe position needed to maximise the inelastic signal from a selected column can be located and monitored during the analysis using the much higher intensity elastic signal. Although object functions for EELS are typically less than 1 {Angstrom} full width at half maximum, this is still an order of magnitude larger than the corresponding object functions for elastic (or diffuse) scattering used to form the Z-contrast image. Therefore the analysis is performed with an effective probe that is significantly broader than that used for the reference Z-contrast image. For a 2.2 {Angstrom} probe the effective probe is of the order of 2.5 {Angstrom}, while for a 1.3 {Angstrom} probe the effective probe is 1.6 {Angstrom}. Such increases in effective probe size can significantly reduce or even eliminate contrast between atomic columns that are visible in the image. However, this is only true if we consider circular collector apertures. Calculations based upon the theory of Maslen and Rossouw (Maslen and Rossouw 1984; Rossouw and Maslen 1984) show that employing an annular aperture can reduce the FWHM of the inelastic object function down to values close 0.1 {Angstrom}. With practical aperture sizes it should be possible to achieve this increased spatial resolution without loosing too much signal.

  14. Androgen Receptor Coactivator ARID4B Is Required for the Function of Sertoli Cells in Spermatogenesis

    PubMed Central

    Zeng, Yang; Pan, I-Wen

    2015-01-01

    Defects in spermatogenesis, a process that produces spermatozoa inside seminiferous tubules of the testis, result in male infertility. Spermatogenic progression is highly dependent on a microenvironment provided by Sertoli cells, the only somatic cells and epithelium of seminiferous tubules. However, genes that regulate such an important activity of Sertoli cells are poorly understood. Here, we found that AT-rich interactive domain 4B (ARID4B), is essential for the function of Sertoli cells to regulate spermatogenesis. Specifically, we generated Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, and showed that the Arid4bSCKO male mice were completely infertile with impaired testis development and significantly reduced testis size. Importantly, severe structural defects accompanied by loss of germ cells and Sertoli cell-only phenotype were found in many seminiferous tubules of the Arid4bSCKO testes. In addition, maturation of Sertoli cells was significantly delayed in the Arid4bSCKO mice, associated with delayed onset of spermatogenesis. Spermatogenic progression was also defective, showing an arrest at the round spermatid stage in the Arid4bSCKO testes. Interestingly, we showed that ARID4B functions as a “coactivator” of androgen receptor and is required for optimal transcriptional activation of reproductive homeobox 5, an androgen receptor target gene specifically expressed in Sertoli cells and critical for spermatogenesis. Together, our study identified ARID4B to be a key regulator of Sertoli cell function important for male germ cell development. PMID:26258622

  15. Smaller tumor size is associated with poor survival in T4b colon cancer

    PubMed Central

    Huang, Ben; Feng, Yang; Mo, Shao-Bo; Cai, San-Jun; Huang, Li-Yong

    2016-01-01

    AIM: To hypothesize that in patients with colon cancer showing heavy intestinal wall invasion without distant metastasis (T4bN0-2M0), small tumor size would correlate with more aggressive tumor behaviors and therefore poorer cancer-specific survival (CSS). METHODS: We analyzed T4bN0-2M0 colon cancer patients in the Surveillance, Epidemiology and End Results (SEER) database. A preliminary analysis of T4bN0-2M0 colon cancer patients at the Fudan University Shanghai Cancer Center is also presented. RESULTS: A total of 1734 T4bN0-2M0 colon cancer patients from the SEER database were included. Kaplan-Meier analysis revealed decreasing CSS with decreasing tumor size (P < 0.001). Subgroup analysis showed a significant association between poorer CSS with smaller tumor size in T4bN0 patients (P = 0.024), and a trend of association in T4bN1 (P = 0.182) and T4bN2 patients (P = 0.191). Multivariate analysis identified tumor size as an independent prognostic factor for CSS in T4bN0-2M0 patients (P = 0.024). Preliminary analysis of Fudan University Shanghai Cancer Center samples suggested the 5-year CSS was 50.0%, 72.9% and 77.1% in patients with tumors ≤ 4.0 cm, 4.0-7.0 cm and ≥ 7.0 cm. CONCLUSION: Smaller tumor size is associated with poorer CSS in the T4bN0-2M0 subset of colon cancer, particularly in the T4bN0M0 subgroup. PMID:27547015

  16. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  17. Cytotoxicity of the Bacillus thuringiensis Cry4B toxin is mediated by the cadherin receptor BT-R₃ of Anopheles gambiae.

    PubMed

    Ibrahim, Mohamed A; Griko, Natalya B; Bulla, Lee A

    2013-07-01

    We demonstrate for the first time the selective cytotoxicity of Bacillus thuringiensis subsp. israelensis Cry4B toxin mediated by BT-R₃ using a cell-based system, which employs High Five insect cells stably expressing BT-R₃. Discovery and validation of BT-R₃ as the Cry4B receptor was accomplished using a web-based computational pipeline platform that facilitates high-throughput insecticidal target identification utilizing the Anopheles gambiae genome. Once the Cry4B toxin binds to the BT-R₃ receptor, a cell death pathway is manifested by sequential cytological changes that include membrane blebbing, cell swelling and lysis. Cry4B toxin associates with cell membrane in both oligomeric and monomeric forms. Monomeric toxin binds specifically to BT-R₃ whereas oligomer interacts with cell membrane non-specifically. Cytotoxicity and cell death are the direct result of binding of toxin monomer to BT-R₃. The oligomeric form of Cry4B toxin is not involved in cell death. Both the location of the toxin-binding region within BT-R₃ and its structural motif are critical to the binding affinity and specificity of the toxin. The toxin-binding region of BT-R₃ appears to be located in EC11, the most membrane proximal EC module within the extracellular domain. It is characterized by the presence of two highly conserved amino acid sequences within their N- and C-termini that flank EC11. These sequences represent signature motifs that mark the toxin-binding function in BT-R₃. The two sequences form two adjacent β-strands within the β-barrel of EC11, the positioning of which is a hallmark of all Cry toxin receptors thus far reported.

  18. CUL4B promotes replication licensing by up-regulating the CDK2-CDC6 cascade.

    PubMed

    Zou, Yongxin; Mi, Jun; Wang, Wenxing; Lu, Juanjuan; Zhao, Wei; Liu, Zhaojian; Hu, Huili; Yang, Yang; Gao, Xiaoxing; Jiang, Baichun; Shao, Changshun; Gong, Yaoqin

    2013-03-18

    Cullin-RING ubiquitin ligases (CRLs) participate in the regulation of diverse cellular processes including cell cycle progression. Mutations in the X-linked CUL4B, a member of the cullin family, cause mental retardation and other developmental abnormalities in humans. Cells that are deficient in CUL4B are severely selected against in vivo in heterozygotes. Here we report a role of CUL4B in the regulation of replication licensing. Strikingly, CDC6, the licensing factor in replication, was positively regulated by CUL4B and contributed to the loading of MCM2 to chromatin. The positive regulation of CDC6 by CUL4B depends on CDK2, which phosphorylates CDC6, protecting it from APC(CDH1)-mediated degradation. Thus, aside being required for cell cycle reentry from quiescence, CDK2 also contributes to pre-replication complex assembly in G1 phase of cycling cells. Interestingly, the up-regulation of CDK2 by CUL4B is achieved via the repression of miR-372 and miR-373, which target CDK2. Our findings thus establish a CUL4B-CDK2-CDC6 cascade in the regulation of DNA replication licensing.

  19. ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

    PubMed Central

    Cabrera, Sandra; Fernández, Álvaro F.; Mariño, Guillermo; Aguirre, Alina; Suárez, María F.; Español, Yaiza; Vega, José A.; Laurà, Rosaria; Fueyo, Antonio; Fernández-García, M. Soledad; Freije, José M.P.; Kroemer, Guido; López-Otín, Carlos

    2013-01-01

    The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency. PMID:23782979

  20. Structure based virtual screening to identify selective phosphodiesterase 4B inhibitors.

    PubMed

    Gangwal, Rahul P; Damre, Mangesh V; Das, Nihar R; Dhoke, Gaurao V; Bhadauriya, Anuseema; Varikoti, Rohith A; Sharma, Shyam S; Sangamwar, Abhay T

    2015-04-01

    Phosphodiesterase 4 (PDE4), is a hydrolytic enzyme, is proposed as a promising target in asthma and chronic obstructive pulmonary disease. PDE4B selective inhibitors are desirable to reduce the dose limiting adverse effect associated with non-selective PDE4B inhibitors. To achieve this goal, ligand based pharmacophore modeling and molecular docking approach is employed. Pharmacophore hypotheses for PDE4B and PDE4D are generated using HypoGen algorithm. The best PDE4B pharmacophore hypothesis (Hypo1_PDE4B) consist of one hydrogen-bond acceptor and two ring aromatic features, whereas PDE4D pharmacophore hypothesis (Hypo1_PDE4D) consist of one hydrogen-bond acceptor, one hydrophobic aliphatic, and two ring aromatic features. The validated pharmacophore hypotheses are used in virtual screening to identify selective PDE4B inhibitors. The hits were screened for their estimated activity, FitValue, and quantitative estimation of drug likeness. After molecular docking analysis, ten hits were purchased for in vitro analysis. Out of these, six hits have shown potent and selective inhibitory activity against PDE4B with IC50 values ranging from 2 to 378nM.

  1. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    PubMed

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.

  2. Eruption column physics

    SciTech Connect

    Valentine, G.A.

    1997-03-01

    In this paper the author focuses on the fluid dynamics of large-scale eruption columns. The dynamics of these columns are rooted in multiphase flow phenomena, so a major part of the paper sets up a foundation on that topic that allows one to quickly assess the inherent assumptions made in various theoretical and experimental approaches. The first part is centered on a set of complex differential equations that describe eruption columns, but the focus is on a general understanding of important physical processes rather than on the mathematics. The author discusses briefly the relative merits and weaknesses of different approaches, emphasizing that the largest advances in understanding are made by combining them. He then focuses on dynamics of steady eruption columns and then on transient phenomena. Finally he briefly reviews the effects of varying behavior of the ambient medium through which an eruption column moves. These final sections will emphasize concepts and a qualitative understanding of eruption dynamics. This paper relies on principles of continuum mechanics and transport processes but does not go into detail on the development of those principles. 36 refs., 36 figs., 3 tabs.

  3. Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis.

    PubMed

    Yin, Yan; Liu, Liren; Yang, Chenyi; Lin, Congxing; Veith, George Michael; Wang, Caihong; Sutovsky, Peter; Zhou, Pengbo; Ma, Liang

    2016-03-25

    CUL4B ubiquitin ligase belongs to the cullin-RING ubiquitin ligase family. Although sharing many sequence and structural similarities, CUL4B plays distinct roles in spermatogenesis from its homologous protein CUL4A. We previously reported that genetic ablation ofCul4ain mice led to male infertility because of aberrant meiotic progression. In the present study, we generated Cul4bgerm cell-specific conditional knock-out (Cul4b(Vasa)),as well asCul4bglobal knock-out (Cul4b(Sox2)) mouse, to investigate its roles in spermatogenesis. Germ cell-specific deletion of Cul4bled to male infertility, despite normal testicular morphology and comparable numbers of spermatozoa. Notably, significantly impaired sperm mobility caused by reduced mitochondrial activity and glycolysis level were observed in the majority of the mutant spermatozoa, manifested by low, if any, sperm ATP production. Furthermore,Cul4b(Vasa)spermatozoa exhibited defective arrangement of axonemal microtubules and flagella outer dense fibers. Our mass spectrometry analysis identified INSL6 as a novel CUL4B substrate in male germ cells, evidenced by its direct polyubiquination and degradation by CUL4B E3 ligase. Nevertheless,Cul4bglobal knock-out males lost their germ cells in an age-dependent manner, implying failure of maintaining the spermatogonial stem cell niche in somatic cells. Taken together, our results show that CUL4B is indispensable to spermatogenesis, and it functions cell autonomously in male germ cells to ensure spermatozoa motility, whereas it functions non-cell-autonomously in somatic cells to maintain spermatogonial stemness. Thus, CUL4B links two distinct spermatogenetic processes to a single E3 ligase, highlighting the significance of ubiquitin modification during spermatogenesis. PMID:26846852

  4. Modeling of two-phase flow instabilities during startup transients utilizing RAMONA-4B methodology

    SciTech Connect

    Paniagua, J.; Rohatgi, U.S.; Prasad, V.

    1996-10-01

    RAMONA-4B code is currently under development for simulating thermal hydraulic instabilities that can occur in Boiling Water Reactors (BWRs) and the Simplified Boiling Water Reactor (SBWR). As one of the missions of RAMONA-4B is to simulate SBWR startup transients, where geysering or condensation-induced instability may be encountered, the code needs to be assessed for this application. This paper outlines the results of the assessments of the current version of RAMONA-4B and the modifications necessary for simulating the geysering or condensation-induced instability. The test selected for assessment are the geysering tests performed by Prof Aritomi (1993).

  5. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

  6. Separation of human breast cancer cells from blood by differential dielectric affinity.

    PubMed Central

    Becker, F F; Wang, X B; Huang, Y; Pethig, R; Vykoukal, J; Gascoyne, P R

    1995-01-01

    Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications. Images Fig. 3 PMID:7846067

  7. TAF4b is required for mouse spermatogonial stem cell development

    PubMed Central

    Lovasco, Lindsay A.; Gustafson, Eric A.; Seymour, Kimberly A.; de Rooij, Dirk G.; Freiman, Richard N.

    2014-01-01

    Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development. PMID:25727968

  8. RDR-4B doppler weather radar with forward looking wind shear detection capability

    NASA Technical Reports Server (NTRS)

    Grasley, Steven S.

    1992-01-01

    The topics are presented in viewgraph form and include the following: Bendix/King atmospheric transport and dispersion (ATAD) position; RDR-4A technical baseline; RTA-4A characteristics; RDR-4 antenna characteristics; modification of RDR-4A to RDR-4B; RDR-4A functional block diagram; RDR-4B characteristics; development/test plan; CV-580 testing capability; CV-580 test results; Continental A300 test configuration; Continental Data Recording Program operational considerations; Continental A300 test results; and display considerations.

  9. VizieR Online Data Catalog: XO-4b 3yr observations with DEMONEX (Villanueva+, 2016)

    NASA Astrophysics Data System (ADS)

    Villanueva, S. Jr; Eastman, J. D.; Gaudi, B. S.

    2016-06-01

    New observations of XO-4b were made using DEdicated MONitor of EXotransits (DEMONEX). DEMONEX monitored bright stars hosting known transiting planets over a 3yr period from 2008 to 2011 in order to provide a homogeneous data set of precise relative photometry for over 40 transiting systems. There are 20 nights of data from 2008 November to 2010 May taken during primary transits of XO-4b. All observations were made in the Sloan z band. (1 data file).

  10. Order Parameter measurements of Chromonic Liquid Crystal Benzopurpurin 4B using polarized Raman scattering

    NASA Astrophysics Data System (ADS)

    Nayani, Karthik; Park, Jung Ok; Srinivasarao, Mohan

    2012-02-01

    Benzopurpurin 4B (BPP4B), a commonly used textile dye, is known to form chromonic liquid crystal phases in aqueous solutions at fairly low concentrations (<0.5 wt%) in comparison with other chromonic liquid crystals. Also the aggregation properties and the structure of the aggregates in aqueous solutions of BPP4B are not well understood. Recently McKitterick et al. reported a study on the aggregation and phase behavior of BPP4B in water.ootnotetextC. B. McKitterick, N. L. Erb-Satullo, N. D. LaRacuente, A. J. Dickson, P. J. Collings. J. Phys. Chem. B. 2010. 114, 1888. Further understanding of the behavior of BPP4B in aqueous solutions can be gained by studying how the order parameter of its liquid crystalline phase varies with some relevant parameters. Planar monodomains of BPP4B were obtained using a flat rectangular capillary. Thermal evolution of the order parameter of these aligned monodomains was carried out using polarized Raman scattering measurements. Further, the concentration dependence and the effect of salt on the order parameter were studied. The variation of the order parameter with the above parameters was correlated to the structure of the aggregates using the UV-Vis absorption data.

  11. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    PubMed Central

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  12. Columns in Clay

    ERIC Educational Resources Information Center

    Leenhouts, Robin

    2010-01-01

    This article describes a clay project for students studying Greece and Rome. It provides a wonderful way to learn slab construction techniques by making small clay column capitols. With this lesson, students learn architectural vocabulary and history, understand the importance of classical architectural forms and their influence on today's…

  13. A Column Dispersion Experiment.

    ERIC Educational Resources Information Center

    Corapcioglu, M. Y.; Koroglu, F.

    1982-01-01

    Crushed glass and a Rhodamine B solution are used in a one-dimensional optically scanned column experiment to study the dispersion phenomenon in porous media. Results indicate that the described model gave satisfactory results and that the dispersion process in this experiment is basically convective. (DC)

  14. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  15. Neuroblastoma patient outcomes, tumor differentiation, and ERK activation are correlated with expression levels of the ubiquitin ligase UBE4B

    PubMed Central

    Woodfield, Sarah E.; Guo, Rong Jun; Liu, Yin; Major, Angela M.; Hollingsworth, Emporia Faith; Indiviglio, Sandra; Whittle, Sarah B.; Mo, Qianxing; Bean, Andrew J.; Ittmann, Michael; Lopez-Terrada, Dolores; Zage, Peter E.

    2016-01-01

    Background UBE4B is an E3/E4 ubiquitin ligase whose gene is located in chromosome 1p36.22. We analyzed the associations of UBE4B gene and protein expression with neuroblastoma patient outcomes and with tumor prognostic features and histology. Methods We evaluated the association of UBE4B gene expression with neuroblastoma patient outcomes using the R2 Platform. We screened neuroblastoma tumor samples for UBE4B protein expression using immunohistochemistry. FISH for UBE4B and 1p36 deletion was performed on tumor samples. We then evaluated UBE4B expression for associations with prognostic factors and with levels of phosphorylated ERK in neuroblastoma tumors and cell lines. Results Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma and with worse outcomes in all patient subgroups. UBE4B protein expression was associated with neuroblastoma tumor differentiation, and decreased UBE4B protein levels were associated with high-risk features. UBE4B protein levels were also associated with levels of phosphorylated ERK. Conclusions We have demonstrated associations between UBE4B gene expression and neuroblastoma patient outcomes and prognostic features. Reduced UBE4B protein expression in neuroblastoma tumors was associated with high-risk features, a lack of differentiation, and with ERK activation. These results suggest UBE4B may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions and that UBE4B expression may mediate neuroblastoma differentiation. PMID:27014418

  16. Insights into K-Ras 4B regulation by post-translational lysine acetylation.

    PubMed

    Knyphausen, Philipp; Lang, Franziska; Baldus, Linda; Extra, Antje; Lammers, Michael

    2016-10-01

    Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

  17. Structural and functional comparison of the non-structural protein 4B in flaviviridae.

    PubMed

    Welsch, Christoph; Albrecht, Mario; Maydt, Jochen; Herrmann, Eva; Welker, Martin Walter; Sarrazin, Christoph; Scheidig, Axel; Lengauer, Thomas; Zeuzem, Stefan

    2007-09-01

    Flaviviridae are evolutionarily related viruses, comprising the hepatitis C virus (HCV), with the non-structural protein 4B (NS4B) as one of the least characterized proteins. NS4B is located in the endoplasmic reticulum membrane and is assumed to be a multifunctional protein. However, detailed structure information is missing. The hydrophobic nature of NS4B is a major difficulty for many experimental techniques. We applied bioinformatics methods to analyse structural and functional properties of NS4B in different viruses. We distinguish a central non-globular membrane portion with four to five transmembrane regions from an N- and C-terminal part with non-transmembrane helical elements. We demonstrate high similarity in sequence and structure for the C-terminal part within the flaviviridae family. A palmitoylation site contained in the C-terminal part of HCV is equally conserved in GB virus B. Furthermore, we identify and characterize an N-terminal basic leucine zipper (bZIP) motif in HCV, which is suggestive of a functionally important interaction site. In addition, we model the interaction of the bZIP region with the recently identified interaction partner CREB-RP/ATF6beta, a human activating transcription factor involved in ER-stress. In conclusion, the versatile structure, together with functional sites and motifs, possibly enables NS4B to adopt a role as protein hub in the membranous web interaction network of virus and host proteins. Important structural and functional properties of NS4B are predicted with implications for ER-stress response, altered gene expression and replication efficacy.

  18. 11. TIMBER COLUMN AND CAST IRON COLUMN CAP IN FIFTH ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. TIMBER COLUMN AND CAST IRON COLUMN CAP IN FIFTH FLOOR WAREHOUSE SPACE. VIEW TO SOUTHWEST. - Commercial & Industrial Buildings, Becker-Hazelton Company Warehouse, 280 Iowa Street, Dubuque, Dubuque County, IA

  19. Successfully downsize trayed columns

    SciTech Connect

    Sloley, A.W.; Fleming, B. )

    1994-03-01

    Techniques for the design and sizing of new trayed distillation columns are abundant in the literature. So, too, are the guidelines for modifying towers for operation beyond their original design range. Reducing capacity of distillation trays merits at least as much consideration. Indeed, lack of knowledge and experience in this area causes many tower failures and misdesigned columns. In this article, the authors present some practical design considerations, based on field experience, for tower trays operating at loadings dramatically lower than normal for a particular design. General considerations cover liquid and vapor hydraulics and flow behavior. Case studies are included for there typical units: a refinery vacuum crude still, a petrochemical superfractionator, and a steam stripper.

  20. Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

    PubMed

    Kang, Hyo Jin; Choe, Weonu; Min, Jeong-Ki; Lee, Young-Mi; Kim, B Moon; Chung, Sang J

    2016-09-30

    The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

  1. High-Pressure Synthesis and Crystal Structure of Ce4B14O27

    PubMed Central

    Hinteregger, Ernst; Perfler, Lukas; Huppertz, Hubert

    2013-01-01

    Ce4B14O27 was synthesized under conditions of 2.6 GPa and 750 °C in a Walker-type multianvil apparatus. The crystal structure was determined on the basis of single-crystal X-ray diffraction data, collected at room temperature, revealing that Ce4B14O27 is isotypic to La4B14O27. Ce4B14O27 crystallizes monoclinically with four formula units in the space group C2/c (No. 15) and the lattice parameters a = 1117.8(2), b = 640.9(2), c = 2531.7(5) pm, and β = 100.2(1)°. The three-dimensional boron-oxygen framework consists of [BO4]5– tetrahedra and trigonal-planar [BO3]3– groups. The structure contains two crystallographically different cerium ions. Furthermore, Raman spectroscopy was performed on single crystals of Ce4B14O27. PMID:25995523

  2. Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

    PubMed

    Cabrera, Sandra; Maciel, Mariana; Herrera, Iliana; Nava, Teresa; Vergara, Fabián; Gaxiola, Miguel; López-Otín, Carlos; Selman, Moisés; Pardo, Annie

    2015-04-01

    Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

  3. TAF4b Promotes Mouse Primordial Follicle Assembly and Oocyte Survival

    PubMed Central

    Grive, Kathryn J.; Seymour, Kimberly A.; Mehta, Rajvi; Freiman, Richard N.

    2014-01-01

    Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development. PMID:24836512

  4. A tumor suppressor function for the lipid phosphatase INPP4B in melanocytic neoplasms.

    PubMed

    Perez-Lorenzo, Rolando; Gill, Kamraan Z; Shen, Che-Hung; Zhao, Feng X; Zheng, Bin; Schulze, Hans-Joachim; Silvers, David N; Brunner, Georg; Horst, Basil A

    2014-05-01

    The phosphoinositide-3 kinase (PI3K) pathway is deregulated in a significant proportion of melanomas, and PI3K pathway activation in combination with constitutively active mitogen-activated protein kinase signaling shows synergistic effects in the process of melanoma tumorigenesis. Recently, a tumor suppressor function for the lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described in breast and prostate cancers, with impact on PI3K signaling output. Given the importance of PI3K pathway activity for melanoma formation and growth, we aimed to assess the role of INPP4B in melanocytic tumors. Our studies in native tumors suggest that decreased INPP4B expression is an event correlating with tumor progression in melanocytic neoplasms. We further demonstrate that INPP4B regulates PI3K/Akt signaling and exerts a tumor suppressor effect, impacting the proliferative, invasive, and tumorigenic capacity of melanoma cells. INPP4B expression in melanocytic neoplasms may therefore have potential as a biomarker for disease progression and as a modulator for the prediction of treatment outcome.

  5. A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors.

    PubMed

    Akin, Debra; Wang, S Keisin; Habibzadegah-Tari, Pouran; Law, Brian; Ostrov, David; Li, Min; Yin, Xiao-Ming; Kim, Jae-Sung; Horenstein, Nicole; Dunn, William A

    2014-01-01

    Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma. PMID:25483883

  6. Purification of beef-heart cytochrome c oxidase by hydrophobic interaction chromatography on octyl-Sepharose CL-4B.

    PubMed

    Rosén, S

    1978-04-12

    1. Hydrophobic interaction chromatography on Octyl-Sepharose CL-4B is used as a new and simple method for the preparation of large amounts of beef-heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). 2. The method involves only one cycle of (NH4)2SO4 fractionation before the material is applied to the column. After washing with 10% cholate and 1.5% Tween 80, elution of the enzyme is accomplished with 1% Triton X-100. 3. The enzyme so prepared contains about 10 nmol heme alpha/mg protein and about 0.2% phospholipid. 4. Characterization of the enzyme has been made with optical and EPR spectroscopy and polyacrylamide gel electrophoresis. The preparation appears by these criteria to be at least as good as other purified enzyme preparations. 5. The turnover rate at infinite cytochrome c concentration in 0.1 M sodium phosphate buffer and 0.5% Tween 80 at pH 6.1 is 80 s-1 per functional unit of the enzyme. A more than three-fold activation could be obtained by the addition of phosphatidylcholine at neutral pH.

  7. Cesium cation affinities and basicities

    NASA Astrophysics Data System (ADS)

    Gal, Jean-François; Maria, Pierre-Charles; Massi, Lionel; Mayeux, Charly; Burk, Peeter; Tammiku-Taul, Jaana

    2007-11-01

    This review focuses on the quantitative data related to cesium cation interaction with neutral or negatively charged ligands. The techniques used for measuring the cesium cation affinity (enthalpies, CCA), and cesium cation basicities (Gibbs free energies, CCB) are briefly described. The quantum chemical calculations methods that were specifically designed for the determination of cesium cation adduct structures and the energetic aspects of the interaction are discussed. The experimental results, obtained essentially from mass spectrometry techniques, and complemented by thermochemical data, are tabulated and commented. In particular, the correlations between cesium cation affinities and lithium cation affinities for the various kinds of ligands (rare gases, polyatomic neutral molecules, among them aromatic compounds and negative ions) serve as a basis for the interpretation of the diverse electrostatic modes of interaction. A brief account of some recent analytical applications of ion/molecule reactions with Cs+, as well as other cationization approaches by Cs+, is given.

  8. "Clickable" agarose for affinity chromatography.

    PubMed

    Punna, Sreenivas; Kaltgrad, Eiton; Finn, M G

    2005-01-01

    Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

  9. Column test-rig facility for column scanning studies

    NASA Astrophysics Data System (ADS)

    Zain, Rasif M.; Roslan, Y.

    2010-03-01

    Distillation columns are considered as one of the most critical components in oil and gas plants. The plant performance depends on the ability of these columns to function as intended. Defective columns may lead to serious consequences to the plant operation, and hence the quality of product. In order to perform any inspection techniques to distillation column for NDT practitioner, the best facility was designed when the adjustable defeats of distillation column test rig has been developed. The paper discussed the development and the function of this facility.

  10. Column test-rig facility for column scanning studies

    NASA Astrophysics Data System (ADS)

    Zain, Rasif M.; Roslan, Y.

    2009-12-01

    Distillation columns are considered as one of the most critical components in oil and gas plants. The plant performance depends on the ability of these columns to function as intended. Defective columns may lead to serious consequences to the plant operation, and hence the quality of product. In order to perform any inspection techniques to distillation column for NDT practitioner, the best facility was designed when the adjustable defeats of distillation column test rig has been developed. The paper discussed the development and the function of this facility.

  11. Lack of Cul4b, an E3 Ubiquitin Ligase Component, Leads to Embryonic Lethality and Abnormal Placental Development

    PubMed Central

    Yuan, Jupeng; Qian, Yanyan; Sun, Wenjie; Zou, Yongxin; Guo, Chenhong; Chen, Bingxi; Shao, Changshun; Gong, Yaoqin

    2012-01-01

    Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse. PMID:22606329

  12. Determination for multiple mycotoxins in agricultural products using HPLC-MS/MS via a multiple antibody immunoaffinity column.

    PubMed

    Zhang, Zhaowei; Hu, Xiaofeng; Zhang, Qi; Li, Peiwu

    2016-05-15

    Mycotoxins usually found in agricultural products such as peanut, corn, and wheat, are a serious threat to human health and their detection requires multiplexed and sensitive analysis methods. Herein, a simultaneous determination for aflatoxin B1, B2, G1, G2, ochratoxin A, zearalanone and T-2 toxin was investigated using high performance liquid chromatography coupled with tandem mass spectrometry in a single run via a home-made multiple immunoaffinity column. Four monoclonal antibodies were produced in our lab against aflatoxins, ochratoxin A, zearalanone and T-2 toxin, respectively, then combined as a pool and bound to Sepharose-4B for affinity chromatography. Seven mycotoxins were effectively extracted from the agricultural product samples by using acetonitrile/water/acetic acid (80:19:1, v/v/v) Then, the extraction was cleanup by multiple immunoaffinity column. This method demonstrated a considerable linear range of 0.30-25, 0.12-20, 0.30-20, 0.12-20, 0.60-30, 0.30-25, and 1.2-40μgkg(-1)and lower limits of detection at 0.1, 0.04, 0.1, 0.04, 0.2, 0.1 and 0.4μgkg(-1) for AFB1, AFB2, AFG1, AFG2, OTA, ZEN and T-2, respectively, in comparison with previously reported methods, as well as excellent recoveries. The mIAC capacity for AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and T-2 were 187, 181, 153, 151, 105, 130, 88ng, respectively. It was found that all of the 7 mycotoxins were present in 90 agricultural product samples. The proposed method meets the requirements for rapid sample preparation and highly sensitive identification of multiple mycotoxins in agricultural product and food safety. This method provides a promising alternative with high throughput and high sensitivity for rapid analysis of seven mycotoxins in the monitoring of food safety. PMID:26948441

  13. Real-time PCR quantification of human complement C4A and C4B genes

    PubMed Central

    Szilagyi, Agnes; Blasko, Bernadett; Szilassy, Denes; Fust, George; Sasvari-Szekely, Maria; Ronai, Zsolt

    2006-01-01

    Background The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. Results A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. Conclusion This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes. PMID:16403222

  14. Protection against Asiatic Taenia solium induced by a recombinant 45W-4B protein.

    PubMed

    Luo, Xuenong; Zheng, Yadong; Hou, Junling; Zhang, Shaohua; Cai, Xuepeng

    2009-02-01

    Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates.

  15. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  16. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  17. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  18. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  19. Site-specific DNA-affinity chromatography of the lac repressor.

    PubMed Central

    Herrick, G

    1980-01-01

    To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity. PMID:7001362

  20. Secondary Structure and Membrane Topology of the Full-Length Dengue Virus NS4B in Micelles.

    PubMed

    Li, Yan; Wong, Ying Lei; Lee, Michelle Yueqi; Li, Qingxin; Wang, Qing-Yin; Lescar, Julien; Shi, Pei-Yong; Kang, CongBao

    2016-09-19

    Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections. PMID:27554985

  1. Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

    PubMed Central

    Shepherd, Malcolm; McSorley, Theresa; Olsen, Aileen E; Johnston, Lee Ann; Thomson, Neil C; Baillie, George S; Houslay, Miles D; Bolger, Graeme B

    2003-01-01

    We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types. PMID:12441002

  2. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  3. SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF NEW SULFONAMIDE ISOXAZOLO[5,4-b]PYRIDINE DERIVATIVES.

    PubMed

    Poręba, Krystyna; Pawlik, Krzysztof; Rembacz, Krzysztof P; Kurowska, Ewa; Matuszyk, Janusz; Długosz, Anna

    2015-01-01

    A series of novel sulfonamide isoxazolo[5,4-b]pyridines were synthesized. The substrates for their synthesis were 3-aminoisoxazolo[5,4-b]pyridine and selected aryl sulfonic chlorides, chlorosulfonic acid and selected amines. Reactions were carried out using the classical and microwave methods. Selected compounds were tested towards antibacterial and antiproliferative activity. The structure of the obtained new derivatives was determined by elemental analysis and acquired IR and 1H NMR spectra. Among the tested compounds: N- isoxazolo[5,4-b]pyridine-3-yl-benzenesulfonamide (2) and N-isoxazolo[5,4-b]pyridine-3-yl-4-methylbenzene-sulfonamide (5) showed antimicrobial activity towards Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) at doses: 125, 250 and 500 µg. Both compounds showed a 50% inhibition of proliferation of breast carcinoma cell line MCF7 at concentrations of 152.56 µg/mL and 160 161.08 µg/mL, respectively. PMID:26647630

  4. DISC1, PDE4B, and NDE1 at the centrosome and synapse

    SciTech Connect

    Bradshaw, Nicholas J.; Ogawa, Fumiaki; Antolin-Fontes, Beatriz; Chubb, Jennifer E.; Carlyle, Becky C.; Christie, Sheila; Claessens, Antoine; Porteous, David J.; Millar, J. Kirsty

    2008-12-26

    Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.

  5. Random UV mutagenesis approach for enhanced biodegradation of sulfonated azo dye, Green HE4B.

    PubMed

    Joshi, Swati M; Inamdar, Shrirang A; Jadhav, Jyoti P; Govindwar, Sanjay P

    2013-03-01

    The objective of the study was to execute mutant bacteria for efficient biodegradation of sulfonated azo dye, Green HE4B (GHE4B). UV irradiation was used to introduce random mutations in Pseudomonas sp. LBC1. Genetic alterations induced by UV irradiation in selected mutant bacteria were confirmed by random amplification of polymorphic DNA technique. The mutant bacteria named as Pseudomonas sp. 1 F reduced the time required for complete degradation of recalcitrant dye GHE4B by 25 % when compared with the wild one. The biodegradation was monitored by UV-Vis spectrophotometric analysis. Activities of enzymes like laccase, lignin peroxidase, veratryl alcohol oxidase, and NADH dichlorophenol indophenol reductase were found to be boosted in mutant bacteria as a consequence of UV-induced mutation. Matrix-assisted laser desorption/ionization-time of flight analysis of differentially expressed proteins of mutant bacteria suggested active role of antioxidant enzymes in the degradation of the dye. The degradation product was analyzed by Fourier transform infrared spectroscopy, high-performance thin-layer chromatography, and gas chromatography-mass spectrometry. Results revealed few variations in the degradation end products of wild-type and mutant bacteria. Phytotoxicity study underlined the safer biodegradation of GHE4B by mutant Pseudomonas sp. 1 F.

  6. IcmQ in the Type 4b secretion system contains a novel NAD+ binding domain

    PubMed Central

    Farelli, Jeremiah D.; Gumbart, James C.; Akey, Ildiko V.; Hempstead, Andrew; Amyot, Whitney; Head, James F.; McKnight, C. James; Isberg, Ralph R.; Akey, Christopher W.

    2013-01-01

    A Type4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. IcmQ associates with IcmR, binds to membranes and has a critical role in the T4bSS. We have now solved a crystal structure of IcmR-IcmQ to further our understanding of this complex. This structure revealed an amphipathic four-helix bundle, formed by IcmR and the N-terminal domain of IcmQ, which is linked to a novel C-terminal domain of IcmQ (Qc) by a linker helix. The Qc domain has structural homology with ADP ribosyltransferase domains in certain bacterial toxins and binds NAD+ with a Kd in the physiological range. Structural homology and molecular dynamics were used to identify an extended NAD+ binding site on Qc and the resulting model was tested by mutagenesis and binding assays. Based on the data, we suggest that IcmR-IcmQ binds to membranes where it may interact with or perhaps modify a protein in the T4bSS when NAD+ is bound. PMID:23850453

  7. 16 CFR 1508.5 - Component spacing test method for § 1508.4(b).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Component spacing test method for § 1508.4(b). 1508.5 Section 1508.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR FULL-SIZE BABY CRIBS § 1508.5 Component spacing test method...

  8. 16 CFR 1508.5 - Component spacing test method for § 1508.4(b).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Component spacing test method for § 1508.4(b). 1508.5 Section 1508.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS REQUIREMENTS FOR FULL-SIZE BABY CRIBS § 1508.5 Component spacing test method...

  9. Atg4b-Dependent Autophagic Flux Alleviates Huntington’s Disease Progression

    PubMed Central

    Proenca, Catia C.; Stoehr, Natacha; Bernhard, Mario; Seger, Shanon; Genoud, Christel; Roscic, Ana; Paganetti, Paolo; Liu, Shanming; Murphy, Leon O.; Kuhn, Rainer; Bouwmeester, Tewis; Galimberti, Ivan

    2013-01-01

    The accumulation of aggregated mutant huntingtin (mHtt) inclusion bodies is involved in Huntigton’s disease (HD) progression. Medium sized-spiny neurons (MSNs) in the corpus striatum are highly vulnerable to mHtt aggregate accumulation and degeneration, but the mechanisms and pathways involved remain elusive. Here we have developed a new model to study MSNs degeneration in the context of HD. We produced organotypic cortico-striatal slice cultures (CStS) from HD transgenic mice mimicking specific features of HD progression. We then show that induction of autophagy using catalytic inhibitors of mTOR prevents MSNs degeneration in HD CStS. Furthermore, disrupting autophagic flux by overexpressing Atg4b in neurons and slice cultures, accelerated mHtt aggregation and neuronal death, suggesting that Atg4b-dependent autophagic flux influences HD progression. Under these circumstances induction of autophagy using catalytic inhibitors of mTOR was inefficient and did not affect mHtt aggregate accumulation and toxicity, indicating that mTOR inhibition alleviates HD progression by inducing Atg4b-dependent autophagic flux. These results establish modulators of Atg4b-dependent autophagic flux as new potential targets in the treatment of HD. PMID:23861892

  10. 20. FOUR 4B17Gs BEING CONVERTED TO F9Cs. Photographic copy of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    20. FOUR 4B-17Gs BEING CONVERTED TO F-9Cs. Photographic copy of historic photograph. Jan.-June 1947 OAMA (original print located at Ogden Air Logistics Center, Hill Air Force Base, Utah). Photographer unknown. - Hill Field, Airplane Repair Hangars No. 1-No. 4, 5875 Southgate Avenue, Layton, Davis County, UT

  11. Identification of an NTPase motif in classical swine fever virus NS4B protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive sense single-stranded RNA virus in the genus Pestivirus of the Flaviviridae family. Here, we have identified, within CSFV non-structural (NS) protein NS4B, conserved sequence el...

  12. A new role for reticulon-4B/NOGO-B in the intestinal epithelial barrier function and inflammatory bowel disease.

    PubMed

    Rodríguez-Feo, Juan Antonio; Puerto, Marta; Fernández-Mena, Carolina; Verdejo, Cristina; Lara, José Manuel; Díaz-Sánchez, María; Álvarez, Emilio; Vaquero, Javier; Marín-Jiménez, Ignacio; Bañares, Rafael; Menchén, Luis

    2015-06-15

    Inflammatory bowel disease (IBD) is characterized by an impaired intestinal barrier function. We aimed to investigate the role of reticulon-4B (RTN-4B/NOGO-B), a structural protein of the endoplasmic reticulum, in intestinal barrier function and IBD. We used immunohistochemistry, confocal microscopy, real-time PCR, and Western blotting to study tissue distribution and expression levels of RTN-4B/NOGO-B in control and IBD samples from mouse and humans. We also targeted RTN-4B/NOGO-B using siRNAs in cultured human intestinal epithelial cell (IECs). Epithelial barrier permeability was assessed by transepithelial electrical resistance (TEER) measurement. RTN-4B/NOGO-B is expressed in the intestine mainly by IECs. Confocal microscopy revealed a colocalization of RTN-4B, E-cadherin, and polymerized actin fibers in tissue and cultured IECs. RTN-4B mRNA and protein expression were lower in the colon of IL-10(-/-) compared with wild-type mice. Colocalization of RTN-4B/E-cadherin/actin was reduced in the colon of IL-10(-/-) mice. Analysis of endoscopic biopsies from IBD patients showed a significant reduction of RTN-4B/NOGO-B expression in inflamed mucosa compared with control. Treatment of IECs with H2O2 reduced TEER values and triggered phosphorylation of RTN-4B in serine 107 residues as well as downregulation of RTN-4B expression. Acute RTN-4B/NOGO-B knockdown by siRNAs resulted in a decreased TEER values and reduction of E-cadherin and α-catenin expression and in the amount of F-actin-rich filaments in IECs. Epithelial RTN-4B/NOGO-B was downregulated in human and experimental IBD. RTN-4B participates in the intestinal epithelial barrier function, most likely via its involvement in E-cadherin, α-catenin expression, and actin cytoskeleton organization at sites of cell-to-cell contacts.

  13. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    PubMed

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C

    1985-05-01

    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  14. SPIRAL CONTACTOR FOR SOLVENT EXTRACTION COLUMN

    DOEpatents

    Cooley, C.R.

    1961-06-13

    The patented extraction apparatus includes a column, perforated plates extending across the column, liquid pulse means connected to the column, and an imperforate spiral ribbon along the length of the column.

  15. Why Hexagonal Basalt Columns?

    PubMed

    Hofmann, Martin; Anderssohn, Robert; Bahr, Hans-Achim; Weiß, Hans-Jürgen; Nellesen, Jens

    2015-10-01

    Basalt columns with their preferably hexagonal cross sections are a fascinating example of pattern formation by crack propagation. Junctions of three propagating crack faces rearrange such that the initial right angles between them tend to approach 120°, which enables the cracks to form a pattern of regular hexagons. To promote understanding of the path on which the ideal configuration can be reached, two periodically repeatable models are presented here involving linear elastic fracture mechanics and applying the principle of maximum energy release rate. They describe the evolution of the crack pattern as a transition from rectangular start configuration to the hexagonal pattern. This is done analytically and by means of three-dimensional finite element simulation. The latter technique reproduces the curved crack path involved in this transition.

  16. Why Hexagonal Basalt Columns?

    PubMed

    Hofmann, Martin; Anderssohn, Robert; Bahr, Hans-Achim; Weiß, Hans-Jürgen; Nellesen, Jens

    2015-10-01

    Basalt columns with their preferably hexagonal cross sections are a fascinating example of pattern formation by crack propagation. Junctions of three propagating crack faces rearrange such that the initial right angles between them tend to approach 120°, which enables the cracks to form a pattern of regular hexagons. To promote understanding of the path on which the ideal configuration can be reached, two periodically repeatable models are presented here involving linear elastic fracture mechanics and applying the principle of maximum energy release rate. They describe the evolution of the crack pattern as a transition from rectangular start configuration to the hexagonal pattern. This is done analytically and by means of three-dimensional finite element simulation. The latter technique reproduces the curved crack path involved in this transition. PMID:26550724

  17. Quantitative analysis of C4Ab and C4Bb binding to the C3b/C4b receptor (CR1, CD35)

    PubMed Central

    REILLY, B D; MOLD, C

    1997-01-01

    Complement-dependent clearance of immune complexes in humans is dependent on the activation and binding of the early components of the classical complement cascade. This prevents immune complex precipitation and promotes binding of the complexes by the C4b/C3b complement receptor CR1 (CD35) found on erythrocytes. The fourth component of human complement is encoded by two closely linked genes within the MHC. These genes give rise to the isotypic forms C4A and C4B, and recent studies suggest that CR1 binds activated C4A (C4Ab) to a greater extent than activated C4B (C4Bb). To study this difference in a more quantitative way the binding reactions between CR1 and C4Ab- and C4Bb-coated immune complexes and between CR1 and soluble dimers of C4Ab (C4Ab2) and C4Bb (C4Bb2) were analysed using the native receptor on human erythrocytes. The binding reaction between immune complexes with equivalent amounts of covalently bound C4Ab or C4Bb and erythrocyte CR1 showed a two-fold higher binding of complexes coated with C4A. Furthermore, erythrocyte CR1 bound C4Ab2 with an apparent four-fold higher affinity (Kd ≍ 1.4 10−7M) than C4Bb2 (Kd ≍ 4–8 10−7M), indicating a preferential binding of CR1 for C4A. PMID:9367418

  18. Knockdown of CUL4B Suppresses the Proliferation and Invasion in Non-Small Cell Lung Cancer Cells.

    PubMed

    Wang, Xuguang; Chen, Zhe

    2016-01-01

    Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, was found to be overexpressed in many types of tumors. However, the expression pattern and role of CUL4B in non-small cell lung cancer (NSCLC) remain largely unknown. Therefore, in the present study, we investigated the role of CUL4B in NSCLC, and the underlying mechanism was also explored. Our results showed that CUL4B was highly expressed in NSCLC cell lines. Silencing CUL4B obviously inhibited proliferation and migration/invasion of NSCLC cells, and it also suppressed the epithelial-mesenchymal transition (EMT) progress in NSCLC cells. Furthermore, knockdown of CUL4B significantly inhibited the expression of β-catenin, cyclin D1, and c-Myc in NSCLC cells. Taken together, these results suggest that knockdown of CUL4B inhibited the proliferation and invasion through suppressing the Wnt/β-catenin signaling pathway in NSCLC cells. Therefore, CUL4B may represent a novel therapeutic target for the treatment of NSCLC. PMID:27656838

  19. Buckling of a holey column.

    PubMed

    Pihler-Puzović, D; Hazel, A L; Mullin, T

    2016-09-14

    We report the results from a combined experimental and numerical investigation of buckling in a novel variant of an elastic column under axial load. We find that including a regular line of centred holes in the column can prevent conventional, global, lateral buckling. Instead, the local microstructure introduced by the holes allows the column to buckle in an entirely different, internal, mode in which the holes are compressed in alternate directions, but the column maintains the lateral reflection symmetry about its centreline. The internal buckling mode can be accommodated within a smaller external space than the global one; and it is the preferred buckling mode over an intermediate range of column lengths for sufficiently large holes. For very short or sufficiently long columns a modification of the classical, global, lateral buckling is dominant. PMID:27501288

  20. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

    PubMed Central

    Tzeng, E; Billiar, T R; Robbins, P D; Loftus, M; Stuehr, D J

    1995-01-01

    Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8524846

  1. Compact electron beam focusing column

    SciTech Connect

    Persaud, Arun; Leung, Ka-Ngo; Reijonen, Jani

    2001-07-13

    A novel design for an electron beam focusing column has been developed at LBNL. The design is based on a low-energy spread multicusp plasma source which is used as a cathode for electron beam production. The focusing column is 10 mm in length. The electron beam is focused by means of electrostatic fields. The column is designed for a maximum voltage of 50 kV. Simulations of the electron trajectories have been performed by using the 2-D simulation code IGUN and EGUN. The electron temperature has also been incorporated into the simulations. The electron beam simulations, column design and fabrication will be discussed in this presentation.

  2. Protection against Asiatic Taenia solium Induced by a Recombinant 45W-4B Protein▿

    PubMed Central

    Luo, Xuenong; Zheng, Yadong; Hou, Junling; Zhang, Shaohua; Cai, Xuepeng

    2009-01-01

    Taenia solium is a great threat not only to human health but also to the pig-raising industry. Oncospheral stage-specific 45W proteins are good candidates for the development of anticysticercosis vaccines. In this study, a recombinant 45W-4B protein was highly produced and used for vaccination. Two animal trials resulted in a significant reduction in parasite burden induced by the definite protein against Asiatic T. solium, up to 97.0% and 98.4%, respectively. These provide informative results for the development of effective 45W-4B vaccines against cysticercosis caused by both Chinese and Mexican T. solium isolates and even by other isolates. PMID:19091992

  3. Optical and Near-UV Observations of the Transiting Extrasolar Planet TrES-4b

    NASA Astrophysics Data System (ADS)

    Smith, Carter-Thaxton; Turner, J.; Carleton, T.; Crawford, B.; Guvenen, B.; Hardegree-Ullman, K.; Small, L.; Towner, A. P.; Walker-LaFollette, A.; Henz, T.

    2013-01-01

    Using the Steward Observatory 61” Kuiper Telescope, The University of Arizona Astronomy Club conducted photometric observations of the transiting extrasolar planet TrES-4b as part of the Exoplanet Observation Project. Observations were made in the Bessell U, Harris B, and Harris R filters. Initial observations were made in 2009, with follow up observations in 2011. Basic data reduction and photometry was done using IRAF and determination of transit parameters was done using Transit Analysis Package (TAP) and JKTEBOP transit modeling code. We present an updated planetary mass, radius, density, surface gravity, Safronov number, equilibrium temperature, orbital distance, and orbital inclination for TrES-4b. In addition, we also searched for asymmetries between the near-UV and optical light curves. This project, started in spring 2009, has introduced many undergraduate students to research and given them valuable experience with data reduction and observation techniques.

  4. High resolution crystal structure of human Dim2/TXNL4B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TXNL4A (thioredoxin like 4A) is an essential protein conserved from yeast to human and is a component of the pre-mRNA splicing machinery. TXNL4B was identified as a TXNL4 family protein that also interacts with prp6, an integral component of the U4/U6•U5 tri-snRNP complex, and was shown to function...

  5. Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

    PubMed Central

    Thornbrough, Joshua M.; Jha, Babal K.; Yount, Boyd; Goldstein, Stephen A.; Li, Yize; Elliott, Ruth; Sims, Amy C.; Baric, Ralph S.; Silverman, Robert H.

    2016-01-01

    ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage C Betacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2′,5′-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage A Betacoronavirus PDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention. PMID:27025250

  6. 45. MAIN MEETING ROOM COLUMNS. Ends of gallery columns identified ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    45. MAIN MEETING ROOM COLUMNS. Ends of gallery columns identified at the time of removal for transfer to the George School for re-erection. The stamp reads, 'REMOVED FROM 12th ST. MTG HSE PHILA 1972'. - Twelfth Street Meeting House, 20 South Twelfth Street, Philadelphia, Philadelphia County, PA

  7. HATS-4b: A dense hot Jupiter transiting a super metal-rich G star

    SciTech Connect

    Jordán, Andrés; Brahm, Rafael; Rabus, M.; Suc, V.; Espinoza, N.; Bakos, G. Á.; Penev, K.; Hartman, J. D.; Csubry, Z.; Bhatti, W.; De Val Borro, M.; Bayliss, D.; Zhou, G.; Mancini, L.; Mohler-Fischer, M.; Ciceri, S.; Csák, B.; Henning, T.; Sato, B.; Buchhave, L.; and others

    2014-08-01

    We report the discovery by the HATSouth survey of HATS-4b, an extrasolar planet transiting a V = 13.46 mag G star. HATS-4b has a period of P ≈ 2.5167 days, mass of M{sub p} ≈ 1.32 M {sub Jup}, radius of R{sub p} ≈ 1.02 R {sub Jup}, and density of ρ {sub p} = 1.55 ± 0.16 g cm{sup –3} ≈1.24 ρ{sub Jup}. The host star has a mass of 1.00 M {sub ☉}, a radius of 0.92 R {sub ☉}, and a very high metallicity [Fe/H]=0.43 ± 0.08. HATS-4b is among the densest known planets with masses between 1 and 2 M {sub J} and is thus likely to have a significant content of heavy elements of the order of 75 M {sub ⊕}. In this paper we present the data reduction, radial velocity measurements, and stellar classification techniques adopted by the HATSouth survey for the CORALIE spectrograph. We also detail a technique for simultaneously estimating vsin i and macroturbulence using high resolution spectra.

  8. DISTRIBUTION OF CH{sub 3}OH IN NGC 1333 IRAS4B

    SciTech Connect

    Sakai, Nami; Yamamoto, Satoshi; Ceccarelli, Cecilia; Bottinelli, Sandrine; Sakai, Takeshi

    2012-07-20

    Distribution of the CH{sub 3}OH (J{sub K} = 2{sub K}-1{sub K}, 96.7 GHz) emission has been investigated toward NGC 1333 IRAS4B, a low-mass Class 0 protostar which harbors a hot corino, with Nobeyama Millimeter Array. The CH{sub 3}OH emission is found to be prominent in the shocked region caused by an impact of the molecular outflow from the protostars. The direction of the outflow which is responsible for the shock seems to be opposite to that of a compact outflow known previously in the CO (J = 2-1), HCN (J = 1-0), H{sub 2}CO (3{sub 12}-2{sub 11}), and CH{sub 3}OH (J{sub K} = 7{sub K}-6{sub K}) emissions, whereas it is the same as that of the faint second outflow found in the H{sub 2}CO emission. This double outflow structure can be interpreted most naturally by the existence of more than two protostars in IRAS4B. On the other hand, a centrally condensed component associated apparently with IRAS4B cannot be recognized in our CH{sub 3}OH observation. Our observation suggests that, in this source, the CH{sub 3}OH (J{sub K} 2{sub K}-1{sub K}) emission preferentially traces the shocked regions rather than the hot corino around the protostar.

  9. Two-parameter twisted quantum affine algebras

    NASA Astrophysics Data System (ADS)

    Jing, Naihuan; Zhang, Honglian

    2016-09-01

    We establish Drinfeld realization for the two-parameter twisted quantum affine algebras using a new method. The Hopf algebra structure for Drinfeld generators is given for both untwisted and twisted two-parameter quantum affine algebras, which include the quantum affine algebras as special cases.

  10. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  11. Dorsal column stimulator applications

    PubMed Central

    Yampolsky, Claudio; Hem, Santiago; Bendersky, Damián

    2012-01-01

    Background: Spinal cord stimulation (SCS) has been used to treat neuropathic pain since 1967. Following that, technological progress, among other advances, helped SCS become an effective tool to reduce pain. Methods: This article is a non-systematic review of the mechanism of action, indications, results, programming parameters, complications, and cost-effectiveness of SCS. Results: In spite of the existence of several studies that try to prove the mechanism of action of SCS, it still remains unknown. The mechanism of action of SCS would be based on the antidromic activation of the dorsal column fibers, which activate the inhibitory interneurons within the dorsal horn. At present, the indications of SCS are being revised constantly, while new applications are being proposed and researched worldwide. Failed back surgery syndrome (FBSS) is the most common indication for SCS, whereas, the complex regional pain syndrome (CRPS) is the second one. Also, this technique is useful in patients with refractory angina and critical limb ischemia, in whom surgical or endovascular treatment cannot be performed. Further indications may be phantom limb pain, chronic intractable pain located in the head, face, neck, or upper extremities, spinal lumbar stenosis in patients who are not surgical candidates, and others. Conclusion: Spinal cord stimulation is a useful tool for neuromodulation, if an accurate patient selection is carried out prior, which should include a trial period. Undoubtedly, this proper selection and a better knowledge of its underlying mechanisms of action, will allow this cutting edge technique to be more acceptable among pain physicians. PMID:23230533

  12. An Undergraduate Column Chromatography Experiment.

    ERIC Educational Resources Information Center

    Danot, M.; And Others

    1984-01-01

    Background information, list of materials needed, and procedures used are provided for an experiment designed to introduce undergraduate students to the theoretical and technical aspects of column chromatography. The experiment can also be shortened to serve as a demonstration of the column chromatography technique. (JN)

  13. Analyze distillation columns with thermodynamics

    SciTech Connect

    Ognisty, T.P. )

    1995-02-01

    In a distillation column, heat supplies the work for separating the components of a feed stream into products. Distillation columns consume some 95% of the total energy used in separations. This amounts to roughly 3% of the energy consumed in the US. Since distillation is so energy intensive and requires significant capital outlays, an endless quest to improve the economics has continued since the beginning of the industry. By analyzing the thermodynamics of a distillation column, an engineer can quantify the thermodynamic efficiency of the process, identify the regions where energy can be better utilized, and define the minimum targets for energy consumption. This article reviews the principles of distillation column thermodynamics and outlines the analysis of lost work profiles and column heat profiles. It then illustrates these concepts through three examples.

  14. Mush Column Magma Chambers

    NASA Astrophysics Data System (ADS)

    Marsh, B. D.

    2002-12-01

    Magma chambers are a necessary concept in understanding the chemical and physical evolution of magma. The concept may well be similar to a transfer function in circuit or time series analysis. It does what needs to be done to transform source magma into eruptible magma. In gravity and geodetic interpretations the causative body is (usually of necessity) geometrically simple and of limited vertical extent; it is clearly difficult to `see' through the uppermost manifestation of the concentrated magma. The presence of plutons in the upper crust has reinforced the view that magma chambers are large pots of magma, but as in the physical representation of a transfer function, actual magma chambers are clearly distinct from virtual magma chambers. Two key features to understanding magmatic systems are that they are vertically integrated over large distances (e.g., 30-100 km), and that all local magmatic processes are controlled by solidification fronts. Heat transfer considerations show that any viable volcanic system must be supported by a vertically extensive plumbing system. Field and geophysical studies point to a common theme of an interconnected stack of sill-like structures extending to great depth. This is a magmatic Mush Column. The large-scale (10s of km) structure resembles the vertical structure inferred at large volcanic centers like Hawaii (e.g., Ryan et al.), and the fine scale (10s to 100s of m) structure is exemplified by ophiolites and deeply eroded sill complexes like the Ferrar dolerites of the McMurdo Dry Valleys, Antarctica. The local length scales of the sill reservoirs and interconnecting conduits produce a rich spectrum of crystallization environments with distinct solidification time scales. Extensive horizontal and vertical mushy walls provide conditions conducive to specific processes of differentiation from solidification front instability to sidewall porous flow and wall rock slumping. The size, strength, and time series of eruptive behavior

  15. Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape.

    PubMed

    Xu, Wentao; Huang, Kunlun; Zhao, Heng; Luo, Yunbo

    2005-06-01

    We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.

  16. New AP4B1 mutation in an African-American child associated with intellectual disability

    PubMed Central

    Lamichhane, Dronacharya

    2013-01-01

    Prevalence of intellectual disability (ID) varies from 1–3%. Genetic causes of ID are being increasingly recognized. Although multiple mutations have been identified as a cause of syndromic ID, the genetic etiology of non-syndromic ID is poorly understood. However, more than 100 loci have been mapped that are associated with non-syndromic ID. There have been a couple of reports of AP4B1 gene mutation causing severe intellectual disability, absent speech, shy character, stereotypic laughter, muscular hypotonia that progressed to spastic paraplegia, microcephaly, foot deformity, decreased muscle mass of the lower limbs, inability to walk, and growth retardation. They had structural brain abnormalities and seizures. The reported cases were from Arab families where consanguineous marriage is common. We encountered an African-American child who presented first at the age of 24 mo with language difficulties and was subsequently found to have moderate to severe intellectual disability by standardized tests. Shortly, he started to have seizures and problems with ambulation. Although he was hypotonic at the time of presentation, legs slowly became spastic at the age of 4 yr. After a thorough work up, he was found to have heterozygous mutation in the AP4B1 gene along with another missense mutation in the same gene. There has been no report of mutation in this gene in the North American population. Although AP4B1 typically is said to be an autosomal recessive disease-causing gene, our case is different in the sense that there are two mutations in the same gene one of which has never been reported before and co-exists with a known disease causing mutation. Yet, the phenotype of the case closely resembles those published previously. PMID:27625858

  17. New AP4B1 mutation in an African-American child associated with intellectual disability.

    PubMed

    Lamichhane, Dronacharya

    2013-12-01

    Prevalence of intellectual disability (ID) varies from 1-3%. Genetic causes of ID are being increasingly recognized. Although multiple mutations have been identified as a cause of syndromic ID, the genetic etiology of non-syndromic ID is poorly understood. However, more than 100 loci have been mapped that are associated with non-syndromic ID. There have been a couple of reports of AP4B1 gene mutation causing severe intellectual disability, absent speech, shy character, stereotypic laughter, muscular hypotonia that progressed to spastic paraplegia, microcephaly, foot deformity, decreased muscle mass of the lower limbs, inability to walk, and growth retardation. They had structural brain abnormalities and seizures. The reported cases were from Arab families where consanguineous marriage is common. We encountered an African-American child who presented first at the age of 24 mo with language difficulties and was subsequently found to have moderate to severe intellectual disability by standardized tests. Shortly, he started to have seizures and problems with ambulation. Although he was hypotonic at the time of presentation, legs slowly became spastic at the age of 4 yr. After a thorough work up, he was found to have heterozygous mutation in the AP4B1 gene along with another missense mutation in the same gene. There has been no report of mutation in this gene in the North American population. Although AP4B1 typically is said to be an autosomal recessive disease-causing gene, our case is different in the sense that there are two mutations in the same gene one of which has never been reported before and co-exists with a known disease causing mutation. Yet, the phenotype of the case closely resembles those published previously. PMID:27625858

  18. 75 FR 41871 - International Conference on Harmonisation; Draft Guidance on Q4B Evaluation and Recommendation of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-19

    ... Register of February 21, 2008 (73 FR 9575). Once finalized, the annex will provide guidance to assist... Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the International Conference on... availability of a draft guidance entitled ``Q4B Evaluation and Recommendation of Pharmacopoeial Texts for...

  19. The predominant species of nonstructural protein 4B in hepatitis C virus-replicating cells is not palmitoylated.

    PubMed

    Paul, David; Bartenschlager, Ralf; McCormick, Christopher

    2015-07-01

    Hepatitis C virus (HCV) represents a significant global health burden. Viral replication is thought to occur in close association with remodelled host cell membranes, with non-structural protein 4B (NS4B) being a key player in this process. NS4B is a poorly characterized integral membrane protein, which has been reported to be palmitoylated at its carboxy-terminal end. In order to extend this observation and to establish a functional role for NS4B palmitoylation, we sought to determine the status of this post-translational modification when the protein was expressed as part of a functional viral replicase. We performed direct metabolic labelling and polyethylene glycol-maleimide palmitoylation reporter assays on NS4B expressed in cells containing subgenomic replicons and infectious viral RNA. In a vaccinia virus-based expression system NS4B palmitoylation was detected in a genotype-dependent manner. However, in spite of the high sensitivity of the methods used, no NS4B palmitoylation was found in physiologically more relevant systems. Thus, NS4B palmitoylation is most likely dispensable for HCV RNA replication. PMID:25740959

  20. Multicomponent Dipolar Cycloaddition Strategy: Combinatorial Synthesis of Novel Spiro-Tethered Pyrazolo[3,4-b]quinoline Hybrid Heterocycles.

    PubMed

    Sumesh, Remani Vasudevan; Muthu, Muthumani; Almansour, Abdulrahman I; Suresh Kumar, Raju; Arumugam, Natarajan; Athimoolam, S; Jeya Yasmi Prabha, E Arockia; Kumar, Raju Ranjith

    2016-05-01

    The stereoselective syntheses of a library of novel spiro-tethered pyrazolo[3,4-b]quinoline-pyrrolidine/pyrrolothiazole/indolizine-oxindole/acenaphthene hybrid heterocycles have been achieved through the 1,3-dipolar cycloaddition of azomethine ylides generated in situ from α-amino acids and 1,2-diketones to dipolarophiles derived from pyrazolo[3,4-b]quinoline derivatives.

  1. The predominant species of nonstructural protein 4B in hepatitis C virus-replicating cells is not palmitoylated

    PubMed Central

    Paul, David

    2015-01-01

    Hepatitis C virus (HCV) represents a significant global health burden. Viral replication is thought to occur in close association with remodelled host cell membranes, with non-structural protein 4B (NS4B) being a key player in this process. NS4B is a poorly characterized integral membrane protein, which has been reported to be palmitoylated at its carboxy-terminal end. In order to extend this observation and to establish a functional role for NS4B palmitoylation, we sought to determine the status of this post-translational modification when the protein was expressed as part of a functional viral replicase. We performed direct metabolic labelling and polyethylene glycol-maleimide palmitoylation reporter assays on NS4B expressed in cells containing subgenomic replicons and infectious viral RNA. In a vaccinia virus-based expression system NS4B palmitoylation was detected in a genotype-dependent manner. However, in spite of the high sensitivity of the methods used, no NS4B palmitoylation was found in physiologically more relevant systems. Thus, NS4B palmitoylation is most likely dispensable for HCV RNA replication. PMID:25740959

  2. Microbial activity in weathering columns.

    PubMed

    García, C; Ballester, A; González, F; Blázquez, M L

    2007-03-22

    The aim of the present work was to evaluate the metabolic activity of the microbial population associated with a pyritic tailing after a column-weathering test. For this purpose, a column 150cm high and 15cm diameter was used. The solid was a tailing with 63.4% pyrite and with minor amounts of Cu, Pb and Zn sulfides (1.4, 0.5 and 0.8%, respectively). The column model was the habitual one for weathering tests: distilled water was added at the top of the column; the water flowed down through tailings and finally was collected at the bottom for chemical and microbiological analysis. Weathering was maintained for 36 weeks. The results showed a significant presence of microbial life that was distributed selectively over the column: sulfur- and iron-oxidizing aerobic bacteria were in the more oxygenated zone; anaerobic sulfur-reducing bacteria were isolated from the samples taken from the anoxic part of the column. Activity testing showed that (oxidizing and reducing) bacteria populations were active at the end of the weathering test. The quality of the water draining from the column was thus the final product of biological oxidation and reduction promoted by the bacteria consortia.

  3. Itinerant ferromagnetism and superconductivity in bct ErRh/sub 4/B/sub 4/

    SciTech Connect

    Chaussy, J.; Genicon, J.L.; Lejay, P.; Odin, J.; Shao, L.Q.; Sulpice, A.; Tournier, R.; Chevalier, B.; Etourneau, J.

    1985-04-15

    The body centered tetragonal ErRh/sub 4/B/sub 4/ compound undergoes an itinerant ferromagnetic transition at T/sub m/ = 21 K characterized by a susceptibility equal to the inverse of the demagnetizing coefficient. The itinerant ferromagnetism does not prevent the simultaneous presence of the superconductivity from 7.7 K down to 0 K. The diamagnetic ac susceptibility can be calculated from the ferromagnetic one. The coexistence of itinerant ferromagnetism and superconductivity among d electrons would be one of the most convincing arguments in favor of ''odd-parity'' superconductivity.

  4. Yersiniosis due to infection by Yersinia pseudotuberculosis 4b in captive meerkats (Suricata suricatta) in Japan.

    PubMed

    Nakamura, Shin-Ichi; Hayashidani, Hideki; Yonezawa, Aya; Suzuki, Isao; Une, Yumi

    2015-09-01

    Two meerkats (Suricata suricatta) housed in the same zoological garden in Japan died due to Yersinia pseudotuberculosis serotype 4b infection. Gross and microscopic lesions included necrotizing enteritis and enlargement of the spleen and liver with multifocal necrosis. Inflammatory cells, primarily neutrophils, and nuclear debris were associated with clusters of Gram-negative bacilli. Additionally, there were aberrant organism forms that were larger than bacilli and appeared as basophilic globular bodies. Immunohistochemical examination showed that the bacilli and globular bodies were strongly positive for Y. pseudotuberculosis O4 antigen. The globular bodies were considered a shape-changed form of Y. pseudotuberculosis, and these morphologically abnormal bacteria could present a diagnostic challenge.

  5. Crystal structure features in a new compound C4B25Mg1.42

    NASA Astrophysics Data System (ADS)

    Konovalikhin, S. V.; Ponomarev, V. I.

    2015-09-01

    The composition of C4B25Mg1.42 crystal obtained by self-propagating high-temperature synthesis was determined using X-ray diffraction. This is the first crystalline structure where all boron atoms in the В12 icosahedron occupy crystallographically independent positions; this circumstance allowed us to analyze the effect of substituents on bond lengths in the icosahedron. The crystal structure features, including the channels filled with disordered Mg atoms and the spread of В—В endo- and exo-bond lengths in the icosahedra, are described. A crystallochemical analysis of pair bonds has been performed for the first time.

  6. Input files with ORNL—mathematical phantoms of the human body for MCNP-4B

    NASA Astrophysics Data System (ADS)

    Krstić, D.; Nikezić, D.

    2007-01-01

    Protection against ionizing radiation requires information on the absorbed doses in organs of the human body. Implantation of many dosimeters in the human body is undesirable (or impossible), so the doses in organs are not measurable and some kind of dose calculation has to be applied. Calculation of doses in organs requests: (a) an exact description of the geometry of organs, (b) the chemical constitution of tissues, and (c) appropriate computer programs. The first two items, (a) and (b), make a so-called "phantom". In another words, the "phantom of a human body" is a mathematical representation of the human body including all other relevant information. All organs are represented with geometrical bodies (like cylinders, ellipsoids, tori, cones etc.), which are described with suitable mathematical equations. A corresponding chemical constitution for various types of organ tissues is also defined. MCNP-4B ( Monte Carlo N- Particle) is often used as transport code. Users of this software prepare an "input file" providing all necessary information for program execution. This information includes: (a) source definition—type of ionizing radiation, energy spectrum, and geometry of the source; (b) target definition—material constitution, geometry, location in respect to the source etc.; (c) characterization of absorbing media between the source and target; (d) output tally, etc. This paper presents input files with "human phantoms" for the MCNP-4B code. The input files with "phantoms" were prepared based on publications issued by the Oak Ridge National Laboratory (ORNL). Seven input files relating to different age groups (newborn, 1, 5, 10, 15 years, as well as, male and female adults) are presented here. A test example and comparison with other data found in literature are also given. Program summaryTitle of program: INPUT FILES, AMALE, AFEMALE, AGE15, AGE10, AGE5, AGE01, NEWB Catalogue identifier:ADYF_v1_0 Program summary URL

  7. Complete Genome Sequence of the Larvicidal Bacterium Lysinibacillus sphaericus Strain OT4b.25

    PubMed Central

    Rey, Andrés; Silva-Quintero, Laura

    2016-01-01

    Lysinibacillus sphaericus OT4b.25 is a native Colombian strain isolated from coleopteran larvae in an oak forest near Bogotá D.C.; this strain has shown high levels of pathogenic activity against Culex quinquefasciatus larvae in laboratory assays compared to that of other members of the same species. Using Pacific Biosciences sequencing technology, we propose a chromosomal contig of 4,665,775 bp that, according to comparative analysis, is highly similar to that of reference strain L. sphaericus C3-41. PMID:27151786

  8. Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns?

    PubMed

    Jones, Andrew; Pravadali-Cekic, Sercan; Dennis, Gary R; Shalliker, R Andrew

    2015-08-19

    Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL. A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance. Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column. PMID:26343427

  9. Optimal design of thermally coupled distillation columns

    SciTech Connect

    Duennebier, G.; Pantelides, C.C.

    1999-01-01

    This paper considers the optimal design of thermally coupled distillation columns and dividing wall columns using detailed column models and mathematical optimization. The column model used is capable of describing both conventional and thermally coupled columns, which allows comparisons of different structural alternatives to be made. Possible savings in both operating and capital costs of up to 30% are illustrated using two case studies.

  10. Identification of Amino Acid Determinants in CYP4B1 for Optimal Catalytic Processing of 4-Ipomeanol

    PubMed Central

    Wiek, Constanze; Schmidt, Eva M; Roellecke, Katharina; Freund, Marcel; Nakano, Mariko; Kelly, Edward J; Kaisers, Wolfgang; Yarov-Yarovoy, Vladimir; Kramm, Christof M; Rettie, Allan E; Hanenberg, Helmut

    2014-01-01

    Mammalian CYP4B1 enzymes are cytochrome P450 monooxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast to the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine at position 427 is unable to bio-activate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYB4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B–C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans. PMID:25247810

  11. One-step purification of lactoperoxidase from bovine milk by affinity chromatography.

    PubMed

    Atasever, Ali; Ozdemir, Hasan; Gulcin, Ilhami; Irfan Kufrevioglu, O

    2013-01-15

    Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

  12. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  13. Engineering antibody affinity and specificity.

    PubMed

    Webster, D M; Roberts, S; Cheetham, J C; Griest, R; Rees, A R

    1988-01-01

    A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed. PMID:3209295

  14. Proton affinities of hydrated molecules

    NASA Astrophysics Data System (ADS)

    Valadbeigi, Younes

    2016-09-01

    Proton affinities (PA) of non-hydrated, M, and hydrated forms, M(H2O)1,2,3, of 20 organic molecules including alcohols, ethers, aldehydes, ketones and amines were calculated by the B3LYP/6-311++G(d,p) method. For homogeneous families, linear correlations were observed between PAs of the M(H2O)1,2,3 and the PAs of the non-hydrated molecules. Also, the absolute values of the hydration enthalpies of the protonated molecules decreased linearly with the PAs. The correlation functions predicted that for an amine with PA < 1100 kJ/mol the PA(M(H2O)) is larger than the corresponding PA, while for an amine with PA > 1100 kJ/mol the PA(M(H2O)) is smaller than the PA.

  15. High speed immuno-affinity chromatography on supports with gigapores and porous glass.

    PubMed

    Schuste, M; Wasserbauer, E; Neubauer, A; Jungbauer, A

    2000-01-01

    Immuno-affinity chromatography exploiting the Ca2+ dependent interaction of the anti-Flag antibody and Flag-tagged proteins has been investigated. The antibody has been immobilized on porous glass beads (Prosep) containing gigapores and on a monolith, the polymethacrylate based Convective Interactive Media (CIM) column at a ligand density of 2 mg/g and 10 mg/ml respectively. The performance of the columns was assessed by applying clarified yeast culture supernatant containing overexpressed Flag-human serum albumin. Dynamic binding capacity and purity was checked at various flow rates ranging from 100 cm/h to 800 cm/h. 95% purity could be obtained. Anti Flag-CIM columns showed a higher unspecific adsorption, requiring a longer wash cycle to obtain the same purity compared to the Prosep column. Anti Flag-CIM columns showed a flow independent performance, which is explained by its monolithic structure. A decreasing dynamic binding capacity with flow was observed with anti-Flag-Prosep columns. Both columns are suited to purify milligrams of protein out of a yeast culture supernatant within a few minutes. We considered them as promising candidates for high throughput screening, where fast purification is a necessity.

  16. Univalent and Bivalent Ligands of Butorphan: Characteristics of the Linking Chain determine the Affinity and Potency of Such Opioid Ligands†

    PubMed Central

    Decker, Michael; Fulton, Brian S.; Zhang, Bin; Knapp, Brian I.; Bidlack, Jean M.; Neumeyer, John L.

    2009-01-01

    Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the μ, δ, and κ opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer-length and structure show potent binding profiles with the most potent compound (4b) having Ki values of 0.47 nM for both the μ and κ opioid receptors, and 4a having Ki values of 0.95 and 0.62 nM for the μ and κ receptors, respectively. Both 4a and 4b were partial agonists at the κ and μ receptors in the [35S]GTPγS binding assay. PMID:19634902

  17. Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes.

    PubMed

    Hodgson, D A

    2000-01-01

    This is the first report of generalized transduction in the gram-positive, food-borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37 degrees C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well-characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (phiLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(phiCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.

  18. Self-regenerating column chromatography

    DOEpatents

    Park, Woo K.

    1995-05-30

    The present invention provides a process for treating both cations and anions by using a self-regenerating, multi-ionic exchange resin column system which requires no separate regeneration steps. The process involves alternating ion-exchange chromatography for cations and anions in a multi-ionic exchange column packed with a mixture of cation and anion exchange resins. The multi-ionic mixed-charge resin column works as a multi-function column, capable of independently processing either cationic or anionic exchange, or simultaneously processing both cationic and anionic exchanges. The major advantage offered by the alternating multi-function ion exchange process is the self-regeneration of the resins.

  19. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, K.S.

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  20. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  1. LIQUID-LIQUID EXTRACTION COLUMNS

    DOEpatents

    Thornton, J.D.

    1957-12-31

    This patent relates to liquid-liquid extraction columns having a means for pulsing the liquid in the column to give it an oscillatory up and down movement, and consists of a packed column, an inlet pipe for the dispersed liquid phase and an outlet pipe for the continuous liquid phase located in the direct communication with the liquid in the lower part of said column, an inlet pipe for the continuous liquid phase and an outlet pipe for the dispersed liquid phase located in direct communication with the liquid in the upper part of said column, a tube having one end communicating with liquid in the lower part of said column and having its upper end located above the level of said outlet pipe for the dispersed phase, and a piston and cylinder connected to the upper end of said tube for applying a pulsating pneumatic pressure to the surface of the liquid in said tube so that said surface rises and falls in said tube.

  2. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  3. Non-affine deformations in polymer hydrogels

    PubMed Central

    Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

    2012-01-01

    Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

  4. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    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.

  5. Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution

    PubMed Central

    Parkinson, Oliver T.; Roellecke, Katharina; Freund, Marcel; Gombert, Michael; Lottmann, Nadine; Steward, Charles A.; Kramm, Christof M.; Yarov-Yarovoy, Vladimir; Rettie, Allan E.; Hanenberg, Helmut

    2015-01-01

    CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5–exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution. PMID:26355749

  6. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  7. Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair.

    PubMed

    Yu, Jun; Fernández-Hernando, Carlos; Suarez, Yajaira; Schleicher, Michael; Hao, Zhengrong; Wright, Paulette L; DiLorenzo, Annarita; Kyriakides, Themis R; Sessa, William C

    2009-10-13

    Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control. PMID:19805174

  8. A new experiment station on beamline 4B7A at Beijing Synchrotron Radiation Facility

    NASA Astrophysics Data System (ADS)

    Zheng, L.; Zhao, Y. D.; Tang, K.; Ma, C. Y.; Hong, C. H.; Han, Y.; Cui, M. Q.; Guo, Z. Y.

    2014-11-01

    A new experiment station was installed on beamline 4B7A at Beijing Synchrotron Radiation Facility (BSRF), making it possible to record X-ray absorption fine structure (XAFS) spectrum in three modes over an energy range from 1750 eV to 6000 eV. A 13-element Si(Li) array detector and a single-element SDD detector were used to acquire data in partial fluorescence yield (PFY) mode. Two low-pressure noble gas ion chambers were adopted for measuring XAFS in transmission mode. In total electron yield (TEY) mode the current of sample is recorded. Solid, wet and liquid samples were suitable for this experimental station. Some representative results obtained from this station were shown and discussed.

  9. BOREAS Level-4b AVHRR-LAC Ten-Day Composite Images: At-sensor Radiance

    NASA Technical Reports Server (NTRS)

    Cihlar, Josef; Chen, Jing; Nickerson, Jaime; Newcomer, Jeffrey A.; Huang, Feng-Ting; Hall, Forrest G. (Editor)

    2000-01-01

    The BOReal Ecosystem-Atmosphere Study (BOREAS) Staff Science Satellite Data Acquisition Program focused on providing the research teams with the remotely sensed satellite data products they needed to compare and spatially extend point results. Manitoba Remote Sensing Center (MRSC) and BOREAS Information System (BORIS) personnel acquired, processed, and archived data from the Advanced Very High Resolution Radiometer (AVHRR) instruments on the National Oceanic and Atmospheric Administration (NOAA-11) and -14 satellites. The AVHRR data were acquired by CCRS and were provided to BORIS for use by BOREAS researchers. These AVHRR level-4b data are gridded, 10-day composites of at-sensor radiance values produced from sets of single-day images. Temporally, the 10- day compositing periods begin 11-Apr-1994 and end 10-Sep-1994. Spatially, the data cover the entire BOREAS region. The data are stored in binary image format files.

  10. Yersiniosis due to infection by Yersinia pseudotuberculosis 4b in captive meerkats (Suricata suricatta) in Japan.

    PubMed

    Nakamura, Shin-Ichi; Hayashidani, Hideki; Yonezawa, Aya; Suzuki, Isao; Une, Yumi

    2015-09-01

    Two meerkats (Suricata suricatta) housed in the same zoological garden in Japan died due to Yersinia pseudotuberculosis serotype 4b infection. Gross and microscopic lesions included necrotizing enteritis and enlargement of the spleen and liver with multifocal necrosis. Inflammatory cells, primarily neutrophils, and nuclear debris were associated with clusters of Gram-negative bacilli. Additionally, there were aberrant organism forms that were larger than bacilli and appeared as basophilic globular bodies. Immunohistochemical examination showed that the bacilli and globular bodies were strongly positive for Y. pseudotuberculosis O4 antigen. The globular bodies were considered a shape-changed form of Y. pseudotuberculosis, and these morphologically abnormal bacteria could present a diagnostic challenge. PMID:26179097

  11. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed.

  12. Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography

    PubMed Central

    Mousavi Hosseini, Kamran; Nasiri, Saleh

    2015-01-01

    Background: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. Methods: PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Results: Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). Results of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). Conclusion: It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII. PMID:26034723

  13. Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B

    PubMed Central

    Rao, Minxi; Smith, Brian C.

    2015-01-01

    ABSTRACT Nitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis. PMID:25944856

  14. Response of Atmospheric-Methane Oxidation to Methane-Flux Manipulation in a Laboratory Soil-Column Experiment

    NASA Astrophysics Data System (ADS)

    Schroth, M. H.; Mignola, I.; Henneberger, R.

    2015-12-01

    Upland soils are an important sink for atmospheric methane (CH4). Uptake of atmospheric CH4 in soils is generally diffusion limited, and is mediated by aerobic CH4 oxidizing bacteria (MOB) that possess a high-affinity form of a key enzyme, allowing CH4 consumption at near-atmospheric concentrations (≤ 1.9 µL/L). As cultivation attempts for these high-affinity MOB have shown little success, there remains much speculation regarding their functioning in different environments. For example, it is frequently assumed that they are highly sensitive to physical disturbance, but their response in activity and abundance to changes in substrate availability remains largely unknown. We present results of a laboratory column experiment conducted to investigate the response in activity and abundance of high-affinity MOB to an increase in CH4 flux. Intact soil cores, collected at a field site where atmospheric CH4 oxidation activity is frequently quantified, were transferred into two 1-m-long, 12-cm-dia. columns. The columns were operated at constant temperature in the dark, their headspace being continuously flushed with air. Diffusive gas-transport conditions were maintained in the reference column, whereas CH4 flux was increased in several steps in the treatment column by inducing advective gas flow using a diaphragm pump. Soil-gas samples periodically collected from ports installed along the length of the columns were analyzed for CH4 content. Together with measurements of soil-water content, atmospheric CH4 oxidation was quantified using the soil-profile method. First results indicate that atmospheric CH4 oxidation activity comparable with the field was maintained in the reference column throughout the experiment. Moreover, high-affinity MOB quickly adjusted to an increase in CH4 flux in the treatment column, efficiently consuming CH4. Quantification of MOB abundance is currently ongoing. Our data provide new insights into controls on atmospheric CH4 oxidation in soils.

  15. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction.

    PubMed

    Karpol, Alon; Kantorovich, Lia; Demishtein, Alik; Barak, Yoav; Morag, Ely; Lamed, Raphael; Bayer, Edward A

    2009-01-01

    Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. PMID:18979459

  16. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  17. Dependence on vitamin K-dependent protein S for eukaryotic cell secretion of the beta-chain of C4b-binding protein.

    PubMed

    Carlsson, Sofia; Dahlbäck, Björn

    2010-10-15

    The anticoagulant vitamin K-dependent protein S (PS) circulates in plasma in two forms, 30% free and 70% being bound to the complement regulatory protein C4b-binding protein (C4BP). The major C4BP isoform consists of 7 α-chains and 1 β-chain (C4BPβ(+)), the chains being linked by disulfide bridges. PS binds to the β-chain with high affinity. In plasma, PS is in molar excess over C4BPβ(+) and due to the high affinity, all C4BPβ(+) molecules contain a bound PS. Taken together with the observation that PS-deficient patients have decreased levels of C4BPβ(+), this raises the question of whether PS is important for secretion of the β-chain from the cell. To test this hypothesis, HEK293 cells were stably and transiently transfected with β-chain cDNA in combinations with cDNAs for PS and/or the α-chain. The concentration of β-chains in the medium increased after co-transfection with PS cDNA, but not by α-chain cDNA, suggesting secretion of the β-chains from the cells to be dependent on concomitant synthesis of PS, but not of the α-chains. Thus, β-chains that were not disulfide-linked to the α-chains were secreted in complex with PS, either as monomers or dimers. Pulse-chase demonstrated that the complexes between PS and β-chain were formed intracellularly, in the endoplasmic reticulum. In conclusion, our results demonstrate that successful secretion of β-chains depends on intracellular complex formation with PS, but not on the α-chains. This provides an explanation for the decreased β-chain levels observed in PS-deficient patients.

  18. Magnetic anisotropy and crystalline electric field effects in RRh{sub 4}B{sub 4} single crystals.

    SciTech Connect

    Zhou, H.; Lambert, S. E.; Maple, M. B.; Dunlap, B. D.; Materials Science Division; Univ. of California at San Diego

    2009-08-01

    Research on polycrystalline RRh{sub 4}B{sub 4} samples has shown that crystalline electric field (CEF) effects play an important role in these compounds. The successful synthesis of single crystal samples of RRh{sub 4}B{sub 4} with R = Y, Sm, Gd, Tb, Dy, Ho, Er, Tm, and Lu has provided an opportunity to further investigate CEF effects in these materials. Magnetization and magnetic susceptibility measurements on the RRh{sub 4}B{sub 4} single crystals revealed strong magnetic anisotropy, and the experimental results could be described well by CEF calculations based on the parameters derived from an analysis of experimental data for ErRh{sub 4}B{sub 4} single crystals. The easy directions of magnetization of these compounds are consistent with the signs of the Stevens factor {alpha}J of the CEF Hamiltonian. A strong influence of magnetic anisotropy on superconductivity was also observed.

  19. Evaluation of an immunoaffinity extraction column for enrichment of adducts between human serum albumin and hexahydrophthalic anhydride in plasma.

    PubMed

    Johannesson, Gunvor A; Kristiansson, Monica H; Jönsson, Bo A G; Lindh, Christian H

    2008-03-01

    An immunoaffinity extraction (IAE) column was prepared for extraction of adducts between human serum albumin (HSA) and hexahydrophthalic anhydride (HHPA). HHPA is a strong sensitizer inducing immunoglobulin E antibodies in vivo. Polyclonal antibodies from a rabbit immunized with keyhole limpet hemocyananin-HHPA conjugate were purified using a Protein A Sepharose gel. To obtain antibodies with optimal affinity towards HHPA-protein adducts, HHPA-specific antibodies were selected using an N-hydroxysuccinimide-Sepharose column coupled with albumin-HHPA conjugate. Antibodies eluted from this column at pH 2.2 were selected to prepare the IAE column. The column was evaluated using 2 mL plasma spiked with HSA-HHPA conjugate. The column was eluted with glycine buffer at pH 2.0. The conjugates in the eluate were hydrolyzed to the corresponding HHP acid and quantified by mass spectrometry. The average recovery of HHPA adducts in 11 experiments was 68% with a coefficient of variation (CV) of 7%. The column's capacity to bind protein-HHPA adducts was found to be linear in the range of 0.15-1.2 nmol conjugate. The evaluation showed that the IAE column had adequate affinity towards the HHPA adducts and that the adducts could be extracted with good recovery and precision from a large volume of plasma.

  20. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    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.

  1. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  2. Sesuvium portulacastrum (L.) L.: a potential halophyte for the degradation of toxic textile dye, Green HE4B.

    PubMed

    Patil, Asmita V; Lokhande, Vinayak H; Suprasanna, Penna; Bapat, Vishwas A; Jadhav, Jyoti P

    2012-05-01

    Sesuvium portulacastrum is a common halophyte growing well in adverse surroundings and is exploited mainly for the environmental protection including phytoremediation, desalination and stabilization of contaminated soil. In the present investigation, attempts have been made on the decolorization of a toxic textile dye Green HE4B (GHE4B) using in vitro grown Sesuvium plantlets. The plantlets exhibited significant (70%) decolorization of GHE4B (50 mg l(-1)) that sustain 200 mM sodium chloride (NaCl) within 5 days of incubation. The enzymatic analysis performed on the root and shoot tissues of the in vitro plantlets subjected to GHE4B decolorization in the presence of 200 mM NaCl showed a noteworthy induction of tyrosinase, lignin peroxidase and NADH-DCIP reductase activities, indicating the involvement of these enzymes in the metabolism of the dye GHE4B. The UV-visible spectrophotometer, HPLC and Fourier Transform Infrared Spectroscopy (FTIR) analyses of the samples before and after decolorization of the dye confirmed the efficient phytotransformation of GHE4B in the presence of 200 mM NaCl. Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of the products revealed the formation of three metabolites such as p -amino benzene, p -amino toluene and 1, 2, 7-amino naphthalene after phytotransformation of GHE4B. Based on the FTIR and GC-MS results, the possible pathway for the biodegradation of GHE4B in the presence of 200 mM NaCl has been proposed. The phytotoxicity experiments confirmed the non-toxicity of the degraded products. The present study demonstrates for the first time the potential of Sesuvium for the efficient degradation of textile dyes and its efficacy on saline soils contaminated with toxic compounds.

  3. Scale-up of native beta-lactoglobulin affinity separation process.

    PubMed

    Vyas, H K; Izco, J M; Jiménez-Flores, R

    2002-07-01

    Affinity separation of beta-lactoglobulin in its native form with all-trans-retinal immobilized on calcium bio-silicate was scaled up and applied to separate it from industrial sweet whey. Three different methods of mixing the modified calcium bio-silicate and whey for the interaction between all-trans-retinal and beta-lactoglobulin were tried at pilot scale. The three methods used were 1) a column packed with calcium bio-silicate, 2) a stirred tank, and 3) a fluidized bed column of calcium bio-silicate particles. Adsorption and desorption of beta-lactoglobulin were carried out at pH 5.1 and 7.0, using 0.01 and 0.1 M phosphate buffers, respectively. The phosphate buffer containing desorbed beta-lactoglobulin was concentrated 20 times using ultrafiltration and then freeze-dried. The packed column, stirred tank, and fluidized bed column produced beta-lactoglobulin with purity of 80, >95, and >95%, and recovery of 0.65, 2.88, and 2.88 g per kilogram of calcium bio-silicate, respectively. The comparative poor purity and recovery of beta-lactoglobulin in the case of the packed column was attributed to insufficient contact between the passing fluids and the calcium bio-silicate during adsorption, desorption, and intermittent washing. The fluidized bed column method, with a gentle mixing action, was considered the best suited for further scale up to the industrial level. PMID:12201513

  4. Purification of the hexokinases by affinity chromatography on sepharose-N-aminoacylglucosamine derivates. Design of affinity matrices from free solution kinetics.

    PubMed Central

    Wright, C L; Warsy, A S; Holroyde, M J; Trayer, I P

    1978-01-01

    The purification is described of rat hepatic hexokinase type III and kidney hexokinase type I on a large scale by using a combination of conventional and affinity techniques similar to those previously used for the purification of rat hepatic glucokinase [Holroyde, Allen, Storer, Warsy, Chesher, Trayer, Cornish-Bowden & Walker (1976) Biochem. J. 153, 363-373] and muscle hexokinase type II [Holroyde & Trayer (1976) FEBS Lett. 62, 215-219]. The key to each purification was the use of a Sepharose-N-aminoacylglucosamine affinity matrix in which a high degree of specificity for a particular hexokinase isoenzyme could be introduced by either varying the length of the aminoacyl spacer and/or varying the ligand concentration coupled to the gel. This was predicted from a study of the free solution kinetic properties of the various N-aminoacylglucosamine derivatives used (N-aminopropionyl, N-aminobutyryl, N-aminohexanoyl and N-aminooctanoyl), synthesized as described by Holroyde, Chesher, Trayer & Walker [(1976) Biochem. J. 153, 351-361]. All derivatives were competitive inhibitors, with respect to glucose, of the hexokinase reaction, and there was a direct correlation between the Ki for a particular derivative and its ability to act as an affinity matrix when immobilized to CNBr-activated Sepharose 4B. Muscle hexokinase type II could be chromatographed on the Sepharose conjugates of all four N-aminoacylglucosamine derivatives, although the N-aminohexanoylglucosamine derivative proved best. This same derivative was readily able to bind hepatic glucokinase and hexokinase type III, but Sepharose-N-amino-octanoyl-glucosamine was better for these enzymes and was the only derivative capable of binding kidney hexokinase type I efficiently. Separate studies with yeast hexokinase showed that again only the Sepharose-N-amino-octanoylglucosamine was capable of acting as an efficient affinity matrix for this enzyme. Implications of these studies in our understanding of affinity

  5. FRACTIONATING COLUMN PRODUCT COLLECTOR CONTROL

    DOEpatents

    Paxson, G.D. Jr.

    1964-03-10

    Means for detecting minute fluid products from a chemical separation column and for advancing a collector tube rack in order to automatically separate and collect successive fractionated products are described. A charge is imposed on the forming drops at the column orifice to create an electric field as the drop falls in the vicinity of a sensing plate. The field is detected by an electrometer tube coupled to the plate causing an output signal to actuate rotation of a collector turntable rack, thereby positioning new collectors under the orifice. The invention provides reliable automatic collection independent of drop size, rate of fall, or chemical composition. (AEC)

  6. Slag columns for upgrading phosphorus removal from constructed wetland effluents.

    PubMed

    Chazarenc, F; Brisson, J; Comeau, Y

    2007-01-01

    The current best option to upgrade constructed wetlands (CWs) for phosphorus (P) retention, in terms of efficiency, cost and simplicity, consists in using media having a strong P affinity. The media can be used either in the planted beds or in a filtration system downstream of the beds. The use of slag filters was shown to be efficient for removing P from wastewater as it represented a slow release source of calcium and hydroxide, favouring the formation of hydroxyapatite. Our study aimed at maximising the P retention capacity of slag filters located at the outlet of CWs since electric arc furnace slag has been shown to inhibit the growth of macrophytes when used in the filtration matrix. Bench-scale columns (Vtot = 6.2 L) filled with various combinations of filter media (slag, granite, limestone) of different sizes (2-5, 5-10, 10-20 mm) were fed on-site during four months with a CW effluent (in mg/L: 30 COD, 30 TSS, 10 Pt). Results showed that the best media combination enabling the maximum o-PO4 retention (more than 80% removal without clogging) consisted in a series of a ternary mix column (slag 5-10 mm, granite 2-5 mm, limestone 5-10 mm) followed by a slag column (slag 5-10 mm). Pilot scale columns (Vtot = 300 L), filled with the best media combination, were installed at the outlet of a 28 m2 CW. These columns showed more than 75% removal efficiency during one year and were designed to be easily replaced each year.

  7. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    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.

  8. Effect of Heating Rate on Soft Magnetic Properties in Nanocrystalline Fe80.5Cu1.5Si4B14 and Fe82Cu1Nb1Si4B12 Alloys

    NASA Astrophysics Data System (ADS)

    Ohta, Motoki; Yoshizawa, Yoshihito

    2009-02-01

    The effect of the heating rate in the annealing process of rapidly quenched Fe80.5Cu1.5Si4B14 and Fe82Cu1Nb1Si4B12 amorphous alloys on their soft magnetic properties is investigated. The iron loss in magnetic flux density region of B > 1.55 T in Fe80.5Cu1.5Si4B14 alloy decreases by high-heating-rate annealing (HA). The coercivity Hc and the saturation magnetic flux density Bs in HA specimen of Fe82Cu1Nb1Si4B12 alloy were about 3 A/m and 1.78 T. By applying HA to high-Fe-concentration amorphous alloys such as Fe82Cu1Nb1Si4B12, Fe82Cu1Nb1Si2B12P2, and Fe80.8Cu1.2Si5B11P2, the nanocrystalline alloys with a high Bs of more than 1.75 T and a low Hc of about 3 A/m are obtained.

  9. Adsorption and thermal decomposition of 2-octylthieno[3,4-b]thiophene on Au(111).

    PubMed

    Park, Joon B; Zong, Kyukwan; Jeon, Il Chul; Hahn, Jae Ryang; Stacchiola, Dario; Starr, David; Müller, Kathrin; Noh, Jaegeun

    2012-10-15

    The adsorption and thermal stability of 2-octylthieno[3,4-b]thiophene (OTTP) on the Au(111) surfaces have been studied using scanning tunneling microscopy (STM), temperature programmed desorption (TPD), and X-ray photoelectron spectroscopy (XPS). UHV-STM studies revealed that the vapor-deposited OTTP on Au(111) generated disordered adlayers with monolayer thickness even at saturation coverage. XPS and TPD studies indicated that OTTP molecules on Au(111) are stable up to 450 K and further heating of the sample resulted in thermal decomposition to produce H(2) and H(2)S via C-S bond scission in the thieno-thiophene rings. Dehydrogenation continues to occur above 600 K and the molecules were ultimately transformed to carbon clusters at 900 K. Highly resolved air-STM images showed that OTTP adlayers on Au(111) prepared from solution are composed of a well-ordered and low-coverage phase where the molecules lie flat on the surface, which can be assigned as a (9×2√33)R5° structure. Finally, based on analysis of STM, TPD, and XPS results, we propose a thermal decomposition mechanism of OTTP on Au(111) as a function of annealing temperature. PMID:22818203

  10. High-temperature mass spectrometry - Vaporization of group 4-B metal carbides. [using Knudsen effusion

    NASA Technical Reports Server (NTRS)

    Stearns, C. A.; Kohl, F. J.

    1974-01-01

    The high temperature vaporization of the metal-carbon systems TiC, ZrC, HfC, and ThC was studied by the Knudsen effusion - mass spectrometric method. For each system the metal dicarbide and tetracarbide molecular species were identified in the gas phase. Relative ion currents of the carbides and metals were measured as a function of temperature. Second- and third-law methods were used to determine enthalpies. Maximum values were established for the dissociation energies of the metal monocarbide molecules TiC, ZrC, HfC, and ThC. Thermodynamic functions used in the calculations are discussed in terms of assumed molecular structures and electronic contributions to the partition functions. The trends shown by the dissociation energies of the carbides of Group 4B are compared with those of neighboring groups and discussed in relation to the corresponding oxides and chemical bonding. The high temperature molecular beam inlet system and double focusing mass spectrometer are described.

  11. Study of Historical 4B/X17 Mega Flare on 28 October 2003 (P58)

    NASA Astrophysics Data System (ADS)

    Uddin, W.; Chandra, R.; Ali, S. S.

    2006-11-01

    wuddin_99@yahoo.com We analysed multi-wavelength data of 28 October 2003 4B/X17.2 class extremely energetic parallel ribbon solar flare, which occurred in NOAA 10486. The flare was well observed in H-alpha at ARIES, Nainital and various space (SOHO, TRACE, RHESSI, WIND etc.) and ground based Observatories. The H-alpha observations show the stretching/detwisting and eruption of helically twisted S shaped (sigmoid) filament in the South-West direction of the active region with bright shock front followed by rapid increase in intensity and area of the gigantic flare. The flare is associated with a bright/fast full halo earth directed CME, strong type II, III and IV radio bursts, an intense proton event and GLE. It seems that the filament eruption triggered the halo CME because the helical structure is clearly visible in the SOHO/LASCO C2, C3 images. This indicates helicity transfer from chromosphere to corona and interplanetary medium. The magnetic field of the flaring region was most complex with high magnetic shear. From the above analysis we feel that the energy buildup/release process of this unique flare support helically twisted magnetic flux rope model.

  12. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  13. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  14. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

    PubMed Central

    Plog, Stephanie; Klymiuk, Nikolai; Binder, Stefanie; Van Hook, Matthew J.; Thoreson, Wallace B.; Gruber, Achim D.; Mundhenk, Lars

    2015-01-01

    The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype. PMID:26474299

  15. CD4(+)B220(+)TCRγδ(+) T cells produce IL-17 in lupus-prone MRL/lpr mice.

    PubMed

    Qiu, Feng; Li, Tingting; Zhang, Kui; Wan, Jun; Qi, Xiaokun

    2016-09-01

    Systemic lupus erythematosus is an autoimmune disease with comprehensive immune cell disorders. Recent studies suggested that pro-inflammatory cytokine IL-17 plays important role in lupus, leaving the cellular sources and their pathogenic and physiologic characters largely unknown. In the current study, by using lupus-prone MRL/lpr mice, we demonstrated that Th17 response prevails in lupus disease regarding significantly accumulated serum IL-17, increased IL-17-producing splenocytes, and elevated phospho-STAT3 in CD4(+) T cells. Intracellular staining revealed that unusual CD4(+)B220(+) T cells are major IL-17-producing cells, whereas conventional CD4(+)B220(-) T cells are major IFN-γ-producing cells. Subsequent studies showed that CD4(+)B220(+) cells contains both αβ and γδ T cells in the spleen and thymus of MRL/lpr mice. Further study showed that around 60% of γδ T cells in MRL/lpr mice co-express both B220 and CD4 on their surface, and are the major RORγt(+) cells in MRL/lpr mice. Finally, CD4(+)B220(+) T cells alone do not proliferate, but could enhance the proliferation and IFN-γ-production of conventional CD4(+)B220(-) T cells. Our findings suggest the pathogenic role of unusual CD4(+)B220(+) T cells in lupus disease in MRL/lpr mice according to their IL-17-producing ability and stimulatory function for conventional CD4(+)B220(-) T cells. PMID:27235595

  16. Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression.

    PubMed

    Aguilo, Francesca; Zhou, Ming-Ming; Walsh, Martin J

    2011-08-15

    Polycomb group proteins (PcG) function as transcriptional repressors of gene expression. The important role of PcG in mediating repression of the INK4b-ARF-INK4a locus, by directly binding to the long noncoding RNA (lncRNA) transcript antisense noncoding RNA in the INK4 locus (ANRIL), was recently shown. INK4b-ARF-INK4a encodes 3 tumor-suppressor proteins, p15(INK4b), p14(ARF), and p16(INK4a), and its transcription is a key requirement for replicative or oncogene-induced senescence and constitutes an important barrier for tumor growth. ANRIL gene is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases. Although lncRNA-mediated regulation of INK4b-ARF-INK4a gene is not restricted to ANRIL, both polycomb repressive complex-1 (PRC1) and -2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism would provide an increased advantage for bypassing senescence, sustaining the requirements for the proliferation of stem and/or progenitor cell populations or inappropriately leading to oncogenesis through the aberrant saturation of the INK4b-ARF-INK4a locus by PcG complexes. In this review, we summarize recent findings on the underlying epigenetic mechanisms that link PcG function with ANRIL, which impose gene silencing to control cellular homeostasis as well as cancer development. PMID:21828241

  17. Long Noncoding RNA, Polycomb, and the Ghosts Haunting INK4b-ARF-INK4a Expression

    PubMed Central

    Aguilo, Francesca; Zhou, Ming-Ming; Walsh, Martin J.

    2012-01-01

    Polycomb group proteins (PcG) function as transcriptional repressors of gene expression. The important role of PcG in mediating repression of the INK4b-ARF-INK4a locus, by directly binding to the long noncoding RNA (lncRNA) transcript antisense noncoding RNA in the INK4 locus (ANRIL), was recently shown. INK4b-ARF-INK4a encodes 3 tumor-suppressor proteins, p15INK4b, p14ARF, and p16INK4a, and its transcription is a key requirement for replicative or oncogene-induced senescence and constitutes an important barrier for tumor growth. ANRIL gene is transcribed in the antisense orientation of the INK4b-ARF-INK4a gene cluster, and different single-nucleotide polymorphisms are associated with increased susceptibility to several diseases. Although lncRNA-mediated regulation of INK4b-ARF-INK4a gene is not restricted to ANRIL, both polycomb repressive complex-1 (PRC1) and -2 (PRC2) interact with ANRIL to form heterochromatin surrounding the INK4b-ARF-INK4a locus, leading to its repression. This mechanism would provide an increased advantage for bypassing senescence, sustaining the requirements for the proliferation of stem and/or progenitor cell populations or inappropriately leading to oncogenesis through the aberrant saturation of the INK4b-ARF-INK4a locus by PcG complexes. In this review, we summarize recent findings on the underlying epigenetic mechanisms that link PcG function with ANRIL, which impose gene silencing to control cellular homeostasis as well as cancer development. PMID:21828241

  18. Preparation of a boronate-functionalized affinity hybrid monolith for specific capture of glycoproteins.

    PubMed

    Yang, F; Mao, J; He, X W; Chen, L X; Zhang, Y K

    2013-06-01

    A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) reaction of an alkyne-boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne-boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg · g(-1) at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.

  19. Affinity labeling and binding of nitrobenzylthionosine (NBTI) to a membrane fraction (MF) of cultured cell lines

    SciTech Connect

    Woffendin, C.; Plagemann, P.G.W.

    1986-05-01

    Equilibrium binding identified high affinity NBTI binding sites (K/sub D/ = 1-3 nM) on the MF's of L929, L1210, P388, S49 and CHO cells. High affinity NBTI binding sites are associated with the nucleoside transporter since none were present in a MF of a transport-deficient mutant of S49 cells (AE1). MF's of Novikoff cells, like intact Novikoff cells, also lacked high affinity NBTI binding sites. MF's of the cell lines were equilibrium labeled with (/sup 3/H)NBTI using photoaffinity conditions and analyzed by SDS-polyacrylamide gel electrophoresis. Radioactivity was specifically incorporated covalently into a 50-70 Kd protein fraction, but the labeled proteins from CHO and L929 cells had a higher apparent molecular weight than those from S49 and P388 cells. In addition, in MF's from some cell lines lower molecular weight components became photoaffinity labeled. Maximum photoaffinity labeling of the MF proteins was observed with much higher (/sup 3/H)NBTI concentrations (100-200 nM) than those saturating the nucleoside transporter. This finding is explained by a reduced affinity of the photoactivated NBTI intermediate(s) for the transporter. When detergent solubilized MF's from cultured cells were chromotographed on a DEAE cellulose column, only 5-10% of the protein, but practically all high affinity NBTI sites, were recovered in the flow through fraction.

  20. Identification of an AAA ATPase VPS4B-Dependent Pathway That Modulates Epidermal Growth Factor Receptor Abundance and Signaling during Hypoxia

    PubMed Central

    Lin, H. Helen; Li, Xu; Chen, Jo-Lin; Sun, Xiuzhu; Cooper, Fariba Norouziyan; Chen, Yun-Ru; Zhang, Wenyu; Chung, Yiyin; Li, Angela; Cheng, Chun-Ting; Yang, Lixin; Deng, XuTao; Liu, Xiyong; Yen, Yun; Johnson, Deborah L.; Shih, Hsiu-Ming; Yang, Austin

    2012-01-01

    VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4B (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic-hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer. PMID:22252323

  1. Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis.

    PubMed

    Hong, Tingting; Chen, Xueping; Xu, Yujing; Cui, Xiaoqin; Bai, Ruihan; Jin, Can; Li, Ruijun; Ji, Yibing

    2016-07-22

    Novel graphene oxide (GO)-modified affinity capillary monoliths were developed employing human serum albumin (HSA) or pepsin as chiral selector. Three types of amino donors for GO immobilization, including ammonium hydroxide (NH4OH), ethanediamine (EDA) and polyethyleneimine (PEI), were applied to explore the effect of spacer arm on enantioseparation. It was observed that HSA-GO-EDA-based affinity capillary monoliths exhibited better chiral recognition ability in comparison with the other two spacer-based monoliths. Under the optimized conditions, the obtained columns revealed satisfactory repeatability concerning column-to-column, run-to-run and interday repeatability. In addition, the impact of GO concentration on enantiomeric separation was also investigated. HSA-GO-EDA-based affinity capillary monoliths provided higher chiral selectivity for nine pairs of enantiomers compared to the columns without GO. Furthermore, the influence of amino donors and GO on proteolytic activity of pepsin-based immobilized enzymatic reactor (IMER) was discussed. Unfortunately, pepsin-GO-PEI-based affinity capillary monoliths possessed the highest protein digestion capacity, which was different from the effect of amino donors on enantiorecognition. Moreover, GO presented as a favorable choice to improve the enzymatic activity of IMER. These results proved that GO-functionalized affinity capillary monoliths have promising potential for chiral separation and proteolysis.

  2. Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis.

    PubMed

    Hong, Tingting; Chen, Xueping; Xu, Yujing; Cui, Xiaoqin; Bai, Ruihan; Jin, Can; Li, Ruijun; Ji, Yibing

    2016-07-22

    Novel graphene oxide (GO)-modified affinity capillary monoliths were developed employing human serum albumin (HSA) or pepsin as chiral selector. Three types of amino donors for GO immobilization, including ammonium hydroxide (NH4OH), ethanediamine (EDA) and polyethyleneimine (PEI), were applied to explore the effect of spacer arm on enantioseparation. It was observed that HSA-GO-EDA-based affinity capillary monoliths exhibited better chiral recognition ability in comparison with the other two spacer-based monoliths. Under the optimized conditions, the obtained columns revealed satisfactory repeatability concerning column-to-column, run-to-run and interday repeatability. In addition, the impact of GO concentration on enantiomeric separation was also investigated. HSA-GO-EDA-based affinity capillary monoliths provided higher chiral selectivity for nine pairs of enantiomers compared to the columns without GO. Furthermore, the influence of amino donors and GO on proteolytic activity of pepsin-based immobilized enzymatic reactor (IMER) was discussed. Unfortunately, pepsin-GO-PEI-based affinity capillary monoliths possessed the highest protein digestion capacity, which was different from the effect of amino donors on enantiorecognition. Moreover, GO presented as a favorable choice to improve the enzymatic activity of IMER. These results proved that GO-functionalized affinity capillary monoliths have promising potential for chiral separation and proteolysis. PMID:27334417

  3. Chromatographic behaviors of proteins on cation-exchange column.

    PubMed

    Li, Rong; Chen, Guo-Liang; Zhao, Wen-Ming

    2004-12-01

    A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process. The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed. PMID:15689030

  4. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  5. Middle East respiratory syndrome coronavirus ORF4b protein inhibits type I interferon production through both cytoplasmic and nuclear targets

    PubMed Central

    Yang, Yang; Ye, Fei; Zhu, Na; Wang, Wenling; Deng, Yao; Zhao, Zhengdong; Tan, Wenjie

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel and highly pathogenic human coronavirus and has quickly spread to other countries in the Middle East, Europe, North Africa and Asia since 2012. Previous studies have shown that MERS-CoV ORF4b antagonizes the early antiviral alpha/beta interferon (IFN-α/β) response, which may significantly contribute to MERS-CoV pathogenesis; however, the underlying mechanism is poorly understood. Here, we found that ORF4b in the cytoplasm could specifically bind to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKKε), suppress the molecular interaction between mitochondrial antiviral signaling protein (MAVS) and IKKε, and inhibit IFN regulatory factor 3 (IRF3) phosphorylation and subsequent IFN-β production. Further analysis showed that ORF4b could also inhibit IRF3 and IRF7-induced production of IFN-β, whereas deletion of the nuclear localization signal of ORF4b abrogated its ability to inhibit IRF3 and IRF7-induced production of IFN-β, but not IFN-β production induced by RIG-I, MDA5, MAVS, IKKε, and TBK-1, suggesting that ORF4b could inhibit the induction of IFN-β in both the cytoplasm and nucleus. Collectively, these results indicate that MERS-CoV ORF4b inhibits the induction of type I IFN through a direct interaction with IKKε/TBK1 in the cytoplasm, and also in the nucleus with unknown mechanism. Viruses have evolved multiple strategies to evade or thwart a host’s antiviral responses. A novel human coronavirus (HCoV), Middle East respiratory syndrome coronavirus (MERS-CoV), is distinguished from other coronaviruses by its high pathogenicity and mortality. However, virulence determinants that distinguish MERS-CoV from other HCoVs have yet to be identified. MERS-CoV ORF4b antagonizes the early antiviral response, which may contribute to MERS-CoV pathogenesis. Here, we report the identification of the interferon (IFN) antagonism mechanism of MERS-CoV ORF4b. MERS-CoV ORF4b inhibits the production

  6. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  7. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  8. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  9. Designing Chaotic Systems by Piecewise Affine Systems

    NASA Astrophysics Data System (ADS)

    Wu, Tiantian; Li, Qingdu; Yang, Xiao-Song

    Based on mathematical analysis, this paper provides a methodology to ensure the existence of homoclinic orbits in a class of three-dimensional piecewise affine systems. In addition, two chaotic generators are provided to illustrate the effectiveness of the method.

  10. Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

    PubMed

    Aoyama, Akihisa; Kurane, Ryuichiro; Matsuura, Akira; Nagai, Kazuo

    2015-01-01

    An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

  11. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  12. UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation*

    PubMed Central

    Sirisaengtaksin, Natalie; Gireud, Monica; Yan, Qing; Kubota, Yoshihisa; Meza, Denisse; Waymire, Jack C.; Zage, Peter E.; Bean, Andrew J.

    2014-01-01

    The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR. PMID:24344129

  13. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  14. Transforming growth factor beta stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclin D1-cdk4 association in human mammary epithelial cells.

    PubMed Central

    Sandhu, C; Garbe, J; Bhattacharya, N; Daksis, J; Pan, C H; Yaswen, P; Koh, J; Slingerland, J M; Stampfer, M R

    1997-01-01

    The effects of transforming growth factor beta (TGF-beta) were studied in closely related human mammary epithelial cells (HMEC), both finite-life-span 184 cells and immortal derivatives, 184A1S, and 184A1L5R, which differ in their cell cycle responses to TGF-beta but express type I and type II TGF-beta receptors and retain TGF-beta induction of extracellular matrix. The arrest-resistant phenotype was not due to loss of cyclin-dependent kinase (cdk) inhibitors. TGF-beta was shown to regulate p15INK4B expression at at least two levels: mRNA accumulation and protein stability. In TGF-beta-arrested HMEC, there was not only an increase in p15 mRNA but also a major increase in p5INK4B protein stability. As cdk4- and cdk6-associated p15INK4B increased during TGF-beta arrest of sensitive cells, there was a loss of cyclin D1, p21Cip1, and p27Kip1 from these kinase complexes, and cyclin E-cdk2-associated p27Kip1 increased. In HMEC, p15INK4B complexes did not contain detectable cyclin. p15INK4B from both sensitive and resistant cells could displace in vitro cyclin D1, p21Cip1, and p27Kip1 from cdk4 isolated from sensitive cells. Cyclin D1 could not be displaced from cdk4 in the resistant 184A1L5R cell lysates. Thus, in TGF-beta arrest, p15INK4B may displace already associated cyclin D1 from cdks and prevent new cyclin D1-cdk complexes from forming. Furthermore, p27Kip1 binding shifts from cdk4 to cyclin E-cdk2 during TGF-beta-mediated arrest. The importance of posttranslational regulation of p15INK4B by TGF-beta is underlined by the observation that in TGF-beta-resistant 184A1L5R, although the p15 transcript increased, p15INK4B protein was not stabilized and did not accumulate, and cyclin D1-cdk association and kinase activation were not inhibited. PMID:9111314

  15. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  16. Investigation of the process chain leading to the development of convection during COP IOP 4b

    NASA Astrophysics Data System (ADS)

    Bauer, H.-S.; Schwitalla, T.; Aoshima, F.; Behrendt, A.; Wulfmeyer, V.

    2012-04-01

    The COPS IOP 4b took place from June 20th to June 21st 2007. It was characterized by widespread convection in the COPS domain. The development was steered by a strong low pressure system southwest of the British Isles. On its eastern side warm and moist subtropical air was directed to central Europe. First convection was triggered over the Vosges Mountains around noon on the 20th of June long before the front approached the COPS region. After a calm early afternoon, severe convection was triggered in wide regions of the COPS region in the evening and moved eastwards to Bavaria during the night to the 21st of June. In contrast to other IOPs, the situation was not captured correctly by most of the involved prediction models, no matter whether they were operated with or without sophisticated data assimilation. Aim of this presentation is to unravel the mechanisms responsible for the triggering of convection and to understand the processes preparing the atmosphere for the development of severe convection during the afternoon and night. For this purpose, many different data sets will be investigated ranging from the high resolution Vienna Enhanced Resolution Analysis (VERA), high resolution radar and satellite images and composites to soundings and data as well as retrieved products from the instruments at the COPS supersites. First impression is that the complicated low-level wind field is the major driver for the preparation of the atmosphere and therefore for the development of convection during the day. The inaccuracies in representing the low level wind field are also expected to be the major reason for the failure of the models to correctly predict the situation.

  17. The Dedicated Monitor of Exotransits (DEMONEX): Seven Transits of XO-4b

    NASA Astrophysics Data System (ADS)

    Villanueva, S., Jr.; Eastman, J. D.; Gaudi, B. S.

    2016-04-01

    The DEdicated MONitor of EXotransits (DEMONEX) was a 20-inch robotic and automated telescope to monitor bright stars hosting transiting exoplanets to discover new planets and improve constraints on the properties of known transiting planetary systems. We present results for the misaligned hot Jupiter XO-4b containing seven new transits from the DEMONEX telescope, including three full and four partial transits. We combine these data with archival light curves and archival radial velocity measurements to derive the host star mass {M}*={1.293}-0.029+0.030 {M}⊙ and radius {R}*={1.554}-0.030+0.042 {R}⊙ , the planet mass {M}P={1.615}-0.099+0.10 {M}{{J}} and radius {R}P={1.317}-0.029+0.040\\\\{R}{{J}}, and a refined ephemeris of P=4.1250687+/- 0.0000024 days and {T}0=2,4547,58.18978+/- 0.00024 {{BJD}}{TDB}. We include archival Rossiter-McLaughlin measurements of XO-4 to infer the stellar spin-planetary orbit alignment of λ =-{40.0}-7.5+8.8 degrees. We test the effects of including various detrend parameters, theoretical and empirical mass-radius relations, and Rossiter-McLaughlin models. We infer that detrending against CCD position and time or airmass can improve data quality but can have significant effects on the inferred values of many parameters—most significantly {R}P/{R}* and the observed central transit times TC. In the case of {R}P/{R}* we find that the systematic uncertainty due to detrending can be three times that of the quoted statistical uncertainties. The choice of mass-radius relation has little effect on our inferred values of the system parameters. The choice of Rossiter-McLaughlin models can have significant effects on the inferred values of v{sin}{I}* and the stellar spin-planet orbit angle λ.

  18. Genome sequence and description of the heavy metal tolerant bacterium Lysinibacillus sphaericus strain OT4b.31

    PubMed Central

    Peña-Montenegro, Tito David; Dussán, Jenny

    2013-01-01

    Lysinibacillus sphaericus strain OT4b.31 is a native Colombian strain having no larvicidal activity against Culex quinquefasciatus and is widely applied in the bioremediation of heavy-metal polluted environments. Strain OT4b.31 was placed between DNA homology groups III and IV. By gap-filling and alignment steps, we propose a 4,096,672 bp chromosomal scaffold. The whole genome (consisting of 4,856,302 bp long, 94 contigs and 4,846 predicted protein-coding sequences) revealed differences in comparison to the L. sphaericus C3-41 genome, such as syntenial relationships, prophages and putative mosquitocidal toxins. Sphaericolysin B354, the coleopteran toxin Sip1A and heavy metal resistance clusters from nik, ars, czc, cop, chr, czr and cad operons were identified. Lysinibacillus sphaericus OT4b.31 has applications not only in bioremediation efforts, but also in the biological control of agricultural pests. PMID:24501644

  19. Serum complement C4b, fibronectin, and prolidase are associated with the pathological changes of pulmonary tuberculosis

    PubMed Central

    2014-01-01

    Background Mycobacterium tuberculosis infection can activate the immune system, leading to characteristic pathological changes such as inflammatory granuloma, caseous necrosis, and cavity formation. Methods Clinical data of 187 cases of pulmonary tuberculosis (PTB) were analyzed using statistical methods, while serum levels of complement C4b (C4b), fibronectin (FN), and prolidase (PEPD) were detected using the ELISA method among the control, minimal PTB, moderate PTB, and advanced PTB groups. Results We found significantly higher levels of serum C4b and PEPD (P = 0.018, P = 0.003), and significantly lower levels of serum FN (P < 0.001) in PTB patients. Furthermore, the serum levels of 3 proteins were significantly different among 3 PTB groups. FN level was significantly higher in the moderate PTB group, compared with patients in the minimal and advanced PTB groups (P < 0.05, P < 0.01). PEPD level was significantly higher in the moderate PTB group, compared with the minimal PTB group (P < 0.05). Analysis of clinical data showed that serum albumin, C-reactive protein (CRP), prealbumin, and C4 were significantly higher (P < 0.05), while serum globulin was significantly lower in patients with PTB (P < 0.001). A significant negative correlation was found between C4b and albumin, prealbumin. On the other hand, a significant positive correlation was found between C4b and globulin, CRP, PEPD, as well as between PEPD and CRP (P < 0.05). Conclusions Our study showed that C4b, FN, and PEPD are associated with tissue damage, granuloma formation, and cavity formation, respectively, in patients with PTB. The present study provides a new experimental basis to understand the pathogenesis and pathological changes of PTB. PMID:24484408

  20. Bioactivation of 4-ipomeanol by CYP4B1: adduct characterization and evidence for an enedial intermediate.

    PubMed

    Baer, Brian R; Rettie, Allan E; Henne, Kirk R

    2005-05-01

    4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromolecules. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liquid chromatography/electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping molecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolation of the biologically derived material provided evidence for an N-substituted cysteinyl pyrrole derivative of IPO, analogous to that characterized previously in model chemical studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s. PMID:15892579

  1. Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection

    PubMed Central

    Kuang, Ersheng; Varsano, Tal; Shu, Vincent Chih-Wen; Qi, Jianfei; Niesman, Ingrid R.; Yang, Huei-Jiun; López-Otín, Carlos; Yang, Wei Yuan; Reed, John C.; Broday, Limor; Nizet, Victor; Ronai, Ze'ev A.

    2012-01-01

    Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy. PMID:23093945

  2. INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells

    SciTech Connect

    Min, Joong Won; Kim, Kwang Il; Kim, Hyun-Ah; Kim, Eun-Kyu; Noh, Woo Chul; Jeon, Hong Bae; Cho, Dong-Hyung; Oh, Jeong Su; Park, In-Chul; Hwang, Sang-Gu; Kim, Jae-Sung

    2013-10-11

    Highlights: •HIF-1α-regulated INPP4B enhances glycolysis. •INPP4B regulates aerobic glycolysis by inducing HK2 via Akt-mTOR pathway. •Blockage of INPP4B and HK2 sensitizes radioresistant laryngeal cancer cells to radiation and anticancer drug. •INPP4B is associated with HK2 in human laryngeal cancer tissues. -- Abstract: Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.

  3. Beam Studies with Electron Columns

    SciTech Connect

    Shiltsev, V.; Valishev, A.; Kuznetsov, G.; Kamerdzhiev, V.; Romanov, A.; /Novosibirsk, IYF

    2009-04-01

    We report preliminary results of experimental studies of 'electron columns' in the Tevatron and in a specialized test setup. In the Tevatron, a beam of 150 GeV protons ionizes residual gas and ionization electrons are stored in an electrostatic trap immersed into strong longitudinal magnetic field. Shifts of proton betatron frequencies are observed. In the test setup, we observe effects pointing to accumulation and escape of ionization electrons.

  4. Development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from s...

  5. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. PMID:26867649

  6. Synthesis of Substituted 2,3,5,6-tetraarylbenzo(1,2-b:5,4-b')difurans

    NASA Technical Reports Server (NTRS)

    Abdul-Aziz, Mahmoud; Auping, Judith V.; Meador, Michael A.

    1995-01-01

    A series of substituted 2,3,5,6-tetraarylbenzo(l,2-b:5,4-b')difurans 1 was synthesized. This synthesis is based upon the photocyclization of 2,5-dibenzoylresorcinol dibenzyl ethers to the corresponding tetrahydrobenzo(1,2-b:5,4-b')difurans. Treatment of the photoproducts with methanesulfonyl chloride in pyridine afforded 1 in overall yields ranging from 30-72%. A number of these compounds have high fluorescence quantum yields (of phi(sub f) = 0.76-0.90), and their fluorescence spectra exhibit large solvatochromic shifts. These compounds may be suitable for use as fluorescent probes.

  7. 3,4,6-Trimethyl-1-phenyl-1H-pyrazolo­[3,4-b]pyridine

    PubMed Central

    Hamri, Salha; Hafid, Abderrafia; Zouihri, Hafid; Lazar, Saïd; Khouili, Mostafa

    2010-01-01

    In the title compound, C15H15N3, the 1H-pyrazolo­[3,4-b]pyridine system and the phenyl ring are each individually planar, with r.m.s. deviations of 0.017 (2) and 0.011 (2) Å, respectively; the dihedral angle between the two aromatic systems is 9.33 (10)°. The crystal packing is stabilized by offset π–π stacking between parallel pyrazolo­[3,4-b]pyridine ring systems [face-to-face distance = 3.449 (6) Å]. PMID:21588287

  8. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers.

  9. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  10. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  11. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  12. Studies on lipase-affinity adsorption using response-surface analysis.

    PubMed

    Kamimura, E S; Medieta, O; Rodrigues, M I; Maugeri, F

    2001-06-01

    Lipases are widely distributed enzymes that can be obtained from animals, plants and micro-organisms. Coupling lipases with a wide range of substrates allows the opportunity for synthesis of optically pure pharmaceutical preparations, flavour compounds and other food additives. Affinity chromatography owes its power as a purification method to specific biological interactions. Response-surface analysis was chosen to study column efficiency. This method allows the understanding of interactions between variables with advantages over conventional methods, which involve changing one variable while fixing others at certain levels. The aim of this work was to study the influence of the ratio bed height/column diameter (L/D) and superficial velocity (V) on the column efficiency. The experimental design involved the two variables, L/D (2-10) and v (1-2 cm/min), at five levels. Lipase was obtained from Geotrichum sp. culture in a complex medium composed of 5% corn-steep liquor, 0.5% NH(4)NO(3) and 1% olive oil at 30 degrees C, with 1VVM (air volume/medium volume per min) aeration and 400 rev./min agitation. Maximum lipase activity was 19 units/ml after almost 9 h of fermentation. This lipase could potentially be used in esterification reactions to increase the content of gamma-linolenic acid and to produce bioaromas for food industries. The adsorption assays were carried out in a fixed-bed column with an affinity adsorbent, which was obtained by reaction of a gel with oleic acid as ligand. Breakthrough curves were obtained for all experiments. It has been shown that the lower the values of both L/D and v, the higher the column efficiency (maximum 65.43%). Also, it was observed from the response surface that the efficiency reached a minimum at an L/D of around 8.

  13. Probing the water and CO snow lines in the young protostar NGC 1333-IRAS4B

    NASA Astrophysics Data System (ADS)

    Anderl, Sibylle; Maret, Sébastien; André, Philippe; Maury, Anaëlle; Belloche, Arnaud; Cabrit, Sylvie; Codella, Claudio; Lefloch, Bertrand

    2015-08-01

    Today, we believe that the onset of life requires free energy, water, and complex, probably carbon-based chemistry. In the interstellar medium, complex organic molecules seem to mostly form in reactions happening on the icy surface of dust grains, such that they are released into the gas phase when the dust is heated. The resulting “snow lines”, marking regions where ices start to sublimate, play an important role for planet growth and bulk composition in protoplanetary disks. However, they can already be observed in the envelopes of the much younger, low-mass Class 0 protostars that are still in their early phase of heavy accretion. The information on the sublimation regions of different kinds of ices can be used to understand the chemistry of the envelope, its temperature and density structure, and may even hint at the history of the accretion process. Accordingly, it is a crucial piece of information in order to get the full picture of how organic chemistry evolves already at the earliest stages of the formation of sun-like stars. As part of the CALYPSO Large Program (http://irfu.cea.fr/Projets/Calypso/), we have obtained observations of C18O, N2H+ and CH3OH towards the Class 0 protostar NGC 1333-IRAS4B with the IRAM Plateau de Bure interferometer at sub-arcsecond resolution. Of these we use the methanol observations as a proxy for the water snow line, assuming methanol is trapped in water ice. The observed anti-correlation of C18O and N2H+, with N2H+ forming a ring around the centrally peaked C18O emission, reveals for the first time the CO snow line in this protostellar envelope, with a radius of ~300 AU. The methanol emission is much more compact than that of C18O, and traces the water snow line with a radius of ~40 AU. We have modeled the emission using a chemical model coupled with a radiative transfer module. We find that the CO snow line appears further inwards than expected from the binding energy of pure CO ices. This may hint at CO being frozen out

  14. Temperature programmable microfabricated gas chromatography column

    DOEpatents

    Manginell, Ronald P.; Frye-Mason, Gregory C.

    2003-12-23

    A temperature programmable microfabricated gas chromatography column enables more efficient chemical separation of chemical analytes in a gas mixture by the integration of a resistive heating element and temperature sensing on the microfabricated column. Additionally, means are provided to thermally isolate the heated column from their surroundings. The small heat capacity and thermal isolation of the microfabricated column improves the thermal time response and power consumption, both important factors for portable microanalytical systems.

  15. Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

    PubMed Central

    Clarke, C M; Knowles, J R

    1977-01-01

    The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed. Images Fig. 4. Fig. 7. PMID:597251

  16. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high-performance affinity chromatography.

    PubMed

    Zhang, Jiwen; Li, Haiyan; Sun, Lixin; Wang, Caifen

    2015-01-01

    The kinetics of the association and dissociation are fundamental kinetic processes for the host-guest interactions (such as the drug-target and drug-excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host-guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the cyclodextrin column, the upload and elution of the samples (non-retained substances and retained solutes) at different flow rates on the cyclodextrin and control column, and data analysis. It has been indicated that cyclodextrin-immobilized chromatography is a cost-efficient high-throughput tool for the measurement of (small molecule) drug-cyclodextrin interactions as well as the dissociation of other supramolecules with relatively weak, fast, and extensive interactions. PMID:25749964

  17. 29 CFR 1926.755 - Column anchorage.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) General requirements for erection stability. (1) All columns shall be anchored by a minimum of 4 anchor rods (anchor bolts). (2) Each column anchor rod (anchor bolt) assembly, including the column-to-base... of anchor rods (anchor bolts). (1) Anchor rods (anchor bolts) shall not be repaired, replaced...

  18. 29 CFR 1926.755 - Column anchorage.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 8 2011-07-01 2011-07-01 false Column anchorage. 1926.755 Section 1926.755 Labor... (CONTINUED) SAFETY AND HEALTH REGULATIONS FOR CONSTRUCTION Steel Erection § 1926.755 Column anchorage. (a) General requirements for erection stability. (1) All columns shall be anchored by a minimum of 4...

  19. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    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

  20. BC(50): a generalized, unifying affinity descriptor.

    PubMed

    Vacca, Alberto; Francesconi, Oscar; Roelens, Stefano

    2012-12-01

    Assessing binding affinities is an unavoidable step that we come across any time interactions between binding species are investigated. A quantitative evaluation of binding affinities relies on the determination of binding constants but, whilst the binding constant fully defines the affinity of a reagent for a ligand when only one complex species is formed, the same is not true when the interacting partners form more than one complex of different stoichiometry, because all complexes contribute to the overall binding affinity. Unfortunately, this situation is the rule rather than the exception in chemical systems, but a generally accepted solution for this issue has not yet been settled. In this Personal Account, we describe the evolution, from the initial idea to a fully developed stage, of a binding descriptor that has been developed with the aim of filling this gap, thereby providing scientists in all fields of chemistry with a unifying tool for the assessment of binding affinities based on the knowledge of the binding constants in systems that involve any number of complex species.

  1. Classification of neocortical interneurons using affinity propagation.

    PubMed

    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.

  2. Classification of neocortical interneurons using affinity propagation.

    PubMed

    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

  3. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  4. 28 CFR Appendix to Part 51 - Jurisdictions Covered Under Section 4(b) of the Voting Rights Act, as Amended

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) of the Voting Rights Act, as Amended Appendix to Part 51 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED Pt. 51, App. Appendix to Part 51—Jurisdictions Covered Under Section 4(b) of the Voting Rights...

  5. Establishment of a cell line panel for the detection of antibodies against human platelet antigen 4b.

    PubMed

    Hayashi, Tomoya; Amakishi, Etsuko; Inoue, Masayasu; Hirayama, Fumiya

    2011-02-01

    Antibodies against human platelet antigens (HPAs) play important roles in thrombocytopenia. In Japan, HPA-4b antibody is frequently responsible for HPA-related neonatal alloimmune thrombocytopenia. A highly sensitive assay using platelets has been developed for the detection of antibodies against HPAs. However, it is difficult to obtain the platelets expressing specific HPAs required for the assay. Therefore, an alternative method not requiring platelets would be helpful to detect antibodies against HPAs. Glycoprotein IIIa (GPIIIa) cDNA encoding HPA-4b was individually co-transduced with that of wild-type GPIIb in K562 cells, which is a non-adherent cell line, using a retroviral vector. The expression of transgene products in cultured cells were observed for over 6 months. Next, to evaluating the sensitivity and specificity of this cell line panel, we performed monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay with a serum previously identified by another method. All HPA-4b antibodies in serum samples were positive, and all serum samples, including normal serum and serum containing HLA antibodies were negative. No difference was observed in the specificity and sensitivity between our method and conventional MAIPA using platelets. The present results indicate that this established cell line panel permits highly sensitive detection of specific antibodies against HPA-4b. PMID:21286877

  6. Role of the inositol polyphosphate-4-phosphatase type II Inpp4b in the generation of ovarian teratomas

    PubMed Central

    Balakrishnan, Ashwini; Chaillet, J. Richard

    2012-01-01

    Teratomas are a unique class of tumors composed of ecto- meso- and endodermal tissues, all foreign to the site of origin. In humans, the most common teratoma is the ovarian teratoma. Not much is known about the molecular and genetic etiologies of these tumors. Female carriers of the Tgkd transgene are highly susceptible to developing teratomas. Ovaries of Tgkd/+ hemizygous female mice exhibit defects in luteinization, with numerous corpora lutea, some of which contain central trapped, fully-grown oocytes. Genetically, Tgkd teratomas originate from mature oocytes that have completed meiosis I, suggesting that Tgkd teratomas originate from these trapped oocytes. The insertion of Tgkd 3′ of the Inpp4b gene is associated with decreased expression of Inpp4b and changes in intracellular PI3 Kinase/AKT signaling in follicular granulosa cells. Because Inpp4b is not expressed in fully-grown wild-type or Tgkd oocytes, these findings suggest that hyperactivation of the PI3K/AKT pathway caused by the decrease in INPP4B in granulosa cells promotes an ovarian environment defective in folliculogenesis and conducive to teratoma formation. PMID:23078915

  7. Occurrence of Optic Neuritis and Cervical Cord Schwannoma with Charcot-Marie-Tooth Type 4B1 Disease.

    PubMed

    Scott, Patrick; Bruwer, Zandre; Al-Kharusi, Khalsa; Meftah, Douja; Al-Murshedi, Fathiya

    2016-05-01

    Charcot-Marie-Tooth neuropathy type 4B1 (CMT4B1) disease is a rare subtype of CMT4 with reported association of facial weakness, vocal cord paresis, chest deformities, and claw hands. We report the unusual occurrence of optic neuritis and cervical cord schwannoma in a male individual with confirmed CMT4B1 disease. Sequencing of the MTMR2 gene revealed a novel nonsense homozygous mutation c.1768C>T (p.Gln590*). The mutation was identified in affected relatives of the proband and a second, apparently unrelated, family. The rare association of optic neuritis or schwannoma with genetically confirmed CMT1A has been individually observed, but never with recessive CMT. To the best of our knowledge, the occurrence of optic neuritis and cervical cord schwannoma in the same patient has never been reported with any form of CMT including CMT4B1. In similar cases, we recommend immediate medical attention to rule out the possibility of schwannomas in patients with all demyelinating CMT subtypes in case of the development of focal neurological signs or acute worsening of clinical status.

  8. KDM4B as a Target for Prostate Cancer: Structural Analysis and Selective Inhibition by a Novel Inhibitor

    PubMed Central

    2015-01-01

    The KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A–KDM4D), which selectively remove the methyl group(s) from tri/dimethylated lysine 9/36 of H3, modulate transcriptional activation and genome stability. The overexpression of KDM4A/KDM4B in prostate cancer and their association with androgen receptor suggest that KDM4A/KDM4B are potential progression factors for prostate cancer. Here, we report the crystal structure of the KDM4B·pyridine 2,4-dicarboxylic acid·H3K9me3 ternary complex, revealing the core active-site region and a selective K9/K36 site. A selective KDM4A/KDM4B inhibitor, 4, that occupies three subsites in the binding pocket is identified by virtual screening. Pharmacological and genetic inhibition of KDM4A/KDM4B significantly blocks the viability of cultured prostate cancer cells, which is accompanied by increased H3K9me3 staining and transcriptional silencing of growth-related genes. Significantly, a substantial portion of differentially expressed genes are AR-responsive, consistent with the roles of KDM4s as critical AR activators. Our results point to KDM4 as a useful therapeutic target and identify a new inhibitor scaffold. PMID:24971742

  9. Occurrence of Optic Neuritis and Cervical Cord Schwannoma with Charcot-Marie-Tooth Type 4B1 Disease

    PubMed Central

    Scott, Patrick; Bruwer, Zandre; Al-Kharusi, Khalsa; Meftah, Douja; Al-Murshedi, Fathiya

    2016-01-01

    Charcot-Marie-Tooth neuropathy type 4B1 (CMT4B1) disease is a rare subtype of CMT4 with reported association of facial weakness, vocal cord paresis, chest deformities, and claw hands. We report the unusual occurrence of optic neuritis and cervical cord schwannoma in a male individual with confirmed CMT4B1 disease. Sequencing of the MTMR2 gene revealed a novel nonsense homozygous mutation c.1768C>T (p.Gln590*). The mutation was identified in affected relatives of the proband and a second, apparently unrelated, family. The rare association of optic neuritis or schwannoma with genetically confirmed CMT1A has been individually observed, but never with recessive CMT. To the best of our knowledge, the occurrence of optic neuritis and cervical cord schwannoma in the same patient has never been reported with any form of CMT including CMT4B1. In similar cases, we recommend immediate medical attention to rule out the possibility of schwannomas in patients with all demyelinating CMT subtypes in case of the development of focal neurological signs or acute worsening of clinical status. PMID:27162595

  10. 75 FR 71145 - San Joaquin River Restoration Program: Reach 4B, Eastside Bypass, and Mariposa Bypass Channel and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-22

    ... Register on September 9, 2009 (74 FR 46453). This revised proposal would include measures for the... Bureau of Reclamation San Joaquin River Restoration Program: Reach 4B, Eastside Bypass, and Mariposa Bypass Channel and Structural Improvements Project, Merced County, CA AGENCY: Bureau of...

  11. 75 FR 40843 - International Conference on Harmonisation; Draft Guidance on Q4B Evaluation and Recommendation of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-14

    ..., which was made available in the Federal Register of February 21, 2008 (73 FR 9575). DATES: Although you... Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the International Conference on... Pharmacopoeial Texts for Use in the ICH Regions; Annex 13: Bulk Density and Tapped Density of Powders...

  12. 75 FR 43172 - International Conference on Harmonisation; Draft Guidance on Q4B Evaluation and Recommendation of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-23

    ... Administration (FDA) is correcting a notice that appeared in the Federal Register of July 14, 2010 (75 FR 40843...-0002, 301-796-9148. SUPPLEMENTARY INFORMATION: In FR Doc. 2010-17055, appearing on page 40843 in the... Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the International Conference...

  13. VizieR Online Data Catalog: TrES-4b RV and Ic curves (Sozzetti+, 2015)

    NASA Astrophysics Data System (ADS)

    Sozzetti, A.; Bonomo, A. S.; Biazzo, K.; Mancini, L.; Damasso, M.; Desidera, S.; Gratton, R.; Lanza, A. F.; Poretti, E.; Rainer, M.; Malavolta, L.; Affer, L.; Barbieri, M.; Bedin, L. R.; Boccato, C.; Bonavita, M.; Borsa, F.; Ciceri, S.; Claudi, R. U.; Gandolfi, D.; Giacobbe, P.; Henning, T.; Knapic, C.; Latham, D. W.; Lodato, G.; Maggio, A.; Maldonado, J.; Marzari, F.; Martinez Fiorenzano, A. F.; Micela, G.; Molinari, E.; Mordasini, C.; Nascimbeni, V.; Pagano, I.; Pedani, M.; Pepe, F.; Piotto, G.; Santos, N.; Scandariato, G.; Shkolnik, E.; Southworth, J.

    2015-06-01

    The TrES-4 system was observed with HARPS-N on 17 individual epochs between March 2013 and July 2014. We carried out Ic-band precision photometric observations of two complete transit events of TrES-4 b with the CAHA 1.23-m on UT 2013 July 6 and UT 2014 June 30. (2 data files).

  14. Genetic determinants for cadmium and arsenic resistance among Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis patients

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Listeria monocytogenes serotype 4b from sporadic listeriosis, heavy metal resistance was primarily encountered in certain clonal groups (ECI, ECII, ECIa). All arsenic-resistant isolates harbored the arsenic resistance cassette previously identified in pLI100; ECIa harbored additional arsenic resi...

  15. Methane gas seepage - Disregard of significant water column filter processes?

    NASA Astrophysics Data System (ADS)

    Schneider von Deimling, Jens; Schmale, Oliver

    2016-04-01

    Marine methane seepage represents a potential contributor for greenhouse gas in the atmosphere and is discussed as a driver for climate change. The ultimate question is how much methane is released from the seafloor on a global scale and what fraction may reach the atmosphere? Dissolved fluxes from methane seepage sites on the seabed were found to be very efficiently reduced by benthic microbial oxidation, whereas transport of free gas bubbles from the seabed is considered to bypass the effective benthic methane filter. Numerical models are available today to predict the fate of such methane gas bubble release to the water column in regard to gas exchange with the ambient water column, respective bubble lifetime and rise height. However, the fate of rising gas bubbles and dissolved methane in the water column is not only governed by dissolution, but is also affected by lateral oceanographic currents and vertical bubble-induced upwelling, microbial oxidation, and physico-chemical processes that remain poorly understood so far. According to this gap of knowledge we present data from two study sites - the anthropogenic North Sea 22/4b Blowout and the natural Coal Oil point seeps - to shed light into two new processes gathered with hydro-acoustic multibeam water column imaging and microbial investigations. The newly discovered processes are hereafter termed Spiral Vortex and Bubble Transport Mechanism. Spiral Vortex describes the evolution of a complex vortical fluid motion of a bubble plume in the wake of an intense gas release site (Blowout, North Sea). It appears very likely that it dramatically changes the dissolution kinetics of the seep gas bubbles. Bubble Transport Mechanism prescribes the transport of sediment-hosted bacteria into the water column via rising gas bubbles. Both processes act as filter mechanisms in regard to vertical transport of seep related methane, but have not been considered before. Spiral Vortex and Bubble Transport Mechanism represent the

  16. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same time,…

  17. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  18. Phosphodiesterase 4B is essential for TH2-cell function and development of airway hyperresponsiveness in allergic asthma

    PubMed Central

    Catherine Jin, S.-L.; Goya, Sho; Nakae, Susumu; Wang, Dan; Bruss, Matthew; Hou, Chiaoyin; Umetsu, Dale; Conti, Marco

    2010-01-01

    Background Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined. Objectives We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and TH2-driven inflammatory responses. Methods Wild-type and PDE4B−/− mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and TH2 cytokine production were determined in cultured bronchial lymph node cells. Results Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased TH2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, TH2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the TH1 cytokine IFN-γ was not affected in PDE4B−/− mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation. Conclusion By relieving a cAMP-negative constraint, PDE4B plays an essential role in TH2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment. PMID:21047676

  19. Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

    PubMed Central

    Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

    2013-01-01

    RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

  20. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed Central

    Kelly, N; Delaney, M; O'Carra, P

    1978-01-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  1. Affinity chromatography of bacterial lactate dehydrogenases.

    PubMed

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  2. European and international collaboration in affinity proteomics.

    PubMed

    Stoevesandt, Oda; Taussig, Michael J

    2012-06-15

    In affinity proteomics, specific protein-binding molecules (a.k.a. binders), principally antibodies, are applied as reagents in proteome analysis. In recent years, advances in binder technologies have created the potential for an unprecedented view on protein expression and distribution patterns in plasma, cells and tissues and increasingly on protein function. Particular strengths of affinity proteomics methods include detecting proteins in their natural environments of cell or tissue, high sensitivity and selectivity for detection of low abundance proteins and exploiting binding actions such as functional interference in living cells. To maximise the use and impact of affinity reagents, it will be essential to create comprehensive, standardised binder collections. With this in mind, the EU FP7 programme AFFINOMICS (http://www.affinomics.org), together with the preceding EU programmes ProteomeBinders and AffinityProteome, aims to extend affinity proteomics research by generating a large-scale resource of validated protein-binding molecules for characterisation of the human proteome. Activity is directed at producing binders to about 1000 protein targets, primarily in signal transduction and cancer, by establishing a high throughput, coordinated production pipeline. An important aspect of AFFINOMICS is the development of highly efficient recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. The programme also involves development of innovative and sensitive technologies for specific detection of target proteins and their interactions, and deployment of binders in proteomics studies of clinical relevance. The need for such binder generation programmes is now recognised internationally, with parallel initiatives in the USA for cancer (NCI) and transcription factors (NIH) and within the Human Proteome Organisation (HUPO). The papers in this volume of New

  3. The dynamics of metric-affine gravity

    SciTech Connect

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

    2011-05-15

    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 theories to

  4. Affine Invariant Character Recognition by Progressive Removing

    NASA Astrophysics Data System (ADS)

    Iwamura, Masakazu; Horimatsu, Akira; Niwa, Ryo; Kise, Koichi; Uchida, Seiichi; Omachi, Shinichiro

    Recognizing characters in scene images suffering from perspective distortion is a challenge. Although there are some methods to overcome this difficulty, they are time-consuming. In this paper, we propose a set of affine invariant features and a new recognition scheme called “progressive removing” that can help reduce the processing time. Progressive removing gradually removes less feasible categories and skew angles by using multiple classifiers. We observed that progressive removing and the use of the affine invariant features reduced the processing time by about 60% in comparison to a trivial one without decreasing the recognition rate.

  5. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  6. Adsorption affinity of anions on metal oxyhydroxides

    NASA Astrophysics Data System (ADS)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  7. New unitary affine-Virasoro constructions

    SciTech Connect

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

    1990-06-20

    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.

  8. Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis.

    PubMed

    Li, Hong-Yi; Deng, Qin-Pei; Zhang, De-Wen; Zhou, Ying-Lin; Zhang, Xin-Xiang

    2010-07-01

    Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affinity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC-labeled aptamers were characterized by HPLC-MS and purified by HPLC. After desalination, the ILITC-labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE-CL) by interface of end column reaction mode, the apparatus of which was home-designed and setup. CE-CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H(2)O(2), were optimized first. The system of thrombin and its 29-mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (K(d)) and the binding site number (n), were calculated. The K(d) obtained was 124.0+/-6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE-CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE-CL apparatus and combination with aptamers for a wide range of targets.

  9. Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of C4b-binding protein.

    PubMed

    Sjöberg, Andreas P; Trouw, Leendert A; McGrath, Fabian D G; Hack, C Erik; Blom, Anna M

    2006-06-15

    C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.

  10. Affinity chromatography of human leukocyte and diploid cell interferons on sepharose-bound antibodies.

    PubMed

    Berg, K; Ogburn, C A; Paucker, K; Mogensen, K E; Cantell, K

    1975-02-01

    Interferons produced in human peripheral leukocytes (LE) and foreskin fibroblast (FS-4) cells were subjected to affinity chromatography on Sepharose-bound globulins from rabbits immunized with these interferons. Anti-LE interferon sera neutralized both interferons, but titers against FS-4 interferon were consistently lower than those against LE interferon. Anti-FS-4 interferon sera neutralized only FS-4 but not LE interferon. Accordingly, affinity columns constructed with anti-FS-4 globulin excluded LE but not FS-4 interferon, whereas those prepared with anti-LE interferon globulin bound and eluted both LE and FS-4 interferons. Purification of native interferons of both types on anti-LE interferon-Sepharose ranged from 680- to 3,600-fold and recoveries from 72 to 126%. Specific activities of eluate pools varied from 4 to 30 times 10-6 reference (B, 69/19) units per milligram protien.

  11. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  12. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  13. A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

    PubMed

    Kaya, Mustafa Oguzhan; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-01-01

    In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH. PMID:25373501

  14. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  15. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    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…

  16. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  17. Aptamer-based organic-silica hybrid affinity monolith prepared via "thiol-ene" click reaction for extraction of thrombin.

    PubMed

    Wang, Zheng; Zhao, Jin-cheng; Lian, Hong-zhen; Chen, Hong-yuan

    2015-06-01

    A novel strategy for preparing aptamer-based organic-silica hybrid monolithic column was developed via "thiol-ene" click chemistry. Due to the large specific surface area of the hybrid matrix and the simplicity, rapidness and high efficiency of "thiol-ene" click reaction, the average coverage density of aptamer on the organic-silica hybrid monolith reached 420 pmol μL(-1). Human α-thrombin can be captured on the prepared affinity monolithic column with high specificity and eluted by NaClO4 solution. N-p-tosyl-Gly-Pro-Arg p-nitroanilide acetate was used as the sensitive chromogenic substrate of thrombin. The thrombin enriched by this affinity column was detected with a detection of limit of 0.01 μM by spectrophotometry. Furthermore, the extraction recovery of thrombin at 0.15 μM in human serum was 91.8% with a relative standard deviation of 4.0%. These results indicated that "thiol-ene" click chemistry provided a promising technique to immobilize aptamer on organic-inorganic hybrid monolith and the easily-assembled affinity monolithic material could be used to realize highly selective recognition of trace proteins.

  18. Preparation of phenylboronate affinity rigid monolith with macromolecular porogen.

    PubMed

    Li, Xiang-Jie; Jia, Man; Zhao, Yong-Xin; Liu, Zhao-Sheng; Akber Aisa, Haji

    2016-03-18

    Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively. PMID:26896914

  19. Surface-modified magnetic colloids for affinity adsorption of immunoglobulins

    NASA Astrophysics Data System (ADS)

    Martins, Fernanda; Pinho, Samantha C.; Zollner, Terezinha C. A.; Zollner, Ricardo L.; de Cuyper, Marcel; Santana, Maria Helena A.

    This work describes the preparation, characterization and in vitro adsorption tests of surface-modified magnetoliposomes for affinity binding of (i) anticardiolipin (isotype G) antibodies and (ii) specific isotype E antibodies generated by hypersensitivity reactions in humans with respiratory allergy. In the first case, cardiolipin embedded in the bilayer of magnetoliposomes was used as specific ligand. In the second case, antigenic proteins present in an extract of Dermatophagoids pteronyssinus and Blomia tropicalis mites were covalently coupled on the surface of magnetoliposomes via a diglycolic spacer arm, and used as specific ligands for IgE. Antibody adsorption was performed in a high-gradient magnetophoresis system, using either sera of healthy individuals or a pool of sera from autoimmune or allergic patients. The selectivity and capacity of the system were quantified by a frontal analysis in a capillary column, and by constructing breakthrough curves. The results show that the highest yield and selectivity were obtained if the ligand was extended into the aqueous layer surrounding the magnetoliposome surface. A 100% selectivity was obtained for adsorption of specific IgE, and 8% for IgG. These results demonstrate the potentialities of both types of surface-modified magnetic biocolloids in the field of in vitro diagnosis tests for allergic or autoimmune conditions.

  20. Preparation of phenylboronate affinity rigid monolith with macromolecular porogen.

    PubMed

    Li, Xiang-Jie; Jia, Man; Zhao, Yong-Xin; Liu, Zhao-Sheng; Akber Aisa, Haji

    2016-03-18

    Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

  1. Aptamer stationary phase for protein capture in affinity capillary chromatography.

    PubMed

    Connor, Adam C; McGown, Linda B

    2006-04-14

    The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.

  2. Report on inspection of compliance with DOE Order 2030.4B at the Savannah River Site

    SciTech Connect

    1997-03-01

    The purpose of this inspection was to evaluate contractor compliance at the Savannah River Site (SRS) with Department of Energy (DOE) Order 2030.4B, {open_quotes}Reporting Fraud, Waste, And Abuse To The Office Of Inspector General.{close_quotes} The specific objective was to determine if the SRS management and operating (M&O) contractors were complying with the requirements in Paragraph 6.c. of DOE Order 2030.4B. These requirements are: (1) annual notification to employees of their duty to report allegations of fraud, waste, abuse, corruption, or mismanagement; (2) display and publish the DOE Office of Inspector General (OIG) Hotline telephone number in common areas of buildings; (3) display and publish the DOE OIG Hotline number in telephone books and newsletters; and (4) notify the OIG cases referred to other law enforcement entities.

  3. Design and synthesis of spirocyclic compounds as HCV replication inhibitors by targeting viral NS4B protein.

    PubMed

    Tai, Vincent W-F; Garrido, Dulce; Price, Daniel J; Maynard, Andrew; Pouliot, Jeffrey J; Xiong, Zhiping; Seal, John W; Creech, Katrina L; Kryn, Luz H; Baughman, Todd M; Peat, Andrew J

    2014-05-15

    Two novel series of spirocyclic piperidine analogs appended to a pyrazolo[1,5-a]pyridine core were designed, synthesized and evaluated for their anti-HCV activity. A series of piperidine ketals afforded dispiro 6p which showed excellent in vitro anti-HCV activities (EC50 of 1.5nM and 1.2nM against genotype 1a and 1b replicons, respectively). A series of piperidine oxazolidinones afforded 27c which showed EC50's of 10.9nM and 6.1nM against 1a and 1b replicons, respectively. Both compounds 6p and 27c bound directly to non-structural NS4B protein in vitro (IC50's=10.2 and 30.4nM, respectively) and exhibited reduced potency in replicons containing resistance mutations encoding changes in the NS4B protein.

  4. Synthesis of novel hydrazone and azole functionalized pyrazolo[3,4-b]pyridine derivatives as promising anticancer agents.

    PubMed

    Nagender, P; Naresh Kumar, R; Malla Reddy, G; Krishna Swaroop, D; Poornachandra, Y; Ganesh Kumar, C; Narsaiah, B

    2016-09-15

    A series of novel pyrazolo[3,4-b]pyridine based target compounds were synthesized starting from the key intermediate ethyl 2-(3-amino-6-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyridin-1-yl)acetate 5 on reaction with hydrazine hydrate followed by reaction with different aldehydes, acid chlorides and isothiocyanates to form hydrazones 7, oxadiazoles 8, 1,2,4 triazoles 10 and thiadiazoles 11 respectively in high yield. All the final compounds were screened for anticancer activity against four human cancer cell lines. Among them, 1,2,4 triazole derivatives showed promising activity and compound 10d is identified as a lead molecule. PMID:27528432

  5. Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

    PubMed

    Ito, Y; Yamasaki, Y; Seno, N; Matsumoto, I

    1986-04-01

    Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin. PMID:3711062

  6. Oscillating water column structural model

    SciTech Connect

    Copeland, Guild; Bull, Diana L; Jepsen, Richard Alan; Gordon, Margaret Ellen

    2014-09-01

    An oscillating water column (OWC) wave energy converter is a structure with an opening to the ocean below the free surface, i.e. a structure with a moonpool. Two structural models for a non-axisymmetric terminator design OWC, the Backward Bent Duct Buoy (BBDB) are discussed in this report. The results of this structural model design study are intended to inform experiments and modeling underway in support of the U.S. Department of Energy (DOE) initiated Reference Model Project (RMP). A detailed design developed by Re Vision Consulting used stiffeners and girders to stabilize the structure against the hydrostatic loads experienced by a BBDB device. Additional support plates were added to this structure to account for loads arising from the mooring line attachment points. A simplified structure was designed in a modular fashion. This simplified design allows easy alterations to the buoyancy chambers and uncomplicated analysis of resulting changes in buoyancy.

  7. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  8. Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

    PubMed Central

    Qu, Lin; McMullan, Laura K.; Rice, Charles M.

    2001-01-01

    Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production. PMID:11602707

  9. 26 CFR 11.402(e)(4)(B)-1 - Election to treat an amount as a lump sum distribution.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 14 2014-04-01 2013-04-01 true Election to treat an amount as a lump sum... EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 § 11.402(e)(4)(B)-1 Election to treat an amount as a lump... is described in section 402(e)(4)(A) and which is not an annuity contract may be treated as a...

  10. 26 CFR 11.402(e)(4)(B)-1 - Election to treat an amount as a lump sum distribution.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 14 2013-04-01 2013-04-01 false Election to treat an amount as a lump sum... EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 § 11.402(e)(4)(B)-1 Election to treat an amount as a lump... is described in section 402(e)(4)(A) and which is not an annuity contract may be treated as a...

  11. 26 CFR 11.402(e)(4)(B)-1 - Election to treat an amount as a lump sum distribution.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 14 2011-04-01 2010-04-01 true Election to treat an amount as a lump sum... EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 § 11.402(e)(4)(B)-1 Election to treat an amount as a lump... is described in section 402(e)(4)(A) and which is not an annuity contract may be treated as a...

  12. 26 CFR 11.402(e)(4)(B)-1 - Election to treat an amount as a lump sum distribution.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 14 2012-04-01 2012-04-01 false Election to treat an amount as a lump sum... EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 § 11.402(e)(4)(B)-1 Election to treat an amount as a lump... is described in section 402(e)(4)(A) and which is not an annuity contract may be treated as a...

  13. 26 CFR 11.402(e)(4)(B)-1 - Election to treat an amount as a lump sum distribution.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Election to treat an amount as a lump sum... EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 § 11.402(e)(4)(B)-1 Election to treat an amount as a lump... is described in section 402(e)(4)(A) and which is not an annuity contract may be treated as a...

  14. Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells.

    PubMed

    Saxena, Ashwini; Bachelor, Martha; Park, Yong H; Carreno, Flavia R; Nedungadi, T Prashant; Cunningham, J Thomas

    2014-10-15

    Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 μM) and by a Src kinase inhibitor PP2 (10 μM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 μM losartan and 10 μM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway. PMID:25080500

  15. A Flight Investigation of the Spinning of the F4B-2 Biplane with Various Loads and Tail Surfaces

    NASA Technical Reports Server (NTRS)

    Scudder, N F; Seidman, Oscar

    1936-01-01

    A flight investigation of the spinning of the F4B-2 single-seat fighter airplane was made for the purpose of finding modifications that would eliminate dangerous spin tendencies exhibited by this type of airplane in service. The effects on steady spins and on recoveries of changing the loading, enlarging the fin areas, changing the elevator plan form, and raising the horizontal surfaces, were determined.

  16. The complement regulator C4b-binding protein analyzed by molecular modeling, bioinformatics and computer-aided experimental design.

    PubMed

    Villoutreix, B O; Blom, A M; Webb, J; Dahlbäck, B

    1999-05-01

    Molecular modeling and bioinformatics have gained recognition as scientific disciplines of importance in the field of biomedical research. Molecular modeling not only allows to predict the three-dimensional structure of a protein but also helps to define its function. Careful incorporation of the experimental findings in the structural/theoretical data provides means to understand molecular mechanisms for highly complex biological systems. C4b-binding protein (C4BP) is composed of one beta-chain and seven alpha-chains essentially built from three- and eight-complement control protein (CCP) modules, respectively, followed by a non-repeat carboxy-terminal region involved in polymerization of the chains. C4BP is involved in the regulation of the complement system and interacts with many molecules such as C4b, Arp, protein S and heparin. Here, we report experimental and computer data obtained for C4BP. Protein modeling together with site directed mutagenesis indicate that R39, R64 and R66 from the C4BP alpha-chain form a key binding site for heparin, suggesting that this region could be of major importance for interaction with C4b. We also propose that the first CCP of the C4BP beta-chain displays a key hydrophobic surface of major importance for the interaction with the coagulation cofactor protein S. PMID:10408373

  17. Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication.

    PubMed

    van Cleef, Koen W R; Overheul, Gijs J; Thomassen, Michael C; Kaptein, Suzanne J F; Davidson, Andrew D; Jacobs, Michael; Neyts, Johan; van Kuppeveld, Frank J M; van Rij, Ronald P

    2013-08-01

    Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by - directly or indirectly - targeting the viral NS4B protein. PMID:23735301

  18. Dithiazolo[5,4-b:4',5'-d]phosphole: a highly luminescent electron-accepting building block.

    PubMed

    He, Xiaoming; Woo, Alva Y Y; Borau-Garcia, Javier; Baumgartner, Thomas

    2013-06-01

    A family of highly emissive dithiazolo[5,4-b:4',5'-d]phospholes has been designed and synthesized. The structures of two trivalent P species, as well as their corresponding P oxides, have been confirmed by X-ray crystallography. The parent dithiazolo[5,4-b:4',5'-d]phosphole oxide exhibits strong blue photoluminescence at λem = 442 nm, with an excellent quantum yield efficiency of ϕPL = 0.81. The photophysical properties of these compounds can be easily tuned by extension of the conjugation and modification of the phosphorus center. Compared with the established dithieno[3,2-b:2',3'-d]phosphole system, the incorporation of electronegative nitrogen atoms leads to significantly lowered frontier orbital energy levels, as validated by both electrochemistry and theoretical calculations, thus suggesting that the dithiazolo[5,4-b:4',5'-d]phospholes are valuable, air-stable, n-type conjugated materials. These new building blocks have been further applied to the construction of an extended oligomer with fluorene. Extension of the dithiazolophosphole core with triazole units through click reactions also provides a suitable N,N-chelating moiety for metal binding and a representative molecular species was successfully used as a selective colorimetric and fluorescent sensor for Cu(II) ions.

  19. The GAPS programme with HARPS-N at TNG. VI. The curious case of TrES-4b

    NASA Astrophysics Data System (ADS)

    Sozzetti, A.; Bonomo, A. S.; Biazzo, K.; Mancini, L.; Damasso, M.; Desidera, S.; Gratton, R.; Lanza, A. F.; Poretti, E.; Rainer, M.; Malavolta, L.; Affer, L.; Barbieri, M.; Bedin, L. R.; Boccato, C.; Bonavita, M.; Borsa, F.; Ciceri, S.; Claudi, R. U.; Gandolfi, D.; Giacobbe, P.; Henning, T.; Knapic, C.; Latham, D. W.; Lodato, G.; Maggio, A.; Maldonado, J.; Marzari, F.; Martinez Fiorenzano, A. F.; Micela, G.; Molinari, E.; Mordasini, C.; Nascimbeni, V.; Pagano, I.; Pedani, M.; Pepe, F.; Piotto, G.; Santos, N.; Scandariato, G.; Shkolnik, E.; Southworth, J.

    2015-03-01

    We update the TrES-4 system parameters using high-precision HARPS-N radial-velocity measurements and new photometric light curves. A combined spectroscopic and photometric analysis allows us to determine a spectroscopic orbit with a semi-amplitude K = 51 ± 3 m s-1. The derived mass of TrES-4b is found to be Mp = 0.49 ± 0.04 MJup, significantly lower than previously reported. Combined with the large radius () inferred from our analysis, TrES-4b becomes the transiting hot Jupiter with the second-lowest density known. We discuss several scenarios to explain the puzzling discrepancy in the mass of TrES-4b in the context of the exotic class of highly inflated transiting giant planets. Based on observations made with the Italian Telescopio Nazionale Galileo (TNG) operated on the island of La Palma by the Fundacion Galileo Galilei of the INAF at the Spanish Observatorio del Roque de los Muchachos of the IAC in the frame of the program Global Architecture of Planetary Systems (GAPS), and with the Zeiss 1.23-m telescope at the German-Spanish Astronomical Center at Calar Alto, Spain. Tables 1 and 3 are available in electronic form at http://www.aanda.org

  20. Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

    PubMed Central

    Periz, Goran; Lu, Jiayin; Zhang, Tao; Kankel, Mark W.; Jablonski, Angela M.; Kalb, Robert; McCampbell, Alexander; Wang, Jiou

    2015-01-01

    Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy. PMID:25837623

  1. Characterization of polyacrylamide based monolithic columns.

    PubMed

    Plieva, Fatima M; Andersson, Jonatan; Galaev, Igor Yu; Mattiasson, Bo

    2004-07-01

    Supermacroporous monolithic polyacrylamide (pAAm)-based columns have been prepared by radical cryo-copolymerization (copolymerization in the moderately frozen system) of acrylamide with functional co-monomer, allyl glycidyl ether (AGE), and cross-linker N,N'-methylene-bis-acrylamide (MBAAm) directly in glass columns (ID 10 mm). The monolithic columns have uniform supermacroporous sponge-like structure with interconnected supermacropores of pore size 5-100 microm. The monoliths can be dried and stored in the dry state. High mechanical stability of the monoliths allowed sterilization by autoclaving. Column-to-column reproducibility of pAAm-monoliths was demonstrated on 5 monolithic columns from different batches prepared under the same cryostructuration conditions.

  2. Characterization of polyacrylamide based monolithic columns.

    PubMed

    Plieva, Fatima M; Andersson, Jonatan; Galaev, Igor Yu; Mattiasson, Bo

    2004-07-01

    Supermacroporous monolithic polyacrylamide (pAAm)-based columns have been prepared by radical cryo-copolymerization (copolymerization in the moderately frozen system) of acrylamide with functional co-monomer, allyl glycidyl ether (AGE), and cross-linker N,N'-methylene-bis-acrylamide (MBAAm) directly in glass columns (ID 10 mm). The monolithic columns have uniform supermacroporous sponge-like structure with interconnected supermacropores of pore size 5-100 microm. The monoliths can be dried and stored in the dry state. High mechanical stability of the monoliths allowed sterilization by autoclaving. Column-to-column reproducibility of pAAm-monoliths was demonstrated on 5 monolithic columns from different batches prepared under the same cryostructuration conditions. PMID:15354560

  3. REDISTRIBUTOR FOR LIQUID-LIQUID EXTRACTION COLUMNS

    DOEpatents

    Bradley, J.G.

    1957-10-29

    An improved baffle plate construction to intimately mix immiscible liquid solvents for solvent extraction processes in a liquid-liquid pulse column is described. To prevent the light and heavy liquids from forming separate continuous homogeneous vertical channels through sections of the column, a baffle having radially placed rectangular louvers with deflection plates opening upon alternate sides of the baffle is placed in the column, normal to the axis. This improvement substantially completely reduces strippiig losses due to poor mixing.

  4. Distillation of light hydrocarbons in packed columns

    SciTech Connect

    Strigle, R.F.

    1985-04-01

    Newly developed design procedures have led to a wider acceptance of packed columns for distillation operations, especially those operating at atmospheric or higher pressures. Based on these new design methods, modern IMTP packing has been used in a wide variety of services to revamp over 300 distillation columns previously equipped with trays. A few of these columns are listed. These revamps were justified by capacity increase and by greater product recovery. In addition, energy savings were realized from reduction of reflux ratio.

  5. LAPTM4B is associated with poor prognosis in NSCLC and promotes the NRF2-mediated stress response pathway in lung cancer cells

    PubMed Central

    Maki, Yuho; Fujimoto, Junya; Lang, Wenhua; Xu, Li; Behrens, Carmen; Wistuba, Ignacio I.; Kadara, Humam

    2015-01-01

    We recently demonstrated that lysosomal protein transmembrane 4 beta (LAPTM4B) is elevated in non-small cell lung cancers (NSCLCs) and in the surrounding premalignant airway field of cancerization. In the present study, we sought to begin to understand the relevance of LAPTM4B expression and signaling to NSCLC pathogenesis. In situ hybridization analysis of LAPTM4B transcript in tissue microarrays comprised of 368 NSCLCs demonstrated that LAPTM4B expression was significantly increased in smoker compared to non-smoker lung adenocarcinoma tumors (P < 0.001) and was significantly associated with poor overall survival (P < 0.05) in adenocarcinoma patients. Knockdown of LAPTM4B expression inhibited cell growth, induced cellular apoptosis and decreased cellular autophagy in serum starved lung cancer cells. Expression profiling coupled with pathways analysis revealed decreased activation of the nuclear factor erythroid 2-like 2 (NRF2) stress response pathway following LAPTM4B knockdown. Further analysis demonstrated that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation as well as increased the expression of NRF2 target genes such as heme oxygenase 1/HMOX1). Our study points to the relevance of LAPTM4B expression to NSCLC pathogenesis as well as to the probable role of LAPTM4B/NRF2 signaling in promoting lung cancer cell survival. PMID:26343532

  6. Association of PDE4B Polymorphisms with Susceptibility to Schizophrenia: A Meta-Analysis of Case-Control Studies

    PubMed Central

    Feng, Yanguo; Cheng, Dejun; Zhang, Chaofeng; Li, Yuchun; Zhang, Zhiying; Wang, Juan; Shi, Yuzhong

    2016-01-01

    Background The PDE4B single nucleotide polymorphisms (SNPs) have been reported to be associated with schizophrenia risk. However, current findings are ambiguous or even conflicting. To better facilitate the understanding the genetic role played by PDE4B in susceptibility to schizophrenia, we collected currently available data and conducted this meta-analysis. Methods A comprehensive electronic literature searching of PubMed, Embase, Web of Science and Cochrane Library was performed. The association between PDE4B SNPs and schizophrenia was evaluated by odds ratios (ORs) and 95% confidence intervals (CIs) under allelic, dominant and recessive genetic models. The random effects model was utilized when high between-study heterogeneity (I2 > 50%) existed, otherwise the fixed effects model was used. Results Five studies comprising 2376 schizophrenia patients and 3093 controls were finally included for meta-analysis. The rs1040716 was statistically significantly associated with schizophrenia risk in Asian and Caucasian populations under dominant model (OR = 0.87, 95% CI: 0.76–0.99, P = 0.04). The rs2180335 was significantly related with schizophrenia risk in Asian populations under allelic (OR = 0.82, 95% CI: 0.72–0.93, P = 0.003) and dominant (OR = 0.75, 95% CI: 0.64–0.88, P < 0.001) models. A significant association was also observed between rs4320761 and schizophrenia in Asian populations under allelic model (OR = 0.87, 95% CI: 0.75–1.00, P = 0.048). In addition, a strong association tendency was found between rs6588190 and schizophrenia in Asian populations under allelic model (OR = 0.87, 95% CI: 0.76–1.00, P = 0.055). Conclusion This meta-analysis suggests that PDE4B SNPs are genetically associated with susceptibility to schizophrenia. However, due to limited sample size, more large-scale, multi-racial association studies are needed to further clarify the genetic association between various PDE4B variants and schizophrenia. PMID:26756575

  7. Interstitial gas effect on vibrated granular columns

    NASA Astrophysics Data System (ADS)

    Pastenes, Javier C.; Géminard, Jean-Christophe; Melo, Francisco

    2014-06-01

    Vibrated granular materials have been intensively used to investigate particle segregation, convection, and heaping. We report on the behavior of a column of heavy grains bouncing on an oscillating solid surface. Measurements indicate that, for weak effects of the interstitial gas, the temporal variations of the pressure at the base of the column are satisfactorily described by considering that the column, despite the observed dilation, behaves like a porous solid. In addition, direct observation of the column dynamics shows that the grains of the upper and lower surfaces are in free fall in the gravitational field and that the dilation is due to a small delay between their takeoff times.

  8. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  9. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  10. Soil column leaching of pesticides.

    PubMed

    Katagi, Toshiyuki

    2013-01-01

    In this review, I address the practical and theoretical aspects of pesticide soil mobility.I also address the methods used to measure mobility, and the factors that influence it, and I summarize the data that have been published on the column leaching of pesticides.Pesticides that enter the unsaturated soil profile are transported downwards by the water flux, and are adsorbed, desorbed, and/or degraded as they pass through the soil. The rate of passage of a pesticide through the soil depends on the properties of the pesticide, the properties of the soil and the prevailing environmental conditions.Because large amounts of many different pesticides are used around the world, they and their degradates may sometimes contaminate groundwater at unacceptable levels.It is for this reason that assessing the transport behavior and soil mobility of pesticides before they are sold into commerce is important and is one indispensable element that regulators use to assess probable pesticide safety. Both elementary soil column leaching and sophisticated outdoor lysimeter studies are performed to measure the leaching potential for pesticides; the latter approach more reliably reflects probable field behavior, but the former is useful to initially profile a pesticide for soil mobility potential.Soil is physically heterogeneous. The structure of soil varies both vertically and laterally, and this variability affects the complex flow of water through the soil profile, making it difficult to predict with accuracy. In addition, macropores exist in soils and further add to the complexity of how water flow occurs. The degree to which soil is tilled, the density of vegetation on the surface, and the type and amounts of organic soil amendments that are added to soil further affect the movement rate of water through soil, the character of soil adsorption sites and the microbial populations that exist in the soil. Parameters that most influence the rate of pesticide mobility in soil are

  11. Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

    PubMed

    Wu, Ming-Chang; Lin, Guan-Hui; Wang, Yuh-Tai; Jiang, Chii-Ming; Chang, Hung-Min

    2005-10-01

    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).

  12. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  13. Effect of column dimension on observed column efficiency in very high pressure liquid chromatography.

    PubMed

    Wu, Naijun; Bradley, Ashley C

    2012-10-26

    The effect of extra-column volume on observed linear velocity was investigated for columns of various internal diameters in very high pressure liquid chromatography. The results showed that the observed linear velocities were approximately 4.5, 9.5, 16.8, and 39.5% lower than the linear velocities corrected for the extra-column volume contribution for 4.6, 3.0, 2.1, and 1.0mm internal diameter columns, respectively. An empirical relationship between extra-column band broadening and extra-column volume was obtained using 50 cm long tubings of various internal diameters. The peak variance from the extra-column volume is near linearly proportional to the square of the extra-column volume for tubings with 0.0635-0.178 mm (0.025-0.07 in.) i.d. using a 50/50 acetonitrile/water mobile phase at flow rates greater than 0.3 mL/min. The effect of column internal diameter and column length on observed efficiency was studied using 50mm columns with four different column internal diameters and 2.1mm i.d columns with three different lengths. The results showed that the observed column efficiencies for 3.0, 2.1, and 1.0mm internal diameter columns were 18, 33, and 73% lower than that for a 4.6mm internal diameter column for benzophenone (k=5.5), respectively. An approximate 20% decrease in theoretical plate number was observed for propiophenone (k=3.3) using a 50 mm × 2.1 mm column packed with 1.7 μm particles compared to a 150 mm × 2.1 mm column packed with 5.0 μm particles, while the former column provided 9 fold faster separation. It is the column to extra column volume ratio instead of absolute extra-column volume that determines the degree of extra-column band-broadening in VHPLC.

  14. Smooth big bounce from affine quantization

    NASA Astrophysics Data System (ADS)

    Bergeron, Hervé; Dapor, Andrea; Gazeau, Jean Pierre; Małkiewicz, Przemysław

    2014-04-01

    We examine the possibility of dealing with gravitational singularities on a quantum level through the use of coherent state or wavelet quantization instead of canonical quantization. We consider the Robertson-Walker metric coupled to a perfect fluid. It is the simplest model of a gravitational collapse, and the results obtained here may serve as a useful starting point for more complex investigations in the future. We follow a quantization procedure based on affine coherent states or wavelets built from the unitary irreducible representation of the affine group of the real line with positive dilation. The main issue of our approach is the appearance of a quantum centrifugal potential allowing for regularization of the singularity, essential self-adjointness of the Hamiltonian, and unambiguous quantum dynamical evolution.

  15. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  16. Artificial Affinity Proteins as Ligands of Immunoglobulins

    PubMed Central

    Mouratou, Barbara; Béhar, Ghislaine; Pecorari, Frédéric

    2015-01-01

    A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized. PMID:25647098

  17. Permeability of self-affine rough fractures

    PubMed

    Drazer; Koplik

    2000-12-01

    The permeability of two-dimensional fractures with self-affine fractal roughness is studied via analytic arguments and numerical simulations. The limit where the roughness amplitude is small compared with average fracture aperture is analyzed by a perturbation method, while in the opposite case of narrow aperture, we use heuristic arguments based on lubrication theory. Numerical simulations, using the lattice Boltzmann method, are used to examine the complete range of aperture sizes, and confirm the analytic arguments. PMID:11138092

  18. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  19. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field.

  20. Overview of affinity biosensors in food analysis.

    PubMed

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field. PMID:16792079

  1. A MEMS Dielectric Affinity Glucose Biosensor

    PubMed Central

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-01-01

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  2. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    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 charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  3. Trematode hemoglobins show exceptionally high oxygen affinity.

    PubMed

    Kiger, L; Rashid, A K; Griffon, N; Haque, M; Moens, L; Gibson, Q H; Poyart, C; Marden, M C

    1998-08-01

    Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

  4. Allelic Variation and Differential Expression of the mSIN3A Histone Deacetylase Complex Gene Arid4b Promote Mammary Tumor Growth and Metastasis

    PubMed Central

    Winter, Scott F.; Lukes, Luanne; Walker, Renard C.; Welch, Danny R.; Hunter, Kent W.

    2012-01-01

    Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer. PMID:22693453

  5. Preparation of an immunoaffinity column and its application in sample cleanup for methandrostenolone residues detection.

    PubMed

    Wang, Yun; Xu, Yan; Zhang, Xun; Wang, Enlan; Dong, Ying

    2011-07-15

    Methandrostenolone (MA) is a steroid used as veterinary medicine on stockbreeding to promote animal growth. The use of MA has been strictly regulated because of its harmful effect on consumers. This paper describes the production of polyclonal antibody (pAb) against MA, the preparation of immunoaffinity column (IAC) and its potential application to the selective extraction of MA residues from animal tissue and feed samples. The produced pAb exhibited good sensitivity to MA with an IC(50) value of 5.6 ng/mL. The cross-reactivity values of the antibody with MA structurally related compounds of testosterone propionate (TP) and trenbolone (TR) were lower than 0.6%. By coupling the produced antibody with CNBr-activated Sepharose 4B, an IAC was prepared. 2% methanol and 80% methanol were selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was approximately 334 ng/mL gel. The average recovery of 20, 40 and 60 ng/mL MA standard solutions from IACs was 97.9% with the relative standard deviation (RSD) among columns of 6.7%. After 3 times of repeated usage, the column capacity and recovery rate still remained 82.0% and 92.6% respectively. The IACs were then challenged with MA-fortified animal tissue and feed samples, recoveries of MA were found to be in the range of 83.5-99.7%. PMID:21703946

  6. Regulator of insulin receptor affinity in rat skeletal muscle sarcolemmal vesicles

    SciTech Connect

    Whitson, R.H.; Barnard, K.J.; Kaplan, S.A.; Itakura, K.

    1986-05-01

    Wheat germ agglutinin (WGA) affinity purification of detergent solubilized insulin receptors (IR) from rat skeletal muscle sarcolemmal vesicles resulted in an apparent increase in total insulin binding activity of greater than 5-fold, suggesting that an inhibitory component had been removed. This was verified when the flow-through fraction from the WGA column was dialyzed and added back to the partially purified receptor. The addition of a 100-fold dilution of the inhibitor preparation caused a 50% reduction in binding to trace amounts of /sup 125/I-insulin. Scatchard analysis revealed that the effect of the inhibitor was to decrease the affinity of the muscle IR. The inhibitor appeared to be tissue specific, inasmuch as the I/sub 50/'s for WGA-purified IR from rat fat and liver were 10 times the I/sub 50/ for muscle IR. The I/sub 50/ for insulin binding to intact IM-9 cells was 30 times the value for muscle IR. The inhibitor eluted in the void volume of Sephadex G-50 columns. Its activity was not destroyed by heating at 90/sup 0/C for 10 minutes, or by prolonged incubation with trypsin or dithiothreitol. The inhibitor described here may have a role in modulating insulin sensitivity in skeletal muscle.

  7. Optimal operation of multivessel batch distillation columns

    SciTech Connect

    Furlonge, H.I.; Pantelides, C.C.; Soerensen, E.

    1999-04-01

    Increased interest in unconventional batch distillation column configurations offers new opportunities for increasing the flexibility and energy efficiency of batch distillation. One configuration of particular interest is the multivessel column, which can be viewed as a generalization of all previously studied batch column configurations. A detailed dynamic model was used for comparing various optimal operating policies for a batch distillation column with two intermediate vessels. A wide variety of degrees of freedom including reflux ratios, product withdrawal rates, heat input to the reboiler, and initial feed distribution were considered. A mixture consisting of methanol, ethanol, n-propanol and n-butanol was studied using an objective function relating to the economics of the column operation. Optimizing the initial distribution of the feed among the vessels improved column performance significantly. For some separations, withdrawing product from the vessels into accumulators was better than total reflux operation in terms of energy consumption. Open-loop optimal operation was also compared to a recently proposed feedback control strategy where the controller parameters are optimized. The energy consumption of a regular column was about twice that of a multivessel column having the same number of stages.

  8. Circulation in gas-slurry column reactors

    SciTech Connect

    Clark, N.; Kuhlman, J.; Celik, I.; Gross, R.; Nebiolo, E.; Wang, Yi-Zun.

    1990-08-15

    Circulation in bubble columns, such as those used in fischer-tropsch synthesis, detracts from their performance in that gas is carried on average more rapidly through the column, and the residence time distribution of the gas in the column is widened. Both of these factors influence mass-transfer operations in bubble columns. Circulation prediction and measurement has been undertaken using probes, one-dimensional models, laser Doppler velocimetry, and numerical modeling. Local void fraction was measured using resistance probes and a newly developed approach to determining air/water threshold voltage for the probe. A tall column of eight inch diameter was constructed of Plexiglas and the distributor plate was manufactured to distribute air evenly through the base of the column. Data were gathered throughout the volume at three different gas throughputs. Bubble velocities proved difficult to measure using twin probes with cross-correlation because of radial bubble movement. A series of three-dimensional mean and RMS bubble and liquid velocity measurements were also obtained for a turbulent flow in a laboratory model of a bubble column. These measurements have been made using a three-component laser Doppler velocimeter (LDV), to determine velocity distributions non-intrusively. Finally, the gas-liquid flow inside a vertically situated circular isothermal column reactor was simulated numerically. 74 refs., 170 figs., 5 tabs.

  9. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples. PMID:27498022

  10. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    PubMed

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  11. Probes for narcotic receptor mediated phenomena. 44. Synthesis of an N-substituted 4-hydroxy-5-(3-hydroxyphenyl)morphan with high affinity and selective μ-antagonist activity

    PubMed Central

    Iyer, Malliga R.; Lee, Yong Sok; Deschamps, Jeffrey R.; Dersch, Christina M.; Rothman, Richard B.; Jacobson, Arthur E.; Rice, Kenner C.

    2012-01-01

    A simple three-step synthesis of 5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-4-ol (3a) was achieved using an osmium tetroxide mediated oxidation of the known intermediate 6. A pyrrolidine-ring variant of 3a (3-(7-(hydroxymethyl)-6-methyl-6-azabicyclo[3.2.1]octan-1-yl)phenol (5)) was isolated when other routes were used. The epimeric hydroxy analogue 4a was synthesized by simple inversion of the stereochemistry at C-4. Both N-methyl (3a and 4a) and N-phenethyl (3b and 4b) derivatives were synthesized. The compounds were examined for their opioid receptor affinity and the N-phenethyl analogue 3b was found to have relatively weak affinity for the μ-opioid receptor (Ki = 74 nM). However, the N-phenethyl analogue of the C-4 epimer, 4b, had about 15 fold higher affinity than 3b and was selective for the μ-opioid receptor (Ki = 4.6 nM). Compound 4b was a moderately potent μ-opioid antagonist (Ke = 12 nM), as determined by [35S]GTP-γ-S assays. Compounds 3b and 4b were energy minimized at the level of B3LYP/6-31G*, and then overlaid onto the 5-phenylmorphan, the (1R,5R,9S)-(−)-enantiomer of 2b (Fig. 1) with the α or β-OH group at the C-9 position. The spatial orientation of the hydroxyl moiety in 3b, 4b, 2a, and 2b is proposed to be the structural requirement for high μ-opioid receptor binding affinity and their agonist or antagonist activity. The modest change in spatial position of the hydroxyl moiety, and not the N-substituent, induced the change from potent agonist to an antagonist of moderate potency. PMID:22341895

  12. Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine.

    PubMed Central

    Smith, G D; Roberts, D V; Daday, A

    1977-01-01

    Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo. PMID:889568

  13. SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus.

    PubMed

    Kia, Sima Kheradmand; Gorski, Marcin M; Giannakopoulos, Stavros; Verrijzer, C Peter

    2008-05-01

    Stable silencing of the INK4b-ARF-INK4a tumor suppressor locus occurs in a variety of human cancers, including malignant rhabdoid tumors (MRTs). MRTs are extremely aggressive cancers caused by the loss of the hSNF5 subunit of the SWI/SNF chromatin-remodeling complex. We found previously that, in MRT cells, hSNF5 is required for p16(INK4a) induction, mitotic checkpoint activation, and cellular senescence. Here, we investigated how the balance between Polycomb group (PcG) silencing and SWI/SNF activation affects epigenetic control of the INK4b-ARF-INK4a locus in MRT cells. hSNF5 reexpression in MRT cells caused SWI/SNF recruitment and activation of p15(INK4b) and p16(INK4a), but not of p14(ARF). Gene activation by hSNF5 is strictly dependent on the SWI/SNF motor subunit BRG1. SWI/SNF mediates eviction of the PRC1 and PRC2 PcG silencers and extensive chromatin reprogramming. Concomitant with PcG complex removal, the mixed lineage leukemia 1 (MLL1) protein is recruited and active histone marks supplant repressive ones. Strikingly, loss of PcG complexes is accompanied by DNA methyltransferase DNMT3B dissociation and reduced DNA methylation. Thus, various chromatin states can be modulated by SWI/SNF action. Collectively, these findings emphasize the close interconnectivity and dynamics of diverse chromatin modifications in cancer and gene control. PMID:18332116

  14. Human X-linked Intellectual Disability Factor CUL4B Is Required for Post-meiotic Sperm Development and Male Fertility

    PubMed Central

    Lin, Chien-Yu; Chen, Chun-Yu; Yu, Chih-Hsiang; Yu, I-Shing; Lin, Shu-Rung; Wu, June-Tai; Lin, Ying-Hung; Kuo, Pao-Lin; Wu, Jui-Ching; Lin, Shu-Wha

    2016-01-01

    In this study, we demonstrate that an E3-ubiquitin ligase associated with human X-linked intellectual disability, CUL4B, plays a crucial role in post-meiotic sperm development. Initially, Cul4bΔ/Y male mice were found to be sterile and exhibited a progressive loss in germ cells, thereby leading to oligoasthenospermia. Adult Cul4b mutant epididymides also contained very low numbers of mature spermatozoa, and these spermatazoa exhibited pronounced morphological abnormalities. In post-meiotic spermatids, CUL4B was dynamically expressed and mitosis of spermatogonia and meiosis of spermatocytes both appeared unaffected. However, the spermatids exhibited significantly higher levels of apoptosis during spermiogenesis, particularly during the acrosome phase through the cap phase. Comparative proteomic analyses identified a large-scale shift between wild-type and Cul4b mutant testes during early post-meiotic sperm development. Ultrastructural pathology studies further detected aberrant acrosomes in spermatids and nuclear morphology. The protein levels of both canonical and non-canonical histones were also affected in an early spermatid stage in the absence of Cul4b. Thus, X-linked CUL4B appears to play a critical role in acrosomal formation, nuclear condensation, and in regulating histone dynamics during haploid male germ cell differentiation in relation to male fertility in mice. Thus, it is possible that CUL4B-selective substrates are required for post-meiotic sperm morphogenesis. PMID:26832838

  15. 15 CFR 744.9 - Restrictions on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... reexports of cameras controlled by ECCN 6A003.b.4.b. 744.9 Section 744.9 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF... on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b. (a) General...

  16. 15 CFR 744.9 - Restrictions on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... reexports of cameras controlled by ECCN 6A003.b.4.b. 744.9 Section 744.9 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF... on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b. (a) General...

  17. 15 CFR 744.9 - Restrictions on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... reexports of cameras controlled by ECCN 6A003.b.4.b. 744.9 Section 744.9 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF... on certain exports and reexports of cameras controlled by ECCN 6A003.b.4.b. (a) General...

  18. Pulmonary cytochrome P450 enzymes belonging to the CYP4B subfamily from an Australian marsupial, the tammar wallaby (Macropus eugenii).

    PubMed

    Milic, Natalie L; Ngo, Suong N T; Marchant, Ceilidh L; Height, Tamara A; McKinnon, Ross A

    2011-01-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported the cloning and characterisation of the koala CYP4A15, the first reported member of the CYP4 family from marsupials, and have demonstrated important species differences in CYP4A activity and tissue expression. In the present study, the cloning of CYP4B1 in the wallaby (Macropus eugenii) and their expression across marsupials is described. Rabbit anti-mouse CYP4B1 antibody detected immunoreactive proteins in lung and liver microsomes from all test marsupials, with relative weak signal detected from the koala, suggesting a species-specific expression. Microsomal 2-aminofluorene bio-activation (a CYP4B1 marker) in wallaby lung was comparable to that of rabbit, with significant higher activities detected in wallaby liver and kidneys compared to rabbit. A 1548bp wallaby lung CYP4B complete cDNA, designated CYP4B1, which encodes a protein of 510 amino acids and shares 72% nucleotide and 69% amino acid sequence identity to human CYP4B1, was cloned by polymerase chain reaction approaches. The results demonstrate the presence of wallaby CYP4B1 that shares several common features with other published CYP4Bs; however the wallaby CYP4B1 cDNA contains four extra amino acid residues at the NH₂-terminal, a fundamentally conserved transmembrane anchor of all eukaryote CYPs.

  19. Modeling of column apparatuses: A review

    SciTech Connect

    Doichinova, M. E-mail: petyabs@yahoo.com; Popova-Krumova, P. E-mail: petyabs@yahoo.com

    2013-12-18

    This paper presents a review of the modeling method on the base of the physical approximations of the mechanics of continua, which have been developed for processes in column apparatuses. This method includes diffusion type of model for modeling of mass transfer with chemical reaction in column apparatuses with and without circulation zones. The diffusion type of model is used for modeling of scale effect in column apparatuses too. The study concluded that the proposal method is possibility for investigation the influence of radial non uniformity of the velocity distribution on the process efficiency, influence of zones breadths on the mass transfer efficiency in the column. The method of the column apparatuses modeling can be used for modeling of physical and chemical absorption, chemical adsorption, homogeneous and heterogeneous (catalytic) chemical reactions, airlift reactors for chemical and photochemical reactions.

  20. Evolution of the local superconducting density of states in ErRh4B4 close to the ferromagnetic transition.

    PubMed

    Crespo, V; Rodrigo, J G; Suderow, H; Vieira, S; Hinks, D G; Schuller, I K

    2009-06-12

    We present local tunneling spectroscopy experiments in the superconducting and ferromagnetic phases of the reentrant superconductor ErRh4B4. The tunneling conductance curves jump from showing normal to superconducting features within a few mK close to the ferromagnetic transition temperature, with a clear hysteretic behavior. Within the ferromagnetic phase, we do not detect any superconducting correlations. Within the superconducting phase we find a peculiar V-shaped density of states at low energies, which is produced by the magnetically modulated phase that coexists with superconductivity just before ferromagnetism sets in. PMID:19658962

  1. Thermo-optical properties of 1H[3,4-b] quinoline films used in electroluminescent devices

    NASA Astrophysics Data System (ADS)

    Jaglarz, Janusz; Kępińska, Mirosława; Sanetra, Jerzy

    2014-06-01

    Electroluminescence cells with H[3,4-b] quinoline layers are promising devices for a blue light emitting EL diode. This work measured the optical reflectance as a function of temperature in copolymers PAQ layers deposited on Si crystalline substrate. Using the extended Cauchy dispersion model of the film refractive index we determined the thermo-optical coefficients for quinoline layers in the temperature range of 76-333 K from combined ellipsometric and spectrofotometric studies. The obtained values of thermo-optical coefficients of thin PAQ film, were negative and ranged in 5-10 × 10-4 [1/K].

  2. Dithieno[3,4-b:3',4'-d]thiophene-annelated antiaromatic planar cyclooctatetraene with olefinic protons.

    PubMed

    Aita, Kazunari; Ohmae, Takeshi; Takase, Masayoshi; Nomura, Kotohiro; Kimura, Hideaki; Nishinaga, Tohru

    2013-07-19

    The design and synthesis of a new planar cyclooctatetraene (COT) with protons directly connected to the COT ring was attained by monoannelation with dithieno[3,4-b:3',4'-d]thiophene. The planar structure of the COT core was unambiguously confirmed by X-ray crystallography. The magnetic antiaromaticity of the COT core was found to be higher than that of the previously synthesized planar COTs with olefinic protons, according to the results of (1)H NMR and absorption spectra as well as NICS calculations.

  3. 6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors targeting hepatitis C virus NS4B.

    PubMed

    Chen, Guangming; Ren, Hongyu; Zhang, Nanjing; Lennox, William; Turpoff, Anthony; Paget, Steven; Li, Chunshi; Almstead, Neil; Njoroge, F George; Gu, Zhengxian; Graci, Jason; Jung, Stephen P; Colacino, Joseph; Lahser, Fred; Zhao, Xin; Weetall, Marla; Nomeir, Amin; Karp, Gary M

    2015-02-15

    A structure-activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series, has excellent potency against the HCV 1b replicon with an EC50 of 2nM and a selectivity index of >5000 with respect to cellular GAPDH RNA. Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats. PMID:25613678

  4. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

    PubMed

    Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H; Grose, Carissa; Jones, Jane E; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G

    2015-11-02

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

  5. Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions.

    PubMed

    Yang, F; Mao, J; He, X W; Chen, L X; Zhang, Y K

    2013-08-01

    In this study, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile one-pot procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane and vinyltrimethoxysilane, organic monomer 3-acrylamidophenylboronic acid and initiator 2,2'-azobisisobutyronitrile together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperatures. After optimizing the preparation conditions, the resulting hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be 2.5 mg g(-1) at pH 7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations of retention time were 1.15 and 4.77 % (n = 5) for run-to-run and column-to-column, respectively. PMID:23807307

  6. Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins.

    PubMed

    Demishtein, Alik; Karpol, Alon; Barak, Yoav; Lamed, Raphael; Bayer, Edward A

    2010-01-01

    Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high-affinity Ca(2+)-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K(A)) of 7.7 × 10(8)M(-1), calculated by a competitive enzyme-linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β-glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody-binding ZZ-domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T-6 as a model system. In addition, the enzymatic activity of a C. thermocellum β-glucosidase, purified using this approach, was tested and found to be similar to that of a β-glucosidase preparation (without the ΔDoc tag) purified using the standard His-tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose-based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca(2+)-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. PMID:21038354

  7. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  8. Aryl thioglycoside-based affinity purification of exo-acting cellulases.

    PubMed

    Piyachomkwan, K; Penner, M H

    1998-01-15

    The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters. PMID:9451508

  9. A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.

    PubMed

    Chen, Chen; El Khoury, Graziella; Zhang, Peiqing; Rudd, Pauline M; Lowe, Christopher R

    2016-04-29

    One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.

  10. Identification of RIP-II toxins by affinity enrichment, enzymatic digestion and LC-MS.

    PubMed

    Fredriksson, Sten-Åke; Artursson, Elisabet; Bergström, Tomas; Östin, Anders; Nilsson, Calle; Åstot, Crister

    2015-01-20

    Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

  11. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

    PubMed

    Kennedy, Jacob J; Yan, Ping; Zhao, Lei; Ivey, Richard G; Voytovich, Uliana J; Moore, Heather D; Lin, Chenwei; Pogosova-Agadjanyan, Era L; Stirewalt, Derek L; Reding, Kerryn W; Whiteaker, Jeffrey R; Paulovich, Amanda G

    2016-02-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  12. A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides

    PubMed Central

    Dremina, Elena S.; Li, Xiaobao; Galeva, Nadezhda A.; Sharov, Victor S.; Stobaugh, John F.; Schöneich, Christian

    2011-01-01

    We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures. PMID:21855526

  13. A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

    PubMed

    Dremina, Elena S; Li, Xiaobao; Galeva, Nadezhda A; Sharov, Victor S; Stobaugh, John F; Schöneich, Christian

    2011-11-15

    We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures. PMID:21855526

  14. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  15. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  16. Identification of Substituted Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands

    PubMed Central

    2013-01-01

    A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators. PMID:23656327

  17. Highly efficient capillary columns packed with superficially porous particles via sequential column packing.

    PubMed

    Treadway, James W; Wyndham, Kevin D; Jorgenson, James W

    2015-11-27

    Highly efficient capillary columns packed with superficially porous particles were created for use in ultrahigh pressure liquid chromatography. Superficially porous particles around 1.5μm in diameter were packed into fused silica capillary columns with 30, 50, and 75μm internal diameters. To create the columns, several capillary columns were serially packed from the same slurry, with packing progress plots being generated to follow the packing of each column. Characterization of these columns using hydroquinone yielded calculated minimum reduced plate heights as low as 1.24 for the most efficient 30μm internal diameter column, corresponding to over 500,000plates/m. At least one highly efficient column (minimum reduced plate height less than 2) was created for all three of the investigated column inner diameters, with the smallest diameter columns having the highest efficiency. This study proves that highly efficient capillary columns can be created using superficially porous particles and shows the efficiency potential of these particles.

  18. Aluminum-based water treatment residual use in a constructed wetland for capturing urban runoff phosphorus: Column study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum-based water treatment residuals (Al-WTR) have a strong affinity to sorb phosphorus. In a proof-of-concept greenhouse column study, Al-WTR was surface-applied at 0, 62, 124, and 248 Mg/ha to 15 cm of soil on top of 46 cm of sand; Al-WTR rates were estimated to capture 0, 10, 20, and 40 year...

  19. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  20. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  1. Measuring an antibody affinity distribution molecule by molecule.

    PubMed

    Temirov, Jamshid P; Bradbury, Andrew R M; Werner, James H

    2008-11-15

    Single molecule fluorescence microscopy was used to observe the binding and unbinding of hapten decorated quantum dots to individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  2. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  3. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  4. Microwaves Scattering by Underdense Inhomogeneous Plasma Column

    NASA Astrophysics Data System (ADS)

    Zhang, Lin; Ouyang, Jiting

    2016-03-01

    The scattering characteristics of microwaves (MWs) by an underdense inhomogeneous plasma column have been investigated. The plasma column is generated by hollow cathode discharge (HCD) in a glass tube filled with low pressure argon. The plasma density in the column can be varied by adjusting the discharge current. The scattering power of X-band MWs by the column is measured at different discharge currents and receiving angles. The results show that the column can affect the properties of scattering wave significantly regardless of its plasma frequency much lower than the incident wave frequency. The power peak of the scattering wave shifts away from 0° to about ±15° direction. The finite-different time-domain (FDTD) method is employed to analyze the wave scattering by plasma column with different electron density distributions. The reflected MW power from a metal plate located behind the column is also measured to investigate the scattering effect on reducing MW reflectivity of a metal target. This study is expected to deepen the understanding of plasma-electromagnetic wave interaction and expand the applications concerning plasma antenna and plasma stealth.

  5. Mechanical interactions of UIS support columns. [LMFBR

    SciTech Connect

    Kennedy, J.M.; Belytschko, T.B.

    1983-01-01

    Code development involving above-core structures (ACS) has recently focused on modeling the complexities of mechanical interactions in the ACS support columns which play a very important role in their behavior. These developments are directed toward two considerations: (1) the prediction of the forces exerted by the column in a hypothetical core-disruptive accident (HCDA) in order that the motion of the ACS can be predicted in a coupled fluid-structure analysis, (2) the calculation of the strains and deformations of the support columns so that situations which lead to complete failure can be identified. Finite element capabilities have been developed to handle various types of plant design for the analysis of coupled hydrodynamics and structural response. Beam elements, which previously represented the support columns were able to account for geometric nonlinearities and material nonlinearities, however, changes in the column cross section were not treated. Therefore, one of the aims of this study was to examine the effect of the change in cross section on the behavior of the support columns. A second effect which has been studied is the behavior of support columns consisting of two concentric cylinders.

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

    PubMed

    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. PMID:25089709

  7. IgA-affinity purification and characterization of the lectin jacalin.

    PubMed

    Roque-Barreira, M C; Praz, F; Halbwachs-Mecarelli, L; Greene, L J; Campos-Neto, A

    1986-01-01

    We describe the use of IgA-Sepharose 4B affinity chromatography to purify the lectin jacalin from saline extracts of Artocarpus integrifolia L. seeds. Elution with 0.8 M D-galactose provides 10-15 mg lectin/50 mg seed protein. Jacalin behaved like a single component on immunoelectrophoresis and a single, somewhat diffuse band was obtained by polyacrylamide gel electrophoresis (PAGE) at pH 4.5. A single peak corresponding to an apparent molecular weight of 43 kDa was obtained by gel filtration on Sephadex G-75 (10 mM phosphate buffered saline (PBS), pH 7.4). On SDS-PAGE +/- 2-mercaptoethanol two bands of apparent molecular weights 11.8 and 14.7 kDa were detected. Jacalin behaved like a protein of apparent molecular weight of 13-14 kDa on Sephadex G-50 eluted with PBS containing 0.2% SDS. These data indicate that the jacalin molecule consists of 3-4 non-identical polypeptide subunits not connected by disulfide bridges. The amino acid composition of IgA affinity-purified jacalin (mol/405 mol amino acids) is Lys (24), His (5), Arg (4), Trp (6), Asx (36), Thr (35), Ser (48), Glx (31), Pro (18), Gly (53), Ala (13), Val (25), Met (3), Ile (23), Leu (25), Tyr (30), Phe (26), which corresponds to a molecular weight of 44.163 kDa.

  8. Avoiding degenerate coframes in an affine gauge approach to quantum gravity

    SciTech Connect

    Mielke, E.W.; McCrea, J.D.; Ne`eman, Y.; Hehl, F.W.

    1993-04-01

    This report discusses the following concepts on quantum gravity: The affine gauge approach; affine gauge transformations versus active differomorphisms; affine gauge approach to quantum gravity with topology change.

  9. Influenza A Virus-Induced Degradation of Eukaryotic Translation Initiation Factor 4B Contributes to Viral Replication by Suppressing IFITM3 Protein Expression

    PubMed Central

    Wang, Song; Chi, Xiaojuan; Wei, Haitao; Chen, Yuhai; Chen, Zhilong; Huang, Shile

    2014-01-01

    ABSTRACT Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. IMPORTANCE Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent

  10. Neural network modeling of distillation columns

    SciTech Connect

    Baratti, R.; Vacca, G.; Servida, A.

    1995-06-01

    Neural network modeling (NNM) was implemented for monitoring and control applications on two actual distillation columns: the butane splitter tower and the gasoline stabilizer. The two distillation columns are in operation at the SARAS refinery. Results show that with proper implementation techniques NNM can significantly improve column operation. The common belief that neural networks can be used as black-box process models is not completely true. Effective implementation always requires a minimum degree of process knowledge to identify the relevant inputs to the net. After background and generalities on neural network modeling, the paper describes efforts on the development of neural networks for the two distillation units.

  11. Development and Characterization of Immobilized Cannabinoid Receptor (CB1/CB2) Open Tubular Column for On-line Screening

    PubMed Central

    Moaddel, R.; Rosenberg, A.; Spelman, K.; Frazier, J.; Frazier, C.; Nocerino, S.; Brizzi, A.; Mugnaini, C.; Wainer, I.W.

    2011-01-01

    Cannabinoid Receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, (KU-812), were immobilized onto the surface of an open tubular capillary to create a CB1/CB2-OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2-OT column can be used to determine the binding affinities (Ki values) for a single compound and to screen individual or a mixture of multiple compounds. The CB1/CB2-OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high affinity phytocannabinoid. PMID:21215722

  12. Evidence of quinonoid structures in the vibrational spectra of thiophene based conducting polymers: Poly(thiophene), poly(thieno[3,4-b]benzene), and poly(thieno[3,4-b]pyrazine)

    NASA Astrophysics Data System (ADS)

    Cuff, Lilee; Kertesz, Miklos

    1997-04-01

    By combining vibrational spectra and ab initio calculations, we obtained a consistent description of the IR and nonresonant Raman spectra, including intensities, of four thiophene based polymers—undoped and heavily doped poly(thiophene) (PTh), undoped poly(thieno[3,4-b]benzene) (PITN), and poly(thieno[3,4-b]pyrazine) (PThP) for the first time. Predicted spectra for poly(thiophene) agree with experiment very well. Based on the calculated force constants and Badger's rule, we also estimated the average inter-ring bond lengths of undoped and doped PTh to be 1.47 and 1.42 Å, respectively. The latter leads to an estimated 33% quinonoid character on average for heavily doped PTh. The average inter-ring bond lengths of undoped PITN and PThP, that are consistent with their vibrational spectra, are estimated to be 1.41, and 1.42 Å, respectively. These values showed that undoped PITN and PThP have quinonoid character close to that of heavily doped PTh. Further, we also estimated that, upon doping the average bond lengths of PTh changed by -0.01, 0.11, and -0.05 Å for intra-ring Cβ-Cβ, Cα-Cβ, and inter-ring bonds, respectively. These bond length changes are significantly different from those of Hartree-Fock-type calculations, reflecting significant correlation contributions and are also in conflict with earlier empirical fits of the vibrational spectrum of the highly doped phase of PTh. However, our results are more in line with the generally accepted picture of an aromatic to quinonoid "transition" of the doping process. Furthermore, the counterintuitive downward frequency shifts in the vibrational spectra of PTh upon doping can be explained by the structural change from an essentially aromatic to a partially quinonoid form.

  13. PDE4B mediates local feedback regulation of β₁-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes.

    PubMed

    Mika, Delphine; Richter, Wito; Westenbroek, Ruth E; Catterall, William A; Conti, Marco

    2014-03-01

    Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their non-redundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in β-adrenergic (βAR) signaling in the heart. Genetic ablation of PDE4B disrupted βAR-induced cAMP transients, as measured by FRET sensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates β1AR-, but not β2AR- or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitation-contraction coupling that reside in the sarcolemmal compartment, including L-type Ca(2+) channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. β1AR- but not β2AR-ligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKA-dependent feedback that controls β1AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca(2+) level and contraction rate.

  14. Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b.

    PubMed

    Rosel, J; Moss, B

    1985-12-01

    We prepared antiserum that reacted with a major core polypeptide of approximately 62,000 daltons (62K polypeptide), designated 4b, and its 74K precursor, designated P4b. A cell-free translation product of vaccinia virus late mRNA that comigrated with P4b was specifically immunoprecipitated. The late mRNA encoding P4b hybridized to restriction fragments derived from the left end of the HindIII A fragment and to a lesser extent from the right side of the HindIII D fragment. A polypeptide that comigrated with P4a, the precursor of another major core polypeptide, was synthesized by mRNA that hybridized to DNA segments upstream of the P4b gene. Complete nucleotide sequence analysis of the P4b gene revealed an open reading frame, entirely within the HindIII A fragment, that was sufficient to encode a 644-amino-acid polypeptide of 73K. The 5' end of the P4b mRNA was located at or just above the translational initiation site.

  15. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  16. High-affinity Cyclic Peptide Matriptase Inhibitors*

    PubMed Central

    Quimbar, Pedro; Malik, Uru; Sommerhoff, Christian P.; Kaas, Quentin; Chan, Lai Y.; Huang, Yen-Hua; Grundhuber, Maresa; Dunse, Kerry; Craik, David J.; Anderson, Marilyn A.; Daly, Norelle L.

    2013-01-01

    The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase. PMID:23548907

  17. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  18. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  19. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  20. The HMG-box transcription factor Sox4b is required for pituitary expression of gata2a and specification of thyrotrope and gonadotrope cells in zebrafish.

    PubMed

    Quiroz, Yobhana; Lopez, Mauricio; Mavropoulos, Anastasia; Motte, Patrick; Martial, Joseph A; Hammerschmidt, Matthias; Muller, Marc

    2012-06-01

    The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshβ), glycoprotein subunit α (gsuα), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshβ and gsuα expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slβ, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshβ and gsuα expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshβ and gsuα expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhβ and fshβ) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary.

  1. Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

    PubMed

    Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

    2013-11-01

    The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

  2. Fate and Transport of Molybdenum Disulfide Nanomaterials in Sand Columns

    PubMed Central

    Lanphere, Jacob D.; Luth, Corey J.; Guiney, Linda M.; Mansukhani, Nikhita D.; Hersam, Mark C.; Walker, Sharon L.

    2015-01-01

    Abstract Research and development of two-dimensional transition metal dichalcogenides (TMDC) (e.g., molybdenum disulfide [MoS2]) in electronic, optical, and catalytic applications has been growing rapidly. However, there is little known regarding the behavior of these particles once released into aquatic environments. Therefore, an in-depth study regarding the fate and transport of two popular types of MoS2 nanomaterials, lithiated (MoS2-Li) and Pluronic PF-87 dispersed (MoS2-PL), was conducted in saturated porous media (quartz sand) to identify which form would be least mobile in aquatic environments. The electrokinetic properties and hydrodynamic diameters of MoS2 as a function of ionic strength and pH were determined using a zeta potential analyzer and dynamic light scattering techniques. Results suggest that the stability is significantly decreased beginning at 10 and 31.6 mM KCl, for MoS2-PL and MoS2-Li, respectively. Transport study results from breakthrough curves, column dissections, and release experiments suggest that MoS2-PL exhibits a greater affinity to be irreversibly bound to quartz surfaces as compared with the MoS2-Li at a similar ionic strength. Derjaguin–Landau–Verwey–Overbeek theory was used to help explain the unique interactions between the MoS2-PL and MoS2-Li surfaces between particles and with the quartz collectors. Overall, the results suggest that the fate and transport of MoS2 is dependent on the type of MoS2 that enters the environment, where MoS2-PL will be least mobile and more likely be deposited in porous media from pluronic–quartz interactions, whereas MoS2-Li will travel greater distances and have a greater tendency to be remobilized in sand columns. PMID:25741176

  3. Rational stabilization of the C-LytA affinity tag by protein engineering.

    PubMed

    Hernández-Rocamora, Víctor M; Maestro, Beatriz; Mollá-Morales, Almudena; Sanz, Jesús M

    2008-12-01

    The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization. PMID:18840883

  4. The accretion column of AE Aqr

    NASA Astrophysics Data System (ADS)

    Rodrigues, Claudia; Costa, D. Joaquim; Luna, Gerardo; Lima, Isabel J.; Silva, Karleyne M. G.; De Araujo, Jose Carlos N.; Coelho, Jaziel

    2016-07-01

    AE Aqr is a magnetic cataclysmic variable, whose white dwarf rotates at the very fast rate of 33 s modulating the flux from high-energies to optical wavelengths. There are many studies of the origin of its emission, which consider emission from a rotating magnetic field or from an accretion column. Recently, MAGIC observations have discarded AE Aqr emission in very high energy gamma-rays discarding non-thermal emission. Furthermore, soft and hard X-ray data from Swift and NuSTAR were fitted using thermal models. Here we present the modelling of AE Aqr X-ray spectra and light curve considering the emission of a magnetic accretion column using the Cyclops code. The model takes into consideration the 3D geometry of the system, allowing to properly represent the white-dwarf auto eclipse, the pre-shock column absorption, and the varying density and temperature of a tall accretion column.

  5. PRTR ion exchange vault column sampling

    SciTech Connect

    Cornwell, B.C.

    1995-03-14

    This report documents ion exchange column sampling and Non Destructive Assay (NDA) results from activities in 1994, for the Plutonium Recycle Test Reactor (PRTR) ion exchange vault. The objective was to obtain sufficient information to prepare disposal documentation for the ion exchange columns found in the PRTR Ion exchange vault. This activity also allowed for the monitoring of the liquid level in the lower vault. The sampling activity contained five separate activities: (1) Sampling an ion exchange column and analyzing the ion exchange media for purpose of waste disposal; (2) Gamma and neutron NDA testing on ion exchange columns located in the upper vault; (3) Lower vault liquid level measurement; (4) Radiological survey of the upper vault; and (5) Secure the vault pending waste disposal.

  6. A Versatile, Automatic Chromatographic Column Packing Device

    ERIC Educational Resources Information Center

    Barry, Eugene F.; And Others

    1977-01-01

    Describes an inexpensive apparatus for packing liquid and gas chromatographic columns of high efficiency. Consists of stainless steel support struts, an Automat Getriebmotor, and an associated three-pulley system capable of 10, 30, and 300 rpm. (MLH)

  7. Fabrication of Graphite/Epoxy Column Elements

    NASA Technical Reports Server (NTRS)

    Bluck, R. M.; Grotbeck, G. H.; Reighard, W. M.

    1983-01-01

    Dimensionally precise columns wound on vertical mandrels. Dry fiber wound on tapered aluminum mandrel and outer sleeve. Winding and injection done at elevated temperature to minimize thermal-expansion problems during curing of resin. Technique used in textile industry.

  8. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach. PMID:27498895

  9. Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

    PubMed

    Bi, Cong; Zheng, Xiwei; Hage, David S

    2016-02-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  10. Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.

    PubMed Central

    Gennis, L S

    1976-01-01

    A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested. PMID:186779

  11. Modeling Tropical Precipitation in a Single Column.

    NASA Astrophysics Data System (ADS)

    Sobel, Adam H.; Bretherton, Christopher S.

    2000-12-01

    A modified formulation of the traditional single column model for representing a limited area near the equator is proposed. This formulation can also be considered a two-column model in the limit as the area represented by one of the columns becomes very large compared to the other. Only a single column is explicitly modeled, but its free tropospheric temperature, rather than its mean vertical velocity, is prescribed. This allows the precipitation and vertical velocity to be true prognostic variables, as in prior analytical theories of tropical precipitation. Two models developed by other authors are modified according to the proposed formulation. The first is the intermediate atmospheric model of J. D. Neelin and N. Zeng, but with the horizontal connections between columns broken, rendering it a set of disconnected column models. The second is the column model of N. O. Rennó, K. A. Emanuel, and P. H. Stone. In the first model, the set of disconnected column models is run with a fixed temperature that is uniform in the Tropics, and insolation, SST, and surface wind speed taken from a control run of the original model. The column models produce a climatological precipitation field that is grossly similar to that of the control run, despite that the circulation implied by the column models is not required to conserve mass. The addition of horizontal moisture advection by the wind from the control run substantially improves the simulation in dry regions. In the second model the sensitivity of the modeled steady-state precipitation and relative humidity to varying SST and wind speed is examined. The transition from shallow to deep convection is simulated in a `Lagrangian' calculation in which the column model is subjected to an SST that increases in time. In this simulation, the onset of deep convection is delayed to a higher SST than in the steady-state case, due to the effect of horizontal moisture advection (viewed in a Lagrangian reference frame). In both of the

  12. Flow in a metal hydride chromatographic column

    SciTech Connect

    Nichols, G.S.

    1990-01-01

    The flow of hydrogen isotopes in a metal hydride chromatographic column is calculated by a one-dimensional finite difference method. The Ergun equation is used to define the gas flow; and equilibrium pressure isotherms are used to define the column holdup. Solid phase loadings are shown to move as a wave front on absorption, but remain more uniform on desorption. 3 refs., 4 figs.

  13. Commander prepares glass columns for electrophoresis experiment

    NASA Technical Reports Server (NTRS)

    1982-01-01

    Commander Jack Lousma prepares on of the glass columns for the electrophoresis test in the middeck area of the Columbia. The experiment, deployed in an L-shaped mode in upper right corner, consists of the processing unit with glass columns in which the separation takes place; a camera (partially obscurred by Lousma's face) to document the process; and a cryogenic freezer to freeze and store the samples after separation.

  14. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

    PubMed

    Buffalo, Cosmo Z; Bahn-Suh, Adrian J; Hirakis, Sophia P; Biswas, Tapan; Amaro, Rommie E; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  15. Magnetic properties and glass formability of Fe61Co10Zr5W4B20 bulk metallic glassy alloy

    NASA Astrophysics Data System (ADS)

    Pawlik, P.; Davies, H. A.; Gibbs, M. R. J.

    2003-10-01

    Amorphous melt spun ribbon samples of Fe61Co10Zr5W4B20 alloy of various thicknesses up to ˜350 μm and suction cast 1 mm diameter rod and 2, 3, and 4 mm outside diameter tube samples of the same alloy were produced. The experimental results for the rod samples indicate a relatively high saturation polarization μ0Ms=0.8 T and a Curie temperature TC=615 K, as well as a very low coercive field Hc=1.4±0.5 A/m with high anisotropy that lowers the permeability of the samples. Thermal analysis by differential scanning calorimetry carried out on ribbon and rod samples revealed a large supercooled liquid region ΔTx of ˜60 K for this alloy.

  16. Spectral properties of 1H-pyrazolo[3,4-b]quinoline substituted with N,N-diethylamine moiety

    NASA Astrophysics Data System (ADS)

    Kolbus, Anna; Grabka, Danuta; Danel, Andrzej; Szary, Karol

    2016-07-01

    Photophysical properties of 6-N,N-diethyl-3-methyl-1-phenyl-1H-pyrazol[3,4-b]quinoline (DEPQ), a potential material for electroluminescent applications, were investigated. The absorption and fluorescence spectra and fluorescence lifetimes were recorded in a great number of solvents with different polarity. The red shifts in absorption and fluorescence maxima with the solvent's polarity were observed. Different trends in values of quantum yield for non-polar and polar solvents suggest two different deactivation ways of the excited state, depending on solvent polarity. DEPQ in non-polar solvents emits from locally excited states while deactivation DEPQ in polar solvents indicates charge transfer (CT) fluorescence. Several electro-optical parameters were also calculated.

  17. Anillin-dependent organization of septin filaments promotes intercellular bridge elongation and Chmp4B targeting to the abscission site.

    PubMed

    Renshaw, Matthew J; Liu, Jinghe; Lavoie, Brigitte D; Wilde, Andrew

    2014-01-01

    The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.

  18. Photocatalytic degradation of Chicago Sky Blue 6B and Benzopurpurin 4B using titanium dioxide thin film.

    PubMed

    Mohammed, Abdul K; McKenzie, Katrina T

    2005-01-01

    Aqueous solutions of azo dyes undergo degradation to form harmless intermediates and colorless products following irradiation by visible light in the presence of titanium dioxide thin films. The dyes that were studied in this work are: Chicago Sky Blue 6B and Benzopurpurin 4B. Results obtained indicated that complete mineralization of the dyes took place under the experimental conditions. There was an increase in conductivity after the complete mineralization experiments possibly indicating the formation of ions such as NO3- and SO4(2-). Chemical oxygen demand (COD) measurements show a decrease in organic matter for both dyes following complete degradation. The effect of how changing experimental conditions such as pH, temperature and starting concentrations of dyes affected the rate of dye degradation was measured. There was an increase in the rate of disappearance of the dye color at lower pH. High concentrations of dye solutions required long degradation time.

  19. Avoid problems during distillation column startups

    SciTech Connect

    Sloley, A.W.

    1996-07-01

    The startup of a distillation column is the end product of the design process. Indeed, startup is the culmination of the theory and practice of designing the column to meet the process objectives. The author will direct most of this discussion towards column revamps due to their inherent complexity; however, the points apply equally to new columns, as well. The most important question that must be answered prior to a startup is how will the distillation system changes affect initial startup, process control of the system, and normal day-to-day operations? How will the operators run the system? Steady-state distillation-column simulations alone cannot provide an authoritative answer and, indeed, engineers` over-reliance on software too often has led them to ignore many practical aspects. Computer modeling, while an important engineering tool, is not reality. Distillation columns are real functioning pieces of equipment that require practical skills to successfully modify. They are not steady-state solutions that result from converged computer simulations. Early planning, coupled with thorough inspections and comprehensive reviews of instrumentation and procedures, can play a key role in assuring smooth startups.

  20. Interpretation of the lime column penetration test

    NASA Astrophysics Data System (ADS)

    Liyanapathirana, D. S.; Kelly, R. B.

    2010-06-01

    Dry soil mix (DSM) columns are used to reduce the settlement and to improve the stability of embankments constructed on soft clays. During construction the shear strength of the columns needs to be confirmed for compliance with technical assumptions. A specialized blade shaped penetrometer known as the lime column probe, has been developed for testing DSM columns. This test can be carried out as a pull out resistance test (PORT) or a push in resistance test (PIRT). The test is considered to be more representative of average column shear strength than methods that test only a limited area of the column. Both PORT and PIRT tests require empirical correlations of measured resistance to an absolute measure of shear strength, in a similar manner to the cone penetration test. In this paper, finite element method is used to assess the probe factor, N, for the PORT test. Due to the large soil deformations around the probe, an Arbitrary Lagrangian Eulerian (ALE) based finite element formulation has been used. Variation of N with rigidity index and the friction at the probe-soil interface are investigated to establish a range for the probe factor.

  1. Gas Chromatograph Method Optimization Trade Study for RESOLVE: 20-meter Column v. 8-meter Column

    NASA Technical Reports Server (NTRS)

    Huz, Kateryna

    2014-01-01

    RESOLVE is the payload on a Class D mission, Resource Prospector, which will prospect for water and other volatile resources at a lunar pole. The RESOLVE payload's primary scientific purpose includes determining the presence of water on the moon in the lunar regolith. In order to detect the water, a gas chromatograph (GC) will be used in conjunction with a mass spectrometer (MS). The goal of the experiment was to compare two GC column lengths and recommend which would be best for RESOLVE's purposes. Throughout the experiment, an Inficon Fusion GC and an Inficon Micro GC 3000 were used. The Fusion had a 20m long column with 0.25mm internal diameter (Id). The Micro GC 3000 had an 8m long column with a 0.32mm Id. By varying the column temperature and column pressure while holding all other parameters constant, the ideal conditions for testing with each column length in their individual instrument configurations were determined. The criteria used for determining the optimal method parameters included (in no particular order) (1) quickest run time, (2) peak sharpness, and (3) peak separation. After testing numerous combinations of temperature and pressure, the parameters for each column length that resulted in the most optimal data given my three criteria were selected. The ideal temperature and pressure for the 20m column were 95 C and 50psig. At this temperature and pressure, the peaks were separated and the retention times were shorter compared to other combinations. The Inficon Micro GC 3000 operated better at lower temperature mainly due to the shorter 8m column. The optimal column temperature and pressure were 70 C and 30psig. The Inficon Micro GC 3000 8m column had worse separation than the Inficon Fusion 20m column, but was able to separate water within a shorter run time. Therefore, the most significant tradeoff between the two column lengths was peak separation of the sample versus run time. After performing several tests, it was concluded that better

  2. Poly(thieno[3,4-b]thiophene)-poly(styrene sulfonate): a low band gap, water dispersible conjugated polymer.

    PubMed

    Lee, Byoungchul; Seshadri, Venkataramanan; Sotzing, Gregory A

    2005-11-01

    Herein we report the oxidative chemical polymerization of thieno[3,4-b]thiophene (T34bT) using several different oxidants including ferric sulfate, ammonium persulfate, and hydrogen peroxide in the presence of poly(styrenesulfonic acid) in water and properties of the resulting poly(thieno[3,4-b]thiophene)-poly(styrenesulfonic acid) (PT34bT-PSS) dispersion. The PT34bT-PSS is rendered a colloidal dispersion in water with a particle size diameter ranging between 180 and 220 nm depending on the oxidant used for polymerization. PT34bT-PSS films have band gaps of ca. 1 eV (1260 nm) as determined by the onset of the pi to pi transition from the vis-NIR spectrum with absorption maxima ranging from 1.4 eV (912 nm) to 1.7 eV (724 nm). The neutral and oxidized forms of PT34bT-PSS prepared from ferric sulfate dispersed in water were blue and lime green, respectively, whereas the neutral and oxidized forms of PT34bT-PSS prepared from ammonium persulfate and hydrogen peroxide were blue and blue-green, respectively. Spectral properties of the PT34bT-PSS dispersion can be tuned by the combination of oxidants. PT34bT-PSS films showed ca. 100% cation dominant ion transport behavior as determined by electrochemical gravimetry with each charge-discharge cycle and the doping level of the polymer was calculated to be 26%. Electrical conductivities for these polymers were found to be dependent on chemical oxidants used and varied from 10(-2) to 10(-4) S/cm. PMID:16262354

  3. Donor Splice-Site Mutation in CUL4B is Likely Cause of X-Linked Intellectual Disability

    PubMed Central

    Londin, Eric R.; Adijanto, Jeffrey; Philp, Nancy; Novelli, Antonio; Vitale, Emilia; Perria, Chiara; Serra, Gigliola; Alesi, Viola; Surrey, Saul; Fortina, Paolo

    2015-01-01

    X-linked intellectual disability is the most common form of cognitive disability in males. Syndromic intellectual disability encompasses cognitive deficits with other medical and behavioral manifestations. Recently, a large family with a novel form of syndromic X-linked intellectual disability was characterized. Eight of 24 members of the family are male and had cognitive dysfunction, short stature, aphasia, skeletal abnormalities, and minor anomalies. To identify the causative gene(s), we performed exome sequencing in three affected boys, both parents, and an unaffected sister. We identified a haplotype consisting of eight variants located in cis within the linkage region that segregated with affected members in the family. Of these variants, two were novel. The first was at the splice-donor site of intron 7 (c.974+1G>T) in the cullin-RING ubiquitin ligase (E3) gene, CUL4B. This variant is predicted to result in failure to splice and remove intron 7 from the primary transcript. The second variant mapped to the 3′-UTR region of the KAISO gene (c.1127T>G). Sanger sequencing validated the variants in these relatives as well as in three affected males and five carriers. The KAISO gene variant was predicted to create a binding site for the microRNAs miR-4999 and miR-4774; however, luciferase expression assays failed to validate increased targeting of these miRNAs to the variant 3′-UTR. This SNP may affect 3′-UTR structure leading to decreased mRNA stability. Our results suggest that the intellectual disability phenotype in this family is caused by aberrant splicing and removal of intron 7 from CUL4B gene primary transcript. PMID:24898194

  4. Structure of a High-Affinity

    SciTech Connect

    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 Res. Inst. /Simon Fraser U. /British Columbia U.

    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

  5. Mass transfer model liquid phase catalytic exchange column simulation applicable to any column composition profile

    SciTech Connect

    Busigin, A.

    2015-03-15

    Liquid Phase Catalytic Exchange (LPCE) is a key technology used in water detritiation systems. Rigorous simulation of LPCE is complicated when a column may have both hydrogen and deuterium present in significant concentrations in different sections of the column. This paper presents a general mass transfer model for a homogenous packed bed LPCE column as a set of differential equations describing composition change, and equilibrium equations to define the mass transfer driving force within the column. The model is used to show the effect of deuterium buildup in the bottom of an LPCE column from non-negligible D atom fraction in the bottom feed gas to the column. These types of calculations are important in the design of CECE (Combined Electrolysis and Catalytic Exchange) water detritiation systems.

  6. A comparison of the properties of polyurethane immobilised Sphagnum moss, seaweed, sunflower waste and maize for the biosorption of Cu, Pb, Zn and Ni in continuous flow packed columns.

    PubMed

    Zhang, Yue; Banks, Charles

    2006-02-01

    The biosorption of Cu, Pb, Zn and Ni from a mixed solution of the metals was investigated in continuous flow packed columns containing polyurethane immobilised biomass. The characteristics and biosorption properties of Sphagnum moss, the brown seaweed Ascophyllum nodosum, waste biomass from the preparation of sunflower oil, and whole plant maize were compared. All the biomass types showed a preference for the sequestration of Pb followed by Cu, with Ni and Zn having roughly equal affinity. With continuous metal loading to the column there was an initial binding of all metals and then a displacement of the lower affinity metals by those with a high affinity. This led to a chromatographic effect in the column with breakthrough concentrations for low-affinity metals higher than the concentration in the feed. A similar phenomenon was found on desorption using acidic solutions where low-affinity metals were desorbed preferentially. The results also indicated that despite competitive displacement of one metal species by another the biomass appeared to succeed in retaining some low-affinity metal species indicating that there may be selective sites present with different affinity characteristics. When using a multi-metal solution with Cu, Pb, Zn and Ni at equal 10 mgl(-1) concentrations as column influent, the total quantities of metal sequestered were: seaweed, 117.3 mg g(-1); sunflower waste, 33.2 mg g(-1); Sphagnum moss, 32.5 mg g(-1); and maize, 2.3 mg g(-1). The use of an acid base potentiometric titration showed a relationship between the number of acid functional groups and biosorption capacity, although this was not proportional for the biomass types studied. It can, however, be used in conjunction with a simple classification of metals into high and low-affinity bands to make a preliminary assessment of a biosorption system.

  7. Effectively nonlocal metric-affine gravity

    NASA Astrophysics Data System (ADS)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  8. Wetting on rough self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Palasantzas, George

    1995-05-01

    In this paper, we present a general investigation of the effective potential for complete wetting on self-affine rough surfaces. The roughness effect is investigated by means of the height-height correlation model in Fourier space ~(1+aξ2q2)-1-H. The parameters H and ξ are, respectively, the roughness exponent and the substrate in-plane correlation length. It is observed that the effect of H on the free interface profile is significant for ξ>ξ) regime is characterized by a power-law scaling ~Y-2.

  9. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  10. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field. PMID:25570332

  11. Evaluation system of negative electron affinity photocathode

    NASA Astrophysics Data System (ADS)

    Fu, Rongguo; Chang, Benkang; Qian, Yunsheng; Wang, Guihua; Zong, Zhiyuan

    2001-10-01

    This article first describes the background of the research and manufacture of evaluation system of Negative Electron Affinity photocathode. This article designs a set of super high vacuum system for activating NEA photocathode on the base of activation theory, the process of design and debugging is given. The system is composed of three parts: super high vacuum system for GaAs material activation, multi-meter testing system, surface analysis system. The system is used for on-line evaluation of activating of NEA photocathode. The technical parameters and structure of the evaluation system of NEA photocathode are given in the paper. The system is finished and experiments are made. At last the picture of the system is given.

  12. Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome.

    PubMed

    Chong, Chan Eng; Lim, Kue Peng; Gan, Chai Phei; Marsh, Christina A; Zain, Rosnah Binti; Abraham, Mannil Thomas; Prime, Stephen S; Teo, Soo-Hwang; Silvio Gutkind, J; Patel, Vyomesh; Cheong, Sok Ching

    2012-08-01

    MAGE proteins have been shown to be good targets for cancer immunotherapy. We demonstrate that MAGED4B is over-expressed in more than 50% of Oral Squamous Cell Carcinoma (OSCC) tissues and the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines that over-express MAGED4B promote migration in vitro, exhibit an increase in cell growth both in vitro and in vivo, and are more resistant to apoptosis compared to control cells. Our data suggest that MAGED4B over-expression is a driver in oral carcinogenesis and argues strongly that this protein may represent a potential therapeutic target in OSCC. PMID:22459352

  13. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  14. Dye affinity cryogels for plasmid DNA purification.

    PubMed

    Çimen, Duygu; Yılmaz, Fatma; Perçin, Işık; Türkmen, Deniz; Denizli, Adil

    2015-11-01

    The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA. PMID:26249596

  15. Column Selection for Biomedical Analysis Supported by Column Classification Based on Four Test Parameters.

    PubMed

    Plenis, Alina; Rekowska, Natalia; Bączek, Tomasz

    2016-01-21

    This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the FKUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the FKUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the FKUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the FKUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis.

  16. Column Selection for Biomedical Analysis Supported by Column Classification Based on Four Test Parameters

    PubMed Central

    Plenis, Alina; Rekowska, Natalia; Bączek, Tomasz

    2016-01-01

    This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the FKUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the FKUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the FKUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the FKUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis. PMID:26805819

  17. Cullin 4B is recruited to tristetraprolin-containing messenger ribonucleoproteins and regulates TNF-α mRNA polysome loading.

    PubMed

    Pfeiffer, Jason R; Brooks, Seth A

    2012-02-15

    TNF-α is a central mediator of inflammation and critical for host response to infection and injury. TNF-α biosynthesis is controlled by transcriptional and posttranscriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-rich element binding protein that regulates the stability of the TNF-α mRNA. Using a screen to identify TTP-interacting proteins, we identified Cullin 4B (Cul4B), a scaffolding component of the Cullin ring finger ligase family of ubiquitin E3 ligases. Short hairpin RNA knockdown of Cul4B results in a significant reduction in TNF-α protein and mRNA in LPS-stimulated mouse macrophage RAW264.7 cells as well as a reduction in TTP protein. TNF-α message t(1/2) was reduced from 69 to 33 min in LPS-stimulated cells. TNF-3' untranslated region luciferase assays utilizing wild-type and mutant TTP-AA (S52A, S178A) indicate that TTP function is enhanced in Cul4B short hairpin RNA cells. Importantly, the fold induction of TNF-α mRNA polysome loading in response to LPS stimulation is reduced by Cul4B knockdown. Cul4B is present on the polysomes and colocalizes with TTP to exosomes and processing bodies, which are sites of mRNA decay. We conclude that Cul4B licenses the TTP-containing TNF-α messenger ribonucleoprotein for loading onto polysomes, and reduction of Cul4B expression shunts the messenger ribonucleoproteins into the degradative pathway.

  18. Interferon-dependent engagement of eukaryotic initiation factor 4B via S6 kinase (S6K)- and ribosomal protein S6K-mediated signals.

    PubMed

    Kroczynska, Barbara; Kaur, Surinder; Katsoulidis, Efstratios; Majchrzak-Kita, Beata; Sassano, Antonella; Kozma, Sara C; Fish, Eleanor N; Platanias, Leonidas C

    2009-05-01

    Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation factor 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-alpha) or IFN-gamma. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene 15 protein (ISG15) or IFN-gamma-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic fibroblasts. In addition, IFN-alpha-inducible ISG15 protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent growth inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases. PMID:19289497

  19. CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility.

    PubMed

    Aravindan, Rolands G; Fomin, Victor P; Naik, Ulhas P; Modelski, Mark J; Naik, Meghna U; Galileo, Deni S; Duncan, Randall L; Martin-Deleon, Patricia A

    2012-08-01

    Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions.

  20. Purification of glycolytic enzymes by using affinity-elution chromatography.

    PubMed Central

    Scopes, R K

    1977-01-01

    1. A systematic procedure for the purification of enzymes by affinity-elution chromatography is described. Enzymes are adsorbed on a cation-exchanger, and eluted with ligands specific for the enzyme concerned. 2. All of the glycolytic and some related enzymes present in rabbit muscle can be purified by the affinity-elution technique. The pH range for adsorption and elution of each enzyme was found, and the effects of minor variations of conditions are described. 3. A description of experimental conditions suitable for affinity elution of each enzyme is given, together with special features relevant to each individual enzyme. 4. Theoretical considerations of affinity elution chromatography are discussed, including its limitations, advantages and disadvantages compared with affinity-adsorption chromatography. Possible developments are suggested to cover enzymes which because of their adsorption characteristics are not at present amenable to affinity-elution procedures. PMID:192194