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Sample records for affinity constant kd

  1. How constant are pesticide Kd values during a rainfall event?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pesticide partitioning between water and sediment (Kd) and surface processes controlling runoff and sediment production determine the magnitude of pesticide losses associated with infiltration, runoff and/or sediment from agricultural fields during a rainfall event. Pesticide Kd values have traditio...

  2. 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

  3. Determination of proton affinities and acidity constants of sugars.

    PubMed

    Feng, Shuting; Bagia, Christina; Mpourmpakis, Giannis

    2013-06-20

    Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions. PMID:23706015

  4. Defect in the membrane expression of high affinity 72-kD Fc gamma receptors on phagocytic cells in four healthy subjects.

    PubMed Central

    Ceuppens, J L; Baroja, M L; Van Vaeck, F; Anderson, C L

    1988-01-01

    Three different receptors for the Fc portion of IgG (FcR) have been characterized on human leukocytes. We have identified four healthy members of one family, whose blood phagocytic cells lack functional 72 kD high-affinity FcRI. Their monocytes were unable to bind the Fc portion of mouse (m)-IgG2a and of monomeric human IgG, and they were unreactive with two anti-FcRI monoclonal antibodies. Thus, FcRI is either absent, expressed at very low density, or is so structurally altered as to be unable to bind both its ligand and the anti-FcRI antibodies. The failure to bind the Fc portion of mIgG2a underlies the previously reported inability of these monocytes to support T cell mitogenesis on OKT3 stimulation. FcRI was not inducible upon incubation of their monocytes or neutrophils in gamma interferon. However, their monocytes were able to bind aggregated human IgG, and to phagocytose IgG-coated particles in vitro. Both functions could be blocked with a monoclonal antibody to the 40-kD low-affinity FcRII and therefore apparently were mediated exclusively through FcRII. This also demonstrates that FcRII can mediate phagocytosis independently. Despite the FcRI defect, these subjects had no circulating immune complexes, no evidence of autoimmune pathology and no increased susceptibility to infections. PMID:2969920

  5. Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

    NASA Astrophysics Data System (ADS)

    Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

    2012-10-01

    NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

  6. Ab initio electron affinity and hyperfine structure constants of ^231Pa:

    NASA Astrophysics Data System (ADS)

    Dinov, Konstantin D.; Beck, Donald R.

    1996-05-01

    We have performed valence shell Relativistic Configuration Interaction calculations(Konstantin D. Dinov and Donald R. Beck, Electron affinity and hyperfine structure constants of Pa^-: 7p attachment.) Submitted to Phys. Rev. A for the Electron Affinity (EA) of ^231Pa. Our result of 0.222 eV for the binding energy of the Pa^- 5f^2 6d 7s^2 7p J=6 state is consistent with the experimental yield(X-L. Zhao, M-J. Nadeau, M.A. Garwan, L.R. Kilius and A.E. Litherland, Nuc. Instr. Meth. B 92), 258-64 (1994). Our result for the hyperfine structure constants of Pa^-, is the first available ab initio result. No other bound states were found for the 7p attachment. We didn't find evidence to support possible 5d attachment in this system. This work extends our previous calculations for the Rare Earth negative ions(K.D. Dinov and D.R. Beck, Phys. Rev. A 52) , 2632-37 (1995); K. Dinov and D.R. Beck, Phys. Rev. A 51 (2), 1680-82 (1995); K. Dinov, D.R. Beck and D. Datta, Phys. Rev. A 50 (2), 1144-48 (1994).

  7. Bayesian Estimation of the Active Concentration and Affinity Constants Using Surface Plasmon Resonance Technology.

    PubMed

    Feng, Feng; Kepler, Thomas B

    2015-01-01

    Surface plasmon resonance (SPR) has previously been employed to measure the active concentration of analyte in addition to the kinetic rate constants in molecular binding reactions. Those approaches, however, have a few restrictions. In this work, a Bayesian approach is developed to determine both active concentration and affinity constants using SPR technology. With the appropriate prior probabilities on the parameters and a derived likelihood function, a Markov Chain Monte Carlo (MCMC) algorithm is applied to compute the posterior probability densities of both the active concentration and kinetic rate constants based on the collected SPR data. Compared with previous approaches, ours exploits information from the duration of the process in its entirety, including both association and dissociation phases, under partial mass transport conditions; do not depend on calibration data; multiple injections of analyte at varying flow rates are not necessary. Finally the method is validated by analyzing both simulated and experimental datasets. A software package implementing our approach is developed with a user-friendly interface and made freely available. PMID:26098764

  8. Bayesian Estimation of the Active Concentration and Affinity Constants Using Surface Plasmon Resonance Technology

    PubMed Central

    Feng, Feng; Kepler, Thomas B.

    2015-01-01

    Surface plasmon resonance (SPR) has previously been employed to measure the active concentration of analyte in addition to the kinetic rate constants in molecular binding reactions. Those approaches, however, have a few restrictions. In this work, a Bayesian approach is developed to determine both active concentration and affinity constants using SPR technology. With the appropriate prior probabilities on the parameters and a derived likelihood function, a Markov Chain Monte Carlo (MCMC) algorithm is applied to compute the posterior probability densities of both the active concentration and kinetic rate constants based on the collected SPR data. Compared with previous approaches, ours exploits information from the duration of the process in its entirety, including both association and dissociation phases, under partial mass transport conditions; do not depend on calibration data; multiple injections of analyte at varying flow rates are not necessary. Finally the method is validated by analyzing both simulated and experimental datasets. A software package implementing our approach is developed with a user-friendly interface and made freely available. PMID:26098764

  9. The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

    PubMed Central

    Zheng, Xiliang; Wang, Jin

    2015-01-01

    We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

  10. The universal statistical distributions of the affinity, equilibrium constants, kinetics and specificity in biomolecular recognition.

    PubMed

    Zheng, Xiliang; Wang, Jin

    2015-04-01

    We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

  11. Studies on 17,24 kD Depleted Photosystem II Membranes : I. Evidences for High and Low Affinity Calcium Sites in 17,24 kD Depleted PSII Membranes from Wheat versus Spinach.

    PubMed

    Cammarata, K V; Cheniae, G M

    1987-07-01

    Analyses were made of the effects of extraction of the 17,24 kilodalton extrinsic proteins from spinach versus wheat photosystem II (PSII) membranes on Ca abundance and O(2) evolution capacity determined in the absence and presence of either Cl(-) or Ca(2+). Extraction of these proteins from spinach PSII routinely diminished steady state O(2) evolution by about 70% when assayed in the presence of sufficient Cl(-). Additionally, O(2) evolution of 17,24 kilodalton-less spinach PSII membranes showed about 2-fold more enhancement by Ca(2+) than by Cl(-) during assay. When the same extraction and assay procedures were applied to wheat PSII membranes, we observed, in contrast to 17,24 kilodalton-less spinach PSII, only about 50% inhibition of O(2) evolution and about 2-fold greater enhancement by Cl(-) than by Ca(2+). Irrespective of differences in the magnitude of enhancement of O(2) evolution by Ca(2+)versus Cl(-) in spinach versus wheat, the K(m) values for Cl(-) (about 1.7 millimolar) and Ca(2+) (about 1.5 millimolar) were similar for both type preparations. The abundance of Ca specifically associated with fully functional PSII (about 2 and about 3 Ca/200 chlorophyll for spinach and wheat, respectively) was diminished to about 1 per 200 chlorophyll upon 17.24 kilodalton protein depletion. Further treatment of wheat 17,24 kilodalton-less PSII in darkness with 2 molar NaCl/1 millimolar ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid/20 micromolar A23187(2) made O(2) evolution highly dependent on Ca(2+) addition, much like the 17,24 kilodalton-less spinach PSII. Analyses of this Ca(2+) effect on O(2) evolution revealed both high (K(m) about 65 micromolar) and low (K(m) about 1.5 millimolar) affinity Ca(2+) sites in wheat 17,24 kilodalton-less PSII. The results suggest that during 17,24 kilodalton extraction by NaCl, spinach PSII is more susceptible than wheat PSII to loss of high affinity Ca and irreversible inhibition of O(2) evolution. PMID:16665485

  12. Evaluation of Taylor dispersion injections: determining kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing.

    PubMed

    Quinn, John G

    2012-02-15

    In label-free biomolecular interaction analysis, a standard injection provides an injection of uniform analyte concentration. An alternative approach exploiting Taylor dispersion produces a continuous analyte titration allowing a full analyte dose response to be recorded in a single injection. The enhanced biophysical characterization that is possible with this new technique is demonstrated using a commercially available surface plasmon resonance-based biosensor. A kinetic interaction model was fitted locally to Taylor dispersion curves for estimation of the analyte diffusion coefficient in addition to affinity/kinetic constants. Statistical confidence in the measured parameters from a single Taylor dispersion injection was comparable to that obtained for global analysis of multiple standard injections. The affinity constants for multisite interactions were resolved with acceptable confidence limits. Importantly, a single analyte injection could be treated as a high-resolution real-time affinity isotherm and was demonstrated using the complex two-site interaction of warfarin with human serum albumin. In all three model interactions tested, the kinetic/affinity constants compared favorably with those obtained from standard kinetic analysis and the estimates of analyte diffusion coefficients were in good agreement with the expected values. PMID:22197422

  13. High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants.

    PubMed

    Yao, Chunhe; Qi, Li; Qiao, Juan; Zhang, Haizhi; Wang, Fuyi; Chen, Yi; Yang, Gengliang

    2010-09-15

    A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of D,L-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of D-tryptophan, with a linear range between 12.0 microM and 979.0 microM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion. PMID:20801337

  14. 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

  15. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    PubMed

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives. PMID:27391930

  16. Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

    SciTech Connect

    Cheng, Chih-Ming; Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan; Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 30043, Taiwan ; Lee, Yuarn-Jang; Wang, Wei-Ting; Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan; Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan ; Hsu, Chien-Ting; Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan ; Tsai, Jing-Shin; Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei 110, Taiwan; Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan ; Wu, Chien-Ming; Ou, Keng-Liang; Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan ; and others

    2011-01-07

    Research highlights: {yields} PD153035 is a DNA intercalator and intercalation occurs only under very low salt concentration. {yields} The minimum distance between adjacent bound PD153035 {approx} 11 bp. {yields} Binding affinity constant for PD153035 is 1.18({+-}0.09) x 10{sup 4} (1/M). {yields} The change of binding free energy of PD153035-DNA interaction is -5.49 kcal mol{sup -1} at 23 {+-} 0.5 {sup o}C. -- Abstract: Accurately predicting binding affinity constant (K{sub A}) is highly required to determine the binding energetics of the driving forces in drug-DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K{sub A} for PD153035, where K{sub A} is determined from the changes in B-form contour length (L) of PD153035-DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K{sub A} = 1.18({+-}0.09) x 10{sup 4} (1/M) at 23 {+-} 0.5 {sup o}C and the minimum distance between adjacent bound PD153035 {approx} 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

  17. From the affinity constant to the half-saturation index: understanding conventional modeling concepts in novel wastewater treatment processes.

    PubMed

    Arnaldos, Marina; Amerlinck, Youri; Rehman, Usman; Maere, Thomas; Van Hoey, Stijn; Naessens, Wouter; Nopens, Ingmar

    2015-03-01

    The "affinity constant" (KS) concept is applied in wastewater treatment models to incorporate the effect of substrate limitation on process performance. As an increasing number of wastewater treatment processes rely on low substrate concentrations, a proper understanding of these so-called constants is critical in order to soundly model and evaluate emerging treatment systems. In this paper, an in-depth analysis of the KS concept has been carried out, focusing on the different physical and biological phenomena that affect its observed value. By structuring the factors influencing half-saturation indices (newly proposed nomenclature) into advectional, diffusional and biological, light has been shed onto some of the apparent inconsistencies present in the literature. Particularly, the importance of non-ideal mixing as a source of variability between observed KS values in different systems has been illustrated. Additionally, discussion on the differences existent between substrates that affect half-saturation indices has been carried out; it has been shown that the observed KS for some substrates will reflect transport or biological limitations more than others. Finally, potential modeling strategies that could alleviate the shortcomings of the KS concept have been provided. These could be of special importance when considering the evaluation and design of emerging wastewater treatment processes. PMID:25576693

  18. Determination of binding constants by affinity capillary electrophoresis, electrospray ionization mass spectrometry and phase-distribution methods

    PubMed Central

    Chen, Zhi; Weber, Stephen G.

    2008-01-01

    Many methods for determining intermolecular interactions have been described in the literature in the past several decades. Chief among them are methods based on spectroscopic changes, particularly those based on absorption or nuclear magnetic resonance (NMR) [especially proton NMR (1H NMR)]. Recently, there have been put forward several new methods that are particularly adaptable, use very small quantities of material, and do not place severe requirements on the spectroscopic properties of the binding partners. This review covers new developments in affinity capillary electrophoresis, electrospray ionization mass spectrometry (ESI-MS) and phasetransfer methods. PMID:19802330

  19. Evaluation of three neutral capillary coatings for the determination of analyte-cyclodextrin binding constants by affinity capillary electrophoresis. Application to N,N'-disubstituted piperazine derivatives.

    PubMed

    Danel, Cécile; Melnyk, Patricia; Azaroual, Nathalie; Larchanché, Paul-Emmanuel; Goossens, Jean-François; Vaccher, Claude

    2016-07-15

    The performances of three neutral static coatings (hydroxypropyl cellulose, polyethylene oxide and poly(N,N-dimethylacrylamide) have been evaluated in order to determine the binding constants of the complexes formed between four polycationic compounds (piperazine derivatives) and four cyclodextrins of pharmaceutical interest (β-CD, HP-β-CD, Me-β-CD and sulfobutyl ether-β-CD) by affinity capillary electrophoresis. The physically-adsorbed poly(N,N-dimethylacrylamide) coating proves to be the more efficient to mask the silanol groups of the capillary wall since the lowest electroosmotic flow was measured for this coating. Moreover, it drastically reduces the adsorption of the compounds since it allows a correct repeatability of their migration time, higher efficiencies of the peaks and no baseline shift. Then, it was verified for four complexes that this coating allows a correct determination of the binding constants avoiding the CD adsorption which is responsible of an undervaluation of binding constants. The highest binding constants are obtained using the anionic sulfobutyl ether-β-CD (SBE-β-CD). The structure of the complex formed between the tacrine derivative and the SBE-β-CD was further investigated through 2D ROESY NMR experiments and structure-binding constant relationships. Results suggest that the inclusion in the SBE-β-CD cavity occurs through the aliphatic ring portion of the tacrine moiety. PMID:27286645

  20. Modeling Taylor dispersion injections: determination of kinetic/affinity interaction constants and diffusion coefficients in label-free biosensing.

    PubMed

    Quinn, John G

    2012-02-15

    A new method based on Taylor dispersion has been developed that enables an analyte gradient to be titrated over a ligand-coated surface for kinetic/affinity analysis of interactions from a minimal number of injections. Taylor dispersion injections generate concentration ranges in excess of four orders of magnitude and enable the analyte diffusion coefficient to be reliably estimated as a fitted parameter when fitting binding interaction models. A numerical model based on finite element analysis, Monte Carlo simulations, and statistical profiling were used to compare the Taylor dispersion method with standard fixed concentration injections in terms of parameter correlation, linearity of parameter error space, and global versus local model fitting. A dramatic decrease in parameter correlations was observed for TDi curves relative to curves from standard fixed concentration injections when surface saturation was achieved. In FCI the binding progress is recorded with respect to injection time, whereas in TDi the second time dependency encoded in the analyte gradient increases resolving power. This greatly lowers the dependence of all parameters on each other and on experimental interferences. When model parameters were fitted locally, the performance of TDis remained comparable to global model fitting, whereas fixed concentration binding response curves yielded unreliable parameter estimates. PMID:22197421

  1. Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

    PubMed

    Aoshima, H; Inoue, Y; Hori, K

    1992-10-01

    Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1337082

  2. Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae.

    PubMed

    Bianchi, Frans; Klooster, Joury S van 't; Ruiz, Stephanie J; Luck, Katja; Pols, Tjeerd; Urbatsch, Ina L; Poolman, Bert

    2016-01-01

    The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels. PMID:27550794

  3. Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae

    PubMed Central

    Bianchi, Frans; Klooster, Joury S. van ‘t; Ruiz, Stephanie J.; Luck, Katja; Pols, Tjeerd; Urbatsch, Ina L.; Poolman, Bert

    2016-01-01

    The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels. PMID:27550794

  4. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

    PubMed

    Kessler, R M; Ansari, M S; de Paulis, T; Schmidt, D E; Clanton, J A; Smith, H E; Manning, R G; Gillespie, D; Ebert, M H

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8. PMID:1831229

  5. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides

    SciTech Connect

    Kessler, R.M.; Ansari, M.S.; de Paulis, T.; Schmidt, D.E.; Clanton, J.A.; Smith, H.E.; Manning, R.G.; Gillespie, D.; Ebert, M.H. )

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. Iodopride (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with (125I)iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, iclopride (KD 0.23 nM) and itopride (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, epidepride (KD 0.057 nM) and ioxipride (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

  6. 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

  7. 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

  8. Structure-based model profiles affinity constant of drugs with hPEPT1 for rapid virtual screening of hPEPT1's substrate.

    PubMed

    Sun, L; Meng, S

    2016-08-01

    The human proton-coupled peptide transporter (hPEPT1) with broad substrates is an important route for improving the pharmacokinetic performance of drugs. Thus, it is essential to predict the affinity constant between drug molecule and hPEPT1 for rapid virtual screening of hPEPT1's substrate during lead optimization, candidate selection and hPEPT1 prodrug design. Here, a structure-based in silico model for 114 compounds was constructed based on eight structural parameters. This model was built by the multiple linear regression method and satisfied all the prerequisites of the regression models. For the entire data set, the r(2) and adjusted r(2) values were 0.74 and 0.72, respectively. Then, this model was used to perform substrate/non-substrate classification. For 29 drugs from DrugBank database, all were correctly classified as substrates of hPEPT1. This model was also used to perform substrate/non-substrate classification for 18 drugs and their prodrugs; this QSAR model also can distinguish between the substrate and non-substrate. In conclusion, the QSAR model in this paper was validated by a large external data set, and all results indicated that the developed model was robust, stable, and can be used for rapid virtual screening of hPEPT1's substrate in the early stage of drug discovery. PMID:27586363

  9. Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis

    PubMed Central

    Bajaj, Madhu S.; Ogueli, Godwin I.; Kumar, Yogesh; Vadivel, Kanagasabai; Lawson, Gregory; Shanker, Sreejesh; Schmidt, Amy E.; Bajaj, S. Paul

    2011-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R. PMID:21115497

  10. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    PubMed

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  11. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  12. A Small, Dilute-Cytoplasm, High-Affinity, Novel Bacterium Isolated by Extinction Culture and Having Kinetic Constants Compatible with Growth at Ambient Concentrations of Dissolved Nutrients in Seawater

    PubMed Central

    Button, D. K.; Robertson, Betsy R.; Lepp, Paul W.; Schmidt, Thomas M.

    1998-01-01

    Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant KA for toluene was low at 1.3 μg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; KA increased with increasing specific affinities. Specific affinities, a°s, were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a°s and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present. PMID:9797308

  13. A sol-gel-integrated protein array system for affinity analysis of aptamer-target protein interaction.

    PubMed

    Ahn, Ji-Young; Kim, Eunkyung; Kang, Jeehye; Kim, Soyoun

    2011-06-01

    A sol-gel microarray system was developed for a protein interaction assay with high activity. Comparing to 2-dimensional microarray surfaces, sol-gel can offer a more dynamic and broad range for proteins. In the present study, this sol-gel-integrated protein array was used in binding affinity analysis for aptamers. Six RNA aptamers and their target protein, yeast TBP (TATA-binding protein), were used to evaluate this method. A TBP-containing sol-gel mixture was spotted using a dispensing workstation under high-humidity conditions and each Cy-3-labeled aptamer was incubated. The dissociation constants (K(d)) were calculated by plotting the fluorescent intensity of the bound aptamers as a function of the TBP concentrations. The K(d) value of the control aptamer was found to be 8 nM, which agrees well with the values obtained using the conventional method, electric mobility shift assay. The sol-gel-based binding affinity measurements fit well with conventional binding affinity measurements, suggesting their possible use as an alternative to the conventional method. In addition, aptamer affinity measurements by the sol-gel-integrated protein chip make it possible to develop a simple high-throughput affinity method for screening high-affinity aptamers. PMID:21749295

  14. Emergent properties of nanosensor arrays: applications for monitoring IgG affinity distributions, weakly affined hypermannosylation, and colony selection for biomanufacturing.

    PubMed

    Reuel, Nigel F; Grassbaugh, Brittany; Kruss, Sebastian; Mundy, J Zachary; Opel, Cary; Ogunniyi, Adebola O; Egodage, Kamal; Wahl, Ramon; Helk, Bernhard; Zhang, Jingqing; Kalcioglu, Z Ilke; Tvrdy, Kevin; Bellisario, Darin O; Mu, Bin; Blake, Steven S; Van Vliet, Krystyn J; Love, J Christopher; Wittrup, Karl Dane; Strano, Michael S

    2013-09-24

    It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG (KD μ = 19 μM, σ(2) = 1000 mM(2)), murine IgG (KD μ = 4.3 nM, σ(2) = 3 μM(2)), and human IgG from CHO cells (KD μ = 2.5 nM, σ(2) = 0.01 μM(2)). Second, we show that an array of nanosensors can uniquely monitor weakly affined analyte interactions via the increased number of observed interactions. One application involves monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example, we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself. PMID:23909808

  15. Kd-Jump: a path-preserving stackless traversal for faster isosurface raytracing on GPUs.

    PubMed

    Hughes, David M; Lim, Ik Soo

    2009-01-01

    Stackless traversal techniques are often used to circumvent memory bottlenecks by avoiding a stack and replacing return traversal with extra computation. This paper addresses whether the stackless traversal approaches are useful on newer hardware and technology (such as CUDA). To this end, we present a novel stackless approach for implicit kd-trees, which exploits the benefits of index-based node traversal, without incurring extra node visitation. This approach, which we term Kd-Jump, enables the traversal to immediately return to the next valid node, like a stack, without incurring extra node visitation (kd-restart). Also, Kd-Jump does not require global memory (stack) at all and only requires a small matrix in fast constant-memory. We report that Kd-Jump outperforms a stack by 10 to 20% and kd-restart by 100%. We also present a Hybrid Kd-Jump, which utilizes a volume stepper for leaf testing and a run-time depth threshold to define where kd-tree traversal stops and volume-stepping occurs. By using both methods, we gain the benefits of empty space removal, fast texture-caching and realtime ability to determine the best threshold for current isosurface and view direction. PMID:19834233

  16. Mutant prenyltransferase-like mitochondrial protein (PLMP) and mitochondrial abnormalities in kd/kd mice

    PubMed Central

    Peng, Min; Jarett, Leonard; Meade, Ray; Madaio, Michael P.; Hancock, Wayne W.; George, Alfred L.; Neilson, Eric G.; Gasser, David L.

    2008-01-01

    Background Mice that are homozygous for the kidney disease (kd) mutation are apparently healthy for the first 8 weeks of life, but spontaneously develop a severe form of interstitial nephritis that progresses to end-stage renal disease (ESRD) by 4 to 8 months of age. By testing for linkage to microsatellite markers, we previously localized the kd gene to a YAC/BAC contig. Methods The sequence of the entire critical region was examined, and candidate genes were identified. These candidate genes were sequenced in both mutant (kd/kd) mice and normal controls. The phenotype was further characterized by immunohistochemistry and electron microscopy. Transgenic mice were constructed that carried the wild-type allele of the prime candidate gene, and this transgene was transferred to a kd/kd background by breeding. Results We have obtained evidence that kd is a mutant allele of a novel gene for a prenyltransferase-like mitochondrial protein (PLMP). This gene is alternatively spliced, with the larger gene product having one domain that resembles transprenyltransferase and another that is similar to geranylgeranyl pyrophosphate synthase. The smaller gene product includes only the first domain. An antiserum to PLMP localizes to mitochondria, and ultrastructural defects are present in the mitochondria of renal tubular epithelial cells, and to a lesser extent, hepatocytes and heart cells from kd/kd mice. In a line of kd/kd mice that carried the wild-type PLMP allele as a transgene, only 1 out of 13 animals expressed the disease by 120 days of age. Conclusion The kd allele codes for a novel protein that localizes to the mitochondria, and the kd/kd mouse has dysmorphic mitochondria in the renal tubular epithelial cells. This mouse is therefore a unique animal model for studying mechanisms that lead to tubulointerstitial nephritis. PMID:15200409

  17. Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

    SciTech Connect

    Pytela, R.; Pierschbacher, M.D.; Ruoslahti, E.

    1985-01-01

    Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.

  18. Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

    PubMed Central

    Dyson, M R; Germaschewski, V; Murray, K

    1995-01-01

    Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities. PMID:7784206

  19. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.

    PubMed

    Brophy, Megan Brunjes; Hayden, Joshua A; Nolan, Elizabeth M

    2012-10-31

    Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His(3)Asp and His(4) sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His(3)Asp and His(4) sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K(d1) = 133 ± 58 pM and K(d2) = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K(d1) ≤ 10 pM and K(d2) ≤ 240 pM in the presence of excess Ca(II). The K(d1) and K(d2) values are assigned to the His(3)Asp and His(4) sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space. PMID:23082970

  20. Bihamiltonian Cohomology of KdV Brackets

    NASA Astrophysics Data System (ADS)

    Carlet, Guido; Posthuma, Hessel; Shadrin, Sergey

    2016-02-01

    Using spectral sequences techniques we compute the bihamiltonian cohomology groups of the pencil of Poisson brackets of dispersionless KdV hierarchy. In particular, this proves a conjecture of Liu and Zhang about the vanishing of such cohomology groups.

  1. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule.

    PubMed

    Friedman, Mikaela; Orlova, Anna; Johansson, Eva; Eriksson, Tove L J; Höidén-Guthenberg, Ingmarie; Tolmachev, Vladimir; Nilsson, Fredrik Y; Ståhl, Stefan

    2008-03-01

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection. PMID:18207161

  2. /sup 125/I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and /sup 125/I-azido-BW-A844U, a photoaffinity label

    SciTech Connect

    Patel, A.; Craig, R.H.; Daluge, S.M.; Linden, J.

    1988-06-01

    3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors; the KI for binding to A2 receptors of human platelet membranes is 2.0 microM (A2/A1 ratio = 8700). Radioiodination of the 3-aminophenethyl group resulted in 125I-BW-A844U, a radioligand that retains high affinity for A1 receptors in bovine brain membranes (KD = 0.14 nM) and to 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate-solubilized receptors (KD = 0.34 nM). Specific binding of 125I-BW-A844U represented greater than 90% of the total binding at the KD. From the association constant (K1 = 5.0 X 10(8) M-1min-1) and the dissociation constant (K-1 = 0.064 min-1), the kinetic KD (K-1/K1) in membranes was calculated to be 0.13 nM. NaCl (1 M) had little effect on the binding affinity of 125I-BW-A844U, in contrast to the large effect of salt on the binding affinity of acidic antagonist radioligands. 8-Sulfophenyltheophylline inhibited radioligand binding with a Hill coefficient of 1.0, indicative of a single affinity binding state for the antagonist. By comparison, two distinct agonist affinity states of A1 receptors for the agonist (R)-phenylisopropyladenosine could be resolved, a high affinity state (62%, KH = 74 pM) and a low affinity state (KL = 3.83 nM). The addition of 0.1 mM guanylylimidodiphosphate converted all receptors to the low affinity state. Addition of NaCl (0.5 M) decreased the fraction of receptors in the high affinity state and increased both KH and KL, suggesting that NaCl alters coupling of receptors to G proteins and influences the conformation of the receptor polypeptide, whether or not the receptor is coupled to a G protein.

  3. 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)

  4. The KdV hierarchy in optics

    NASA Astrophysics Data System (ADS)

    Horsley, S. A. R.

    2016-08-01

    There is a well explored relationship between quantum mechanical scattering from a potential and the Korteweg–de Vries (KdV) equation of fluid dynamics: if the potential is ‘evolved’ according to the KdV equation then it will have the same reflectivity and transmissivity as a function of energy, for each snapshot in time. In this work we explore this connection in optics, where the permittivity plays the role of the potential. We begin by deriving the relationship between the Helmholtz equation and the KdV equation in terms of the current induced in a material when a permittivity profile is changed slightly. It is then shown that the KdV equation can be used to design a plethora of bounded complex potentials that are relfectionless from both sides for all angles of incidence, and planar periodic media that exhibit a real Bloch vector for all angles of propagation. Finally we apply the KdV equation to reduce the reflection of a wave from an interface between two media of differing refractive indices.

  5. Human cord blood contains an IGM antibody to the 41KD flagellar antigen of Borrelia burgdorferi.

    PubMed

    Cooke, W D; Orr, A S; Wiseman, B L; Rouse, S B; Murray, W C; Ranck, S G

    1993-10-01

    Natural antibodies are the IgM products of fetal and neonatal B cells. These are germline encoded low affinity antibodies with multiple specificities to self and exogenous antigens. Lyme borreliosis is the disease resulting from infection with the spirochete, Borrelia burgdorferi. The humoral response to this organism is brisk, directed at multiple proteins, and persistent. Antibody to the 41kd flagellar antigen is found early in disease, but may also be found in non-exposed individuals. These properties suggest that the anti-41kd antibody may be a natural antibody. We report here the finding of an IgM anti-41kd reactivity in 29% of cord blood samples from patients in an area non-endemic for Lyme disease. The results are consistent with the hypothesis that antibody to flagellin may be a germline encoded natural antibody, and could be important in the immunopathogenesis of Lyme arthritis and other arthritides. PMID:8211003

  6. Optical properties of KD*P modulators

    NASA Technical Reports Server (NTRS)

    West, E. A.; Bhatia, S. S.

    1990-01-01

    Longitudinal KD*P modulators are used in ground-based solar magnetographs to eliminate seeing effects. Although the modulators can be used as variable retarders, the optical properties when zero voltage is applied influences the performance on instruments requiring very accurate polarization measurements. Measurements at the Marshall Space Flight Center are discussed in terms of the optical properties of KD*P modulators when zero voltage is applied. The measurements can be used to predict the modulation characteristics of the devices and to determine the polarization accuracy that can be expected from the vector magnetograph.

  7. [Analysis of the impact of heparin on the affinity of high mobility group box-1 protein and the receptor of advanced glycation end products by surface plasmon resonance technology].

    PubMed

    Ling, Yan; Wang, Chun-You; Yang, Zhi-Yong

    2009-11-01

    To investigate the affinity constants of heparin with high mobility group protein 1(HMGB1) and HMGB1 with the receptor of advanced glycation end products (RAGE) and to analyze the impact of heparin on the affinity of HMGB1 and RAGE, the standard BIAcore amine coupling chemistry protocol using EDC and NHS was employed for immobilizing. Surface plasmon resonance biosensor technology was used to detect the affinity constants of heparin/HMGB1, HMGB1/RAGE and heparin/ RAGE. Binding analysis was used to investigate the impact of heparin on the affinity of HMGB1 and RAGE. After the immobilization, 9 000 and 5 000 RU rise of HMGB1 and RAGE respectively were obtained. These meant that the immobilized values of HMGB1 and RAGE were about 9 and 5 ng x mm(-2) respectively. The kinetic constants were k(a) = 1.78 x 10(5) L x mol(-1) x s(-1), kd = 8.02 x 10(-4) s(-1), and the affinity constants were KA = 2.22 x 10(8) L x mol(-1), the equilibrium dissociation constant K(D) = 4.5 x 10(-9) mol x L(-1) for heparin and HMGB1; while the kinetic constants were k(a) = 1.85 x 10(3) L x mol(-1) x s(-1), k(d) = 1.81 x 10(-4) s(-1), K(A) = 1.02 x 10(7) L x mol(-1), K(D) = 9.77 x 10(-8) mol x L(-1) for HMGB1 and RAGE; there was very low affinity of heparin with RAGE. The highest concentration of 10 000 u x L(-1) of heparin in this experiment did not reach the saturation with HMGB1. After 50 mg x L(-1) of HMGB1 was mixed with heparin of 50, 100, 1 000, 10 000 u x L(-1), the combining amount of HMGB1 and RAGE declined from 100 to 50 RU. But there were no significant differences between different concentrations of heparin. It was concluded that heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE. In addition, this impact was not in a dose-dependent manner. PMID:20101991

  8. Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

    PubMed

    Song, Yanling; Shi, Yuanzhi; Li, Xingrui; Ma, Yanli; Gao, Mingxuan; Liu, Dan; Mao, Yu; Zhu, Zhi; Lin, Hui; Yang, Chaoyong

    2016-08-16

    Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening. PMID:27454185

  9. A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

    PubMed

    Enander, Karin; Dolphin, Gunnar T; Liedberg, Bo; Lundström, Ingemar; Baltzer, Lars

    2004-05-17

    A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry. PMID:15146511

  10. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

    PubMed Central

    Day, Christopher J.; Tran, Elizabeth N.; Semchenko, Evgeny A.; Tram, Greg; Hartley-Tassell, Lauren E.; Ng, Preston S. K.; King, Rebecca M.; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A.; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P.

    2015-01-01

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  11. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

    PubMed

    Day, Christopher J; Tran, Elizabeth N; Semchenko, Evgeny A; Tram, Greg; Hartley-Tassell, Lauren E; Ng, Preston S K; King, Rebecca M; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P

    2015-12-29

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  12. Nonlocal Symmetry and its Applications in Perturbed mKdV Equation

    NASA Astrophysics Data System (ADS)

    Ren, Bo; Lin, Ji

    2016-06-01

    Based on the modified direct method, the variable-coefficient perturbed mKdV equation is changed to the constant-coefficient perturbed mKdV equation. The truncated Painlevé method is applied to obtain the nonlocal symmetry of the constant-coefficient perturbed mKdV equation. By introducing one new dependent variable, the nonlocal symmetry can be localized to the Lie point symmetry. Thanks to the localization procedure, the finite symmetry transformation is presented by solving the initial value problem of the prolonged systems. Furthermore, many explicit interaction solutions among different types of solutions such as solitary waves, rational solutions, and Painlevé II solutions are obtained using the symmetry reduction method to the enlarged systems. Two special concrete soliton-cnoidal interaction solutions are studied in both analytical and graphical ways.

  13. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors

    PubMed Central

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (KD) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  14. Structure of the phylloquinone-binding (Q phi) site in green plant photosystem I reaction centers: the affinity of quinones and quinonoid compounds for the Q phi site.

    PubMed

    Iwaki, M; Itoh, S

    1991-06-01

    The dissociation constants (Kd) between the phylloquinone-binding site (designated as the Q phi site) and 23 quinones and 2 quinonoid compounds were measured in spinach photosystem I reaction centers. Kd values were calculated from the dependency of the recovery of the flash-induced stable oxidation of the primary donor chlorophyll P700 in the phylloquinone-extracted reaction center on the concentration of added compounds. The binding free energy, calculated from the Kd value of quinones with nonpolar substituted groups, linearly depended on their partition coefficients between water and cyclohexane, but only if their molecular sizes are smaller than anthraquinone. The quinones with larger molecular sizes showed a lower affinity than expected from their hydrophobicities. This suggests that the quinone-binding domain is hydrophobic and that its size is similar to that of anthraquinone. The interaction other than the hydrophobic one was also estimated to stabilize the binding by -5.7 kcal/mol for alkylated quinones. Deletion of one of the carbonyls of p-quinones significantly decreased the binding affinity. This suggests a hydrogen bond or a pi-pi electronic interaction between quinone and the Q phi site. Effects of halogens and amino substitutions on the binding affinity were also studied. The structure of the quinone-binding site in the photosystem I reaction center is deduced from these results. PMID:2036403

  15. Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase12

    PubMed Central

    Yu, Ying; Mu, Helen He; Wasserman, Bruce P.; Carman, George M.

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme. PMID:11154342

  16. Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

    PubMed

    Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

    2012-11-01

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential. PMID:22918538

  17. 3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

    PubMed

    Werle, E; Lenz, T; Strobel, G; Weicker, H

    1988-07-01

    The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off PMID:2853303

  18. Complex solitary waves and soliton trains in KdV and mKdV equations

    NASA Astrophysics Data System (ADS)

    Modak, Subhrajit; Singh, Akhil Pratap; Panigrahi, Prasanta Kumar

    2016-06-01

    We demonstrate the existence of complex solitary wave and periodic solutions of the Korteweg-de Vries (KdV) and modified Korteweg-de Vries (mKdV) equations. The solutions of the KdV (mKdV) equation appear in complex-conjugate pairs and are even (odd) under the simultaneous actions of parity (𝓟) and time-reversal (𝓣) operations. The corresponding localized solitons are hydrodynamic analogs of Bloch soliton in magnetic system, with asymptotically vanishing intensity. The 𝓟𝓣-odd complex soliton solution is shown to be iso-spectrally connected to the fundamental sech2 solution through supersymmetry. Physically, these complex solutions are analogous to the experimentally observed grey solitons of non-liner Schödinger equation, governing the dynamics of shallow water waves and hence may also find physical verification.

  19. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1. PMID:27173568

  20. On the binding affinity of macromolecular interactions: daring to ask why proteins interact

    PubMed Central

    Kastritis, Panagiotis L.; Bonvin, Alexandre M. J. J.

    2013-01-01

    Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of these interactions, is translated into physico-chemical terms in the dissociation constant (Kd), the latter being an experimental measure that determines whether an interaction will be formed in solution or not. Predicting binding affinity from structural models has been a matter of active research for more than 40 years because of its fundamental role in drug development. However, all available approaches are incapable of predicting the binding affinity of protein–protein complexes from coordinates alone. Here, we examine both theoretical and experimental limitations that complicate the derivation of structure–affinity relationships. Most work so far has concentrated on binary interactions. Systems of increased complexity are far from being understood. The main physico-chemical measure that relates to binding affinity is the buried surface area, but it does not hold for flexible complexes. For the latter, there must be a significant entropic contribution that will have to be approximated in the future. We foresee that any theoretical modelling of these interactions will have to follow an integrative approach considering the biology, chemistry and physics that underlie protein–protein recognition. PMID:23235262

  1. Theoretical analysis of kinetic effects on the quantitative comparison of K(d) values and contaminant retardation factors.

    PubMed

    Tinnacher, Ruth M; Honeyman, Bruce D

    2010-10-21

    Distribution coefficients (K(d) values) describe contaminant partitioning between liquids and solids for linear sorption at equilibrium conditions. If experimentally-determined K(d) values do not represent sorption equilibria, errors are introduced in contaminant transport models. These errors may be further propagated when K(d) values are used to compare contaminant mobility under different chemical solution conditions. Our theoretical analysis based on pseudo-first order sorption kinetics shows that, independent if two systems have the same or different sorption kinetics, relative comparisons of K(d) values and retardation factors are always affected by sorption times under non-equilibrium conditions. The time-frames required for attaining constant K(d) values are not only dependent on kinetic sorption characteristics, but also the equilibrium K(d) values approached. The type of kinetic errors introduced is affected by the specific differences in sorption kinetics and equilibrium K(d) values between the two systems. For systems with the same sorption kinetics, relative increases or decreases in contaminant velocities are always underestimated. In case of different kinetics, either an under- or overestimation of relative differences seems possible. Experimental sorption times should aim to equilibrate the system with the highest K(d) value for systems with comparable kinetics, and the system with the slowest sorption kinetics for different kinetics. PMID:20864208

  2. Affine hypersurfaces with parallel difference tensor relative to affine α-connection

    NASA Astrophysics Data System (ADS)

    Li, Cece

    2014-12-01

    Li and Zhang (2014) studied affine hypersurfaces of R n + 1 with parallel difference tensor relative to the affine α-connection ∇ (α), and characterized the generalized Cayley hypersurfaces by K n - 1 ≠ 0 and ∇ (α) K = 0 for some nonzero constant α, where the affine α-connection ∇ (α) of information geometry was introduced on affine hypersurface. In this paper, by a slightly different method we continue to study affine hypersurfaces with ∇ (α) K = 0, if α = 0 we further assume that the Pick invariant vanishes and affine metric is of constant sectional curvature. It is proved that they are either hyperquadrics or improper affine hypersphere with flat indefinite affine metric, the latter can be locally given as a graph of a polynomial of at most degree n + 1 with constant Hessian determinant. In particular, if the affine metric is definite, Lorentzian, or its negative index is 2, we complete the classification of such hypersurfaces.

  3. Quantification of transcription factor-DNA binding affinity in a living cell

    PubMed Central

    Belikov, Sergey; Berg, Otto G.; Wrange, Örjan

    2016-01-01

    The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [3H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. PMID:26657626

  4. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug. PMID:26851087

  5. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  6. Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

    NASA Technical Reports Server (NTRS)

    Conley, C. A.; Fowler, V. M.

    1999-01-01

    PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

  7. Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species

    PubMed Central

    Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J.; Boonstra, Rudy

    2015-01-01

    Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, Kd) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in Kd (range 2.0–7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The Kd values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5–5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the Kd in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists. PMID:27293705

  8. One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli.

    PubMed

    Yang, Hailin; Fang, Yanan; Wang, Zhizhen; Zhang, Ling

    2015-06-01

    3α-Hydroxysteroid dehydrogenase (3α-HSD), from Comamonas Testosterone, catalyze reversibly the oxidoreduction of 3α-hydroxyl groups of the steroid hormones. The gene encoding 3α-HSD (hsdA) from Comamonas Testosterone was expressed in Escherchia coli BL21 (DE3). A protocol for recovering 3α-HSD based on affinity strategy was designed and employed. Deoxycholic acid was chosen as the affinity ligand, and it was linked to Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. With this specific affinity medium, the enzyme recovery process consisted of only one chromatography step to capture 3α-HSD. The target protein, analyzed on HPLC Agilent SEC-5 column, was of 94% pure among the captured protein, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 8.8% of crude extracted proteins; the recovery yield of 3α-HSD was 73.2%. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ∼56kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 4.5μg/g medium and 21.3mg/g medium, respectively. PMID:25913427

  9. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  10. A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 degrees C and in ileum at 29 degrees C and 37 degrees C.

    PubMed Central

    Barlow, R B; Berry, K J; Glenton, P A; Nilolaou, N M; Soh, K S

    1976-01-01

    1 The affinity of 17 compounds for muscarine-sensitive acetylcholine receptors in atrial pacemaker cells and ileum of the guinea-pig has been measured at 29 degrees C in Ringer-Locke solution. Measurements were also made at 37 degrees C with 7 of them. 2 Some of the compounds had much higher affinity for the receptors in the ileum than for those in the atria. For the most selective compound, 4-diphenylacetoxy-N-methylpiperidine methiodide, the difference was approximately 20-fold. The receptors in the atria are therefore different the structure from those in the ileum. 3 The effect of temperature on affinity are not the same for all the compounds, tested indicating different enthalpies and entropies of adsorption and accounting for some of the difficulty experienced in predicting the affinity of new compounds. PMID:1000135

  11. A set of robust fluorescent peptide probes for quantification of Cu(ii) binding affinities in the micromolar to femtomolar range.

    PubMed

    Young, Tessa R; Wijekoon, Chathuri J K; Spyrou, Benjamin; Donnelly, Paul S; Wedd, Anthony G; Xiao, Zhiguang

    2015-03-01

    Reliable quantification of copper binding affinities and identification of the binding sites provide a molecular basis for an understanding of the nutritional roles and toxic effects of copper ions. Sets of chromophoric probes are now available that can quantify Cu(i) binding affinities from nanomolar to attomolar concentrations on a unified scale under in vitro conditions. Equivalent probes for Cu(ii) are lacking. This work reports development of a set of four fluorescent dansyl peptide probes (DP1-4) that can quantify Cu(ii) binding affinities from micromolar to femtomolar concentrations, also on a unified scale. The probes were constructed by conjugation of a dansyl group to four short peptides of specific design. Each was characterised by its dissociation constant KD, its pH dependence and the nature of its binding site. One equivalent of Cu(ii) is bound by the individual probes that display different and well-separated affinities at pH 7.4 (log KD = -8.1, -10.1, -12.3 and -14.1, respectively). Intense fluorescence is emitted at λmax ∼ 550 nm upon excitation at ∼330 nm. Binding of Cu(ii) quenches the fluorescence intensity linearly until one equivalent of Cu(ii) is bound. Multiple approaches and multiple affinity standards were employed to ensure reliability. Selected examples of application to well-characterised Cu(ii) binding peptides and proteins are presented. These include Aβ16 peptides, two naturally occurring Cu(ii)-chelating motifs in human serum and cerebrospinal fluid with sequences GHK and DAHK and two copper binding proteins, CopC from Pseudomonas syringae and PcoC from Escherichia coli. Previously reported affinities are reproduced, demonstrating that peptides DP1-4 form a set of robust and reliable probes for Cu(ii) binding to peptides and protein targets. PMID:25715324

  12. Crossover behavior between KdV and mKdV equations in a cold plasma with negative ions

    SciTech Connect

    Grecu, D.; Visinescu, Anca; Carstea, A.S.

    2004-10-04

    It is well known that the KdV equation describes the behavior of ion-acoustic waves in a cold plasma. In the presence of negative ions, if their concentration satisfies a certain condition (critical concentration) the relevant equation is the modified KdV equation. The transition between these two regimes is studied from several points of view. The multiple scales analysis is extended to higher order and the role played by the next equations in the corresponding hierarchies KdV and mKdV is discussed.

  13. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  14. The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

    PubMed Central

    Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

    2015-01-01

    The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates. PMID:25658443

  15. 7 CFR 29.1080 - Variegated dark red (KD).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Variegated dark red (KD). 29.1080 Section 29.1080..., 13, 14 and Foreign Type 92) § 29.1080 Variegated dark red (KD). A dark brownish-red discoloration... over extended periods of time. Any leaf of which 20 percent or more of its surface is dark...

  16. 7 CFR 29.1080 - Variegated dark red (KD).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Variegated dark red (KD). 29.1080 Section 29.1080..., 13, 14 and Foreign Type 92) § 29.1080 Variegated dark red (KD). A dark brownish-red discoloration... over extended periods of time. Any leaf of which 20 percent or more of its surface is dark...

  17. 7 CFR 29.1080 - Variegated dark red (KD).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Variegated dark red (KD). 29.1080 Section 29.1080..., 13, 14 and Foreign Type 92) § 29.1080 Variegated dark red (KD). A dark brownish-red discoloration... over extended periods of time. Any leaf of which 20 percent or more of its surface is dark...

  18. 7 CFR 29.1080 - Variegated dark red (KD).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Variegated dark red (KD). 29.1080 Section 29.1080..., 13, 14 and Foreign Type 92) § 29.1080 Variegated dark red (KD). A dark brownish-red discoloration... over extended periods of time. Any leaf of which 20 percent or more of its surface is dark...

  19. 7 CFR 29.1080 - Variegated dark red (KD).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Variegated dark red (KD). 29.1080 Section 29.1080..., 13, 14 and Foreign Type 92) § 29.1080 Variegated dark red (KD). A dark brownish-red discoloration... over extended periods of time. Any leaf of which 20 percent or more of its surface is dark...

  20. Comparison of in situ uranium KD values with a laboratory determined surface complexation model

    USGS Publications Warehouse

    Curtis, G.P.; Fox, P.; Kohler, M.; Davis, J.A.

    2004-01-01

    Reactive solute transport simulations in groundwater require a large number of parameters to describe hydrologic and chemical reaction processes. Appropriate methods for determining chemical reaction parameters required for reactive solute transport simulations are still under investigation. This work compares U(VI) distribution coefficients (i.e. KD values) measured under field conditions with KD values calculated from a surface complexation model developed in the laboratory. Field studies were conducted in an alluvial aquifer at a former U mill tailings site near the town of Naturita, CO, USA, by suspending approximately 10 g samples of Naturita aquifer background sediments (NABS) in 17-5.1-cm diameter wells for periods of 3 to 15 months. Adsorbed U(VI) on these samples was determined by extraction with a pH 9.45 NaHCO3/Na2CO3 solution. In wells where the chemical conditions in groundwater were nearly constant, adsorbed U concentrations for samples taken after 3 months of exposure to groundwater were indistinguishable from samples taken after 15 months. Measured in situ K D values calculated from the measurements of adsorbed and dissolved U(VI) ranged from 0.50 to 10.6 mL/g and the KD values decreased with increasing groundwater alkalinity, consistent with increased formation of soluble U(VI)-carbonate complexes at higher alkalinities. The in situ K D values were compared with KD values predicted from a surface complexation model (SCM) developed under laboratory conditions in a separate study. A good agreement between the predicted and measured in situ KD values was observed. The demonstration that the laboratory derived SCM can predict U(VI) adsorption in the field provides a critical independent test of a submodel used in a reactive transport model. ?? 2004 Elsevier Ltd. All rights reserved.

  1. Weak affinity chromatography as a new approach for fragment screening in drug discovery.

    PubMed

    Duong-Thi, Minh-Dao; Meiby, Elinor; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2011-07-01

    Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM-10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC-MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening. PMID:21352794

  2. Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.

    PubMed

    Wright, S C; Wei, Q S; Zhong, J; Zheng, H; Kinder, D H; Larrick, J W

    1994-12-01

    We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis. PMID:7964487

  3. Rational Solutions for Lattice Potential KdV Equation and Two Semi-discrete Lattice Potential KdV Equations

    NASA Astrophysics Data System (ADS)

    Feng, Wei; Zhao, Songlin; Shi, Ying

    2016-02-01

    By imposing reduction conditions on rational solutions for a system involving the Hirota-Miwa equation, rational solutions for lattice potential KdV equation are constructed. Besides, the rational solutions for two semi-discrete lattice potential KdV equations are also considered. All these rational solutions are in the form of Schur function type.

  4. Improved generalized F-expansion method for the time fractional modified KdV(fmKdV) equation

    NASA Astrophysics Data System (ADS)

    Sonmezoglu, Abdullah

    2016-06-01

    In this article, an improved generalized F-expansion method is used for solving the time fractional modified KdV(fmKdV) equation. Using this approach new Jacobi elliptic function solutions are obtained. This method can be suitable for solving other nonlinear fractional differential equations.

  5. Binding of L-(/sup 3/H)nicotine to a single class of high affinity sites in rat brain membranes

    SciTech Connect

    Lippiello, P.M.; Fernandes, K.G.

    1986-05-01

    The binding of optically pure L-(/sup 3/H)nicotine to rat brain membrane preparations was studied using a rapid filtration method. The binding properties observed depended on the method used for tissue isolation. The most consistent results were obtained with membranes prepared in the presence of protease inhibitors, without divalent cations. Binding was saturable, reversible, and stereospecific. Scatchard analysis revealed a single class of high affinity sites with an average KD of 2 nM and a Bmax of approximately 200 fmol/mg of protein. The Hill coefficient was near unity. The KD calculated from the kinetic rate constants for association (k1 = 0.012 min-1 nM-1) and dissociation (k-1 = 0.04 min-1) was around 3 nM, in good agreement with the dissociation constant determined from equilibrium binding. In competition studies, cholinergic agonists were generally the most effective in inhibiting L-(/sup 3/H)nicotine binding, whereas antagonists were relatively ineffective. The D-isomer of nicotine was about 60-fold less potent than the L-isomer in inhibiting binding. The results were unaffected by temperature, with the exception that Bmax was somewhat lower at 37 degrees. The equilibrium binding properties of these sites were essentially identical in adult male and female brain. However, Bmax was lower in fetal brain tissue. The present findings are consistent with the idea that there is a single class of high affinity nicotinic binding sites in rat brain with cholinoceptive properties.

  6. High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

    PubMed Central

    Wittenberg, Nathan J.; Im, Hyungsoon; Xu, Xiaohua; Wootla, Bharath; Watzlawik, Jens; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

    2012-01-01

    Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (KD ~0.9 nM) for natural autoantibodies. This is approximately one to two orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair. PMID:22762372

  7. High kinetic stability of Zn(II) coordinated by the tris(histidine) unit of carbonic anhydrase towards solvolytic dissociation studied by affinity capillary electrophoresis.

    PubMed

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko

    2016-08-01

    Solvolytic dissociation rate constants (kd) of bovine carbonic anhydrase II (CA) and its metallovariants (M-CAs, M=Co(II), Ni(II), Cu(II), Zn(II), and Cd(II)) were estimated by a ligand substitution reaction, which was monitored by affinity capillary electrophoresis to selectively detect the undissociated CAs in the reaction mixture. Using EDTA as the competing ligand for Zn-CA, the dissociation followed the unimolecular nucleophilic substitution (SN1) mechanism with kd=1.0×10(-7)s(-1) (pH7.4, 25°C). The corresponding solvolysis half-life (t1/2) was 80days, showing the exceptionally high kinetic stability of t Zn-CA, in contrast to the highly labile [Zn(II)(H2O)6](2+), where the water exchange rate (kex) is high. This behavior is attributed to the tetrahedral coordination geometry supported by the tris(histidine) unit (His3) of CA. In the case of Co-CA, it showed a somewhat larger kd value (5.7×10(-7)s(-1), pH7.4, 25°C) even though it shares the same tetrahedral coordination environment with Zn-CA, suggesting that the d(7) electronic configuration of Co(II) in the transition state of the dissociation is stabilized by the ligand field. Among M-CAs, only Ni-CA showed a bimolecular nucleophilic substitution (SN2) reaction path in its reaction with EDTA, implying that the large coordination number (6) of Ni(II) in Ni-CA allows EDTA to form an EDTA-Ni-CA intermediate. Overall, kd values roughly correlated with kex values among M-CAs, with the kd value of Zn-CA deviating strongly from the trend and highlighting the exceptionally high kinetic stabilization of Zn-CA by the His3 unit. PMID:27235274

  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. Decreasing Kd uncertainties through the application of thermodynamic sorption models.

    PubMed

    Domènech, Cristina; García, David; Pękala, Marek

    2015-09-15

    Radionuclide retardation processes during transport are expected to play an important role in the safety assessment of subsurface disposal facilities for radioactive waste. The linear distribution coefficient (Kd) is often used to represent radionuclide retention, because analytical solutions to the classic advection-diffusion-retardation equation under simple boundary conditions are readily obtainable, and because numerical implementation of this approach is relatively straightforward. For these reasons, the Kd approach lends itself to probabilistic calculations required by Performance Assessment (PA) calculations. However, it is widely recognised that Kd values derived from laboratory experiments generally have a narrow field of validity, and that the uncertainty of the Kd outside this field increases significantly. Mechanistic multicomponent geochemical simulators can be used to calculate Kd values under a wide range of conditions. This approach is powerful and flexible, but requires expert knowledge on the part of the user. The work presented in this paper aims to develop a simplified approach of estimating Kd values whose level of accuracy would be comparable with those obtained by fully-fledged geochemical simulators. The proposed approach consists of deriving simplified algebraic expressions by combining relevant mass action equations. This approach was applied to three distinct geochemical systems involving surface complexation and ion-exchange processes. Within bounds imposed by model simplifications, the presented approach allows radionuclide Kd values to be estimated as a function of key system-controlling parameters, such as the pH and mineralogy. This approach could be used by PA professionals to assess the impact of key geochemical parameters on the variability of radionuclide Kd values. Moreover, the presented approach could be relatively easily implemented in existing codes to represent the influence of temporal and spatial changes in geochemistry

  10. Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

    PubMed Central

    Piper, J W; Swerlick, R A; Zhu, C

    1998-01-01

    Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5). PMID:9449350

  11. The Lectin Frontier Database (LfDB), and data generation based on frontal affinity chromatography.

    PubMed

    Hirabayashi, Jun; Tateno, Hiroaki; Shikanai, Toshihide; Aoki-Kinoshita, Kiyoko F; Narimatsu, Hisashi

    2015-01-01

    Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience. PMID:25580689

  12. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

    PubMed

    Kusano-Arai, Osamu; Fukuda, Rie; Kamiya, Wakana; Iwanari, Hiroko; Hamakubo, Takao

    2016-07-01

    The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface. PMID:27095060

  13. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry.

    PubMed

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope. Graphical Abstract ᅟ. PMID:27067900

  14. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  15. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-04-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  16. Failure of the three compartment model to describe the pharmacokinetics in brain of a high affinity substituted benzamide.

    PubMed

    Votaw, J R; Kessler, R M; de Paulis, T

    1993-11-01

    The applicability of using the standard 3-compartment model to describe the neuropharmacokinetics of a high affinity substituted benzamide was investigated. We performed the following experiments using the [18F]-5-(3-fluoropropyl) analog of epidepride ([18F]5-FPrEpid), a potent dopamine D2 receptor antagonist: constant left ventricular infusion, first-pass clearance, varying ligand specific activity, and displacing bound ligand with varying amounts of unlabelled ligand. Taken together, the information from these experiments rigorously tests the standard 3-compartment model. The obtained data and predictions from the model of the kinetic behavior of the ligand are inconsistent. The measured and model predicted dissociation rate (measured koff = 0.065 min-1, model prediction koff = 0.007 min-1) and the equilibrium dissociation constant (measured KD = 0.14 nM, model prediction KD = 2.2 nM) differ by an order of magnitude. Furthermore, the model cannot be used to accurately estimate the receptor density. We postulate that the synapse geometry and physical relationship between receptors are necessary components of a model that describes the pharmacokinetics of [18F]5-FPrEpid. PMID:8278896

  17. DC bias modulation characteristics of longitudinal KD*P modulators

    NASA Astrophysics Data System (ADS)

    West, Ed; Wilkins, Nathan

    1992-12-01

    A test program designed to study the DC modulation characteristics of longitudinal KD*P modulators and to determine what electrode structure might improve the performance of these devices is presented which was developed in the Marshall Space Flight Center. The physical constraints of these devices and the necessary electrical characteristics that the KD*P modulators must have to be used in the MSFC polarimeter are discussed.

  18. On third Poisson structure of KdV equation

    SciTech Connect

    Gorsky, A.; Marshakov, A.; Orlov, A.

    1995-12-01

    The third Poisson structure of the KdV equation in terms of canonical {open_quote}free fields{close_quote} and the reduced WZNW model is discussed. We prove that it is {open_quotes}diagonalized{close_quotes} in the Lagrange variables which were used before in the formulation of 2d gravity. We propose a quantum path integral for the KdV equation based on this representation.

  19. Concanavalin A - Polysaccharides Binding Affinity Analysis Using A Quartz Crystal Microbalance

    PubMed Central

    COULIBALY, FOHONA SOUMAHILA; YOUAN, BI-BOTTI C.

    2014-01-01

    There is no comparative data available on the binding constants of Concanavalin A (Con A) and glycogen and Con A-mannan using Quartz Crystal Microbalance (QCM), cost and time efficient system for biosensor analysis. It is hypothesized that a QCM can be used in its flow injection mode to monitor the binding affinity of polysaccharides to an immobilized lectin, Con A. The biosensor is prepared by immobilizing Con A on a 5 MHz gold crystal by carbodiimide crosslinking chemistry. The attachment efficiency was monitored by Fourier Transform Infrared Spectroscopy. Equilibrium association and dissociation constants describing Con A-polysaccharides interaction are determined in a saturation binding experiment, where increasing concentrations of polysaccharides are run on a Con A-immobilized gold crystal surface, and the frequency shifts recorded on the frequency counter. The molecular weights (MW) of glycogen from Oyster and mannan from Saccharomyces cerevisiae were determined by size exclusion chromatography. The MW for glycogen and mannan are 604±0.002 kDa and 54±0.002 kDa, respectively. The equilibrium association and dissociation constants for ConA-glycogen and Con A-mannan interactions are KA = 3.93 ± 0.7 × 106 M−1 / KD = 0.25 ± 0.06 μM and KA = 3.46 ± 0.22 × 105 M−1 / KD = 2.89 μM ± 0.20 (n = 3), respectively. Their respective frequency and motional resistance shifts relationship (ΔF/ΔR) are 37.29±1.55 and 34.86±0.85 Hz.Ω−1 (n=3), which support the validity of Sauerbrey’s rigidity approximation. This work suggests that Con A-mannan complex could be potentially utilized delivery and the targeting of glucose-rich substances and glycoproteins when fast drug release is desired. PMID:24768820

  20. Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

    PubMed Central

    Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R.; Botana, Luis M.

    2013-01-01

    Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. PMID:24379088

  1. Boundary conditions for General Relativity on AdS3 and the KdV hierarchy

    NASA Astrophysics Data System (ADS)

    Pérez, Alfredo; Tempo, David; Troncoso, Ricardo

    2016-06-01

    It is shown that General Relativity with negative cosmological constant in three spacetime dimensions admits a new family of boundary conditions being labeled by a nonnegative integer k. Gravitational excitations are then described by "boundary gravitons" that fulfill the equations of the k-th element of the KdV hierarchy. In particular, k = 0 corresponds to the Brown-Henneaux boundary conditions so that excitations are described by chiral movers. In the case of k = 1, the boundary gravitons fulfill the KdV equation and the asymptotic symmetry algebra turns out to be infinite-dimensional, abelian and devoid of central extensions. The latter feature also holds for the remaining cases that describe the hierarchy ( k > 1). Our boundary conditions then provide a gravitational dual of two noninteracting left and right KdV movers, and hence, boundary gravitons possess anisotropic Lifshitz scaling with dynamical exponent z = 2 k + 1. Remarkably, despite spacetimes solving the field equations are locally AdS, they possess anisotropic scaling being induced by the choice of boundary conditions. As an application, the entropy of a rotating BTZ black hole is precisely recovered from a suitable generalization of the Cardy formula that is compatible with the anisotropic scaling of the chiral KdV movers at the boundary, in which the energy of AdS spacetime with our boundary conditions depends on z and plays the role of the central charge. The extension of our boundary conditions to the case of higher spin gravity and its link with different classes of integrable systems is also briefly addressed.

  2. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  3. Fast and high-affinity binding of B-lymphotropic papovavirus to human B-lymphoma cell lines.

    PubMed Central

    Herrmann, M; Oppenländer, M; Pawlita, M

    1995-01-01

    Binding of B-lymphotropic papovavirus (LPV) to host cells differing in susceptibility to viral infection was determined by a newly established, direct, nonradioactive virus binding assay, which allows quantitative description of the binding characteristics by receptor saturation and Scatchard analysis. LPV binding to the highly susceptible human B-lymphoma cell line BJA-B K88 is specific, saturable, and noncooperative. Binding occurs very fast, with an association rate constant (k1) of 6.7 x 10(7) M-1s-1, and is of high affinity, with a dissociation constant (Kd) of 2.9 x 10(-12) M; and the virus-receptor complex is stable, with a half life of 70 min. The binding affinities of receptors on four other highly, moderately, or weakly susceptible human B-lymphoma cell lines were similar, with up to twofold variation around a mean Kd value of 3 x 10(-12) M, suggesting the presence of the same LPV receptor on all of these cell lines. This view is further supported by the finding that in all cases a terminal sialic acid is necessary for LPV binding. Tunicamycin has been shown to drastically induce LPV susceptibility and LPV binding in weakly and moderately susceptible B-lymphoma cell lines (O.T. Keppler, M. Herrmann, M. Oppenländer, W. Meschede, and M. Pawlita, J. Virol. 68:6933-6939, 1994). The hypothesis that the constitutively expressed and tunicamycin-induced LPV receptors are identical is strengthened by our finding that both receptor types displayed the same high affinity. LPV susceptibility of different B-lymphoma cell lines was correlated with receptor number but not with receptor affinity. The numbers of receptors per cell on highly and moderately susceptible cell lines ranged from 2,000 to 400 and were directly proportional to LPV susceptibility. This indicates that the number of high-affinity receptors per cell is a key regulating factor for the LPV host range. PMID:7474091

  4. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  5. Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl.

    PubMed Central

    Murthy, J. V.; Kim, H. H.; Hanesworth, V. R.; Hugdahl, J. D.; Morejohn, L. C.

    1994-01-01

    Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a

  6. Effect of Copper and Zinc on the Single Molecule Self-Affinity of Alzheimer’s Amyloid-β Peptides

    PubMed Central

    Hane, Francis T.; Hayes, Reid; Lee, Brenda Y.; Leonenko, Zoya

    2016-01-01

    The presence of trace concentrations of metallic ions, such as copper and zinc, has previously been shown to drastically increase the aggregation rate and neurotoxicity of amyloid-β (Aβ), the peptide implicated in Alzheimer’s disease (AD). The mechanism of why copper and zinc accelerate Aβ aggregation is poorly understood. In this work, we use single molecule force spectroscopy (SMFS) to probe the kinetic and thermodynamic parameters (dissociation constant, Kd, kinetic dissociation rate, koff, and free energy, ΔG) of the dissociation of an Aβ dimer, the amyloid species which initiates the amyloid cascade. Our results show that nanomolar concentrations of copper do not change the single molecule affinity of Aβ to another Aβ peptide in a statistically significant way, while nanomolar concentrations of zinc decrease the affinity of Aβ-Aβ by an order of magnitude. This suggests that the binding of zinc ion to Aβ may interfere with the binding of Aβ-Aβ, leading to a lower self-affinity. PMID:26808970

  7. Beyond the KdV: Post-explosion development.

    PubMed

    Ostrovsky, L; Pelinovsky, E; Shrira, V; Stepanyants, Y

    2015-09-01

    Several threads of the last 25 years' developments in nonlinear wave theory that stem from the classical Korteweg-de Vries (KdV) equation are surveyed. The focus is on various generalizations of the KdV equation which include higher-order nonlinearity, large-scale dispersion, and a non-local integral dispersion. We also discuss how relatively simple models can capture strongly nonlinear dynamics and how various modifications of the KdV equation lead to qualitatively new, non-trivial solutions and regimes of evolution observable in the laboratory and in nature. As the main physical example, we choose internal gravity waves in the ocean for which all these models are applicable and have genuine importance. We also briefly outline the authors' view of the future development of the chosen lines of nonlinear wave theory. PMID:26428573

  8. Beyond the KdV: Post-explosion development

    NASA Astrophysics Data System (ADS)

    Ostrovsky, L.; Pelinovsky, E.; Shrira, V.; Stepanyants, Y.

    2015-09-01

    Several threads of the last 25 years' developments in nonlinear wave theory that stem from the classical Korteweg-de Vries (KdV) equation are surveyed. The focus is on various generalizations of the KdV equation which include higher-order nonlinearity, large-scale dispersion, and a non-local integral dispersion. We also discuss how relatively simple models can capture strongly nonlinear dynamics and how various modifications of the KdV equation lead to qualitatively new, non-trivial solutions and regimes of evolution observable in the laboratory and in nature. As the main physical example, we choose internal gravity waves in the ocean for which all these models are applicable and have genuine importance. We also briefly outline the authors' view of the future development of the chosen lines of nonlinear wave theory.

  9. In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

    PubMed Central

    Li, Bing; Fouts, Ashley E; Stengel, Katharina; Luan, Peng; Dillon, Michael; Liang, Wei-Ching; Feierbach, Becket; Kelley, Robert F; Hötzel, Isidro

    2014-01-01

    Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid

  10. Computational and ESR studies of electron attachment to decafluorocyclopentane, octafluorocyclobutane, and hexafluorocyclopropane: electron affinities of the molecules and the structures of their stable negative ions as determined from 13C and 19F hyperfine coupling constants.

    PubMed

    ElSohly, Adel M; Tschumper, Gregory S; Crocombe, Richard A; Wang, Jih Tzong; Williams, Ffrancon

    2005-08-01

    High-resolution ESR spectra of the ground-state negative ions of hexafluorocyclopropane (c-C3F6*-), octafluorocyclobutane (c-C4F8*-), and decafluorocyclopentane (c-C5F10*-) are reported and their isotropic 19F hyperfine coupling constants (hfcc) of 198.6 +/- 0.4 G, 147.6 +/- 0.4 G, and 117.9 +/- 0.4 G, respectively, are in inverse ratio to the total number of fluorine atoms per anion. Together with the small value of 5.2 +/- 0.4 G determined for the isotropic 13C hfcc of c-C4F8*-, these results indicate that in each case the singly occupied molecular orbital (SOMO) is delocalized over the equivalent fluorines and possesses a nodal plane through the carbon atoms of a time-averaged D(nh) structure. A series of quantum chemical computations were carried out to further characterize these anions and their neutral counterparts. Both the B3LYP density functional and second-order Møller-Plesset perturbation theory (MP2) indicate that c-C3F6*- adopts a D(3h) geometry and a (2)A2'' ground electronic state, that c-C4F8*- adopts a D(4h) geometry and a (2)A2u ground electronic state, and that c-C5F10*- adopts a C(s) structure and a (2)A' electronic state. Moreover, the 19F hyperfine coupling constants computed with the MP2 method and a high quality triple-zeta basis set are within 1% of the experimental values. Also, the values computed for the 13C hfcc of c-C4F8*- are consistent with the experimental value of 5.2 G. Therefore, in keeping with the ESR results, these negative ions derived from first-row elements can be characterized as pi* species. In addition, the hypervalency of these perfluorocycloalkane radical anions has been clarified. PMID:16045345

  11. High affinity ( sup 3 H)glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    SciTech Connect

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D. )

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea (3H) glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of (3H) glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer (3H)glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of (3H)glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats.

  12. Measurement of Bmax and Kd with the glycine transporter 1 radiotracer ¹⁸F-MK6577 using a novel multi-infusion paradigm.

    PubMed

    Xia, Yan; Zheng, Ming-Qiang; Holden, Daniel; Lin, Shu-fei; Kapinos, Michael; Ropchan, Jim; Gallezot, Jean-Dominique; Huang, Yiyun; Carson, Richard E

    2015-12-01

    Glycine is a co-agonist of glutamate at the NMDA receptor. Glycine transporter 1 (GlyT1) inhibitors are reported to be potential therapeutic agents for schizophrenia. (18)F-MK6577 is a new positron emission tomography (PET) radiotracer useful for imaging brain GlyT1 and its occupancy in humans. We devised a novel multi-infusion paradigm of radiolabeled and unlabeled compound and an iterative linear/nonlinear alternating fitting method to allow for the determination of in vivo affinity (Kd) and target concentration (Bmax) images, constraining Kd to be uniform across the brain. This paradigm was tested with (18)F-MK6577 in baboons. Voxel-based analysis produced high quality Bmax images and reliable Kd estimates, and also suggested that the nondisplaceable distribution volume (VND) is not uniform throughout the brain. In vivo GlyT1 Kd was estimated to be 1.87 nmol/L for (18)F-MK6577, and the rank order of GlyT1 distribution measured in the baboon brain was: high in the brainstem (133 nmol/L), medium in the cerebellum (83 nmol/L), and low in the cortex (30 nmol/L). These in vivo Kd and Bmax values agreed well with those determined in vitro, thus validating our novel multi-infusion approach. PMID:26198176

  13. FAST TRACK COMMUNICATION Solving the ultradiscrete KdV equation

    NASA Astrophysics Data System (ADS)

    Willox, Ralph; Nakata, Yoichi; Satsuma, Junkichi; Ramani, Alfred; Grammaticos, Basile

    2010-12-01

    We show that a generalized cellular automaton, exhibiting solitonic interactions, can be explicitly solved by means of techniques first introduced in the context of the scattering problem for the KdV equation. We apply this method to calculate the phase-shifts caused by interactions between the solitonic and non-solitonic parts into which arbitrary initial states separate in time.

  14. Supersymmetry and conservation laws in the KdV system

    SciTech Connect

    Roy, P.K. ); Lahiri, A. ); Bagchi, B. )

    1990-07-20

    The role of supersymmetry in the KdV-MKdV hierarchy of conservation laws is examined. Among all the fifth order evolution equations, the Lax equation turns out to be the only one which is mapped into its modified partner through the pair of supersymmetric transformations.

  15. Conformational kinetics reveals affinities of protein conformational states

    PubMed Central

    Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.

    2015-01-01

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states. PMID:26162682

  16. Modelling radionuclide transport in highly heterogeneous media and under variable hydrochemical conditions using a "dynamic Kd" approach

    NASA Astrophysics Data System (ADS)

    Trinchero, Paolo; Painter, Scott; Ebrahimi, Hedieh; Koskinen, Lasse; Molinero, jorge; Selroos, Jan-Olof

    2015-04-01

    Due to the high heterogeneity of fractured media and the ubiquitous lack of a complete site characterization, deterministic simulations of radionuclide transport in fractured rocks are notoriously highly uncertain. This uncertainty is usually addressed using stochastic methods; e.g. the connectivity structure of the medium is described using multiple realizations of Discrete Fracture Networks (DFN), which are then combined to particle tracking simulations. In these formulations, many complex geochemical retention processes are typically lumped into a single parameter: the distribution coefficient (Kd). This approach relies on an important assumption: the Kd values are constant in time. This hypothesis is critical under long-term geochemical changes as it is known that the distribution coefficient depends on the pH, redox conditions and major chemistry of the system. In this work, we present a novel methodology that combines the robustness of stochastic methods with an explicit description of water-solute-rock interaction processes. The reconciliation of all these is achieved by using a dynamic Kd approach. The hydrogeochemical evolution of the site of study is first computed using long-term and large-scale mechanistic reactive transport simulations. The simulated hydrochemical conditions are then used to generate a complete database of Kd values, which represent the hydrochemical conditions in every position and time of the model domain. Then, MARFA (Painter and Mancillas, 2009) is used to carry out Time Domain Random Walk (TDRW) simulations of radionuclide transport. In these simulations, Kd values are dynamically updated using the afore-mentioned database. The results (i.e. radionuclide breakthrough curves) bring the signature of the underlying changes in the background geochemistry.

  17. Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).

    PubMed

    EL-Sharif, Hazim F; Hawkins, Daniel M; Stevenson, Derek; Reddy, Subrayal M

    2014-08-01

    Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface. PMID:24950144

  18. Characterization of the ER-Targeted Low Affinity Ca(2+) Probe D4ER.

    PubMed

    Greotti, Elisa; Wong, Andrea; Pozzan, Tullio; Pendin, Diana; Pizzo, Paola

    2016-01-01

    Calcium ion (Ca(2+)) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca(2+) store and the free Ca(2+) concentration ([Ca(2+)]) within its lumen ([Ca(2+)]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca(2+) sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca(2+) probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca(2+), low dynamic range, and an affinity for the cation that is too high for the levels of [Ca(2+)] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca(2+) affinity and dynamic range for monitoring [Ca(2+)] variations within the ER. As an example, resting [Ca(2+)]ER have been evaluated in a known paradigm of altered ER Ca(2+) homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca(2+) affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca(2+) content in cells expressing mutated PS2, compared to controls. PMID:27598166

  19. Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

    PubMed

    Kosar-Hashemi, Behjat; Irwin, Jennifer A; Higgins, Jody; Rahman, Sadequr; Morell, Matthew K

    2006-05-01

    A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function. PMID:16645949

  20. KD4v: comprehensible knowledge discovery system for missense variant

    PubMed Central

    Luu, Tien-Dao; Rusu, Alin; Walter, Vincent; Linard, Benjamin; Poidevin, Laetitia; Ripp, Raymond; Moulinier, Luc; Muller, Jean; Raffelsberger, Wolfgang; Wicker, Nicolas; Lecompte, Odile; Thompson, Julie D.; Poch, Olivier; Nguyen, Hoan

    2012-01-01

    A major challenge in the post-genomic era is a better understanding of how human genetic alterations involved in disease affect the gene products. The KD4v (Comprehensible Knowledge Discovery System for Missense Variant) server allows to characterize and predict the phenotypic effects (deleterious/neutral) of missense variants. The server provides a set of rules learned by Induction Logic Programming (ILP) on a set of missense variants described by conservation, physico-chemical, functional and 3D structure predicates. These rules are interpretable by non-expert humans and are used to accurately predict the deleterious/neutral status of an unknown mutation. The web server is available at http://decrypthon.igbmc.fr/kd4v. PMID:22641855

  1. On the Convexity of the KdV Hamiltonian

    NASA Astrophysics Data System (ADS)

    Kappeler, Thomas; Maspero, Alberto; Molnar, Jan; Topalov, Peter

    2016-08-01

    Motivated by perturbation theory, we prove that the nonlinear part {H^{*}} of the KdV Hamiltonian {H^{kdv}}, when expressed in action variables {I = (In)_{n ≥slant 1}}, extends to a real analytic function on the positive quadrant {ℓ2+({N})} of {ℓ2({N})} and is strictly concave near {0}. As a consequence, the differential of {H^{*}} defines a local diffeomorphism near 0 of {ℓ_{{C}}2({N})}. Furthermore, we prove that the Fourier-Lebesgue spaces {{F}{L}^{s,p}} with {-1/2 ≤slant s ≤slant 0} and {2 ≤slant p < ∞}, admit global KdV-Birkhoff coordinates. In particular, it means that {ℓ2_+({N})} is the space of action variables of the underlying phase space {{F}{L}^{-1/2,4}} and that the KdV equation is globally in time {C0}-well-posed on {{F}{L}^{-1/2,4}}.

  2. Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

    PubMed Central

    Klein, M G; Simon, B J; Szucs, G; Schneider, M F

    1988-01-01

    To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds. PMID:3395664

  3. Does the tissue concentration in receptor binding studies change the affinity of the labelled ligand?

    PubMed

    Ensing, K; De Zeeuw, R A

    1984-12-14

    When the tissue concentration in a radioreceptor assay for anticholinergic drugs was varied in order to obtain optimum conditions, and the receptor concentration Cr and the equilibrium dissociation constant KD were determined by Scatchard analysis, the KD increased with increasing tissue concentrations. This phenomenon was considered as an artefact caused by non-specific binding of the labelled ligand to constituents of the receptor preparation which were not completely retained on the glass-fibre filters used for the separation of bound and free fraction of radio-labelled ligand. The increase in KD in these experiments could be described with a mathematical model of the binding experiments. PMID:6514542

  4. Evaluation of kinetic constants of biomolecular interaction on optical surface plasmon resonance sensor with Newton Iteration Method

    NASA Astrophysics Data System (ADS)

    Zhao, Yuanyuan; Jiang, Guoliang; Hu, Jiandong; Hu, Fengjiang; Wei, Jianguang; Shi, Liang

    2010-10-01

    In the immunology, there are two important types of biomolecular interaction: antigens-antibodies and receptors-ligands. Monitoring the response rate and affinity of biomolecular interaction can help analyze the protein function, drug discover, genomics and proteomics research. Moreover the association rate constant and dissociation rate constant of receptors-ligands are the important parameters for the study of signal transmission between cells. Recent advances in bioanalyzer instruments have greatly simplified the measurement of the kinetics of molecular interactions. Non-destructive and real-time monitoring the response to evaluate the parameters between antigens and antibodies can be performed by using optical surface plasmon resonance (SPR) biosensor technology. This technology provides a quantitative analysis that is carried out rapidly with label-free high-throughput detection using the binding curves of antigens-antibodies. Consequently, the kinetic parameters of interaction between antigens and antibodies can be obtained. This article presents a low cost integrated SPR-based bioanalyzer (HPSPR-6000) designed by ourselves. This bioanalyzer is mainly composed of a biosensor TSPR1K23, a touch-screen monitor, a microprocessor PIC24F128, a microflow cell with three channels, a clamp and a photoelectric conversion device. To obtain the kinetic parameters, sensorgrams may be modeled using one of several binding models provided with BIAevaluation software 3.0, SensiQ or Autolab. This allows calculation of the association rate constant (ka) and the dissociation rate constant (kd). The ratio of ka to kd can be used to estimate the equilibrium constant. Another kind is the analysis software OriginPro, which can process the obtained data by nonlinear fitting and then get some correlative parameters, but it can't be embedded into the bioanalyzer, so the bioanalyzer don't support the use of OriginPro. This paper proposes a novel method to evaluate the kinetic parameters

  5. 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

  6. Residual Symmetry and Explicit Soliton-Cnoidal Wave Interaction Solutions of the (2+1)-Dimensional KdV-mKdV Equation

    NASA Astrophysics Data System (ADS)

    Cheng, Wenguang; Li, Biao

    2016-04-01

    The truncated Painlevé method is developed to obtain the nonlocal residual symmetry and the Bäcklund transformation for the (2+1)-dimensional KdV-mKdV equation. The residual symmetry is localised after embedding the (2+1)-dimensional KdV-mKdV equation to an enlarged one. The symmetry group transformation of the enlarged system is computed. Furthermore, the (2+1)-dimensional KdV-mKdV equation is proved to be consistent Riccati expansion (CRE) solvable. The soliton-cnoidal wave interaction solution in terms of the Jacobi elliptic functions and the third type of incomplete elliptic integral is obtained by using the consistent tanh expansion (CTE) method, which is a special form of CRE.

  7. On loop equations in KdV exactly solvable string theory

    SciTech Connect

    Dalley, S. . Joseph Henry Labs.)

    1992-05-10

    In this paper, the non-perturbative behavior of macroscopic loop amplitudes in the exactly solvable string theories based on the KdV hierarchies is considered. Loop equations are presented for the real non-perturbative solutions living on the spectral half-line, allowed by the most general string equation ({bar P}, Q) = Q, where {bar P} generates scale transformations. In general the end of the half-line (the wall) is a non-perturbative parameter whose role is that of boundary cosmological constant. The properties are compared with the perturbative behavior and solutions of (P,Q) = 1. Detailed arguments are given for the (2,2m {minus} 1) models while generalization to the other (p,q) minimal models and c = 1 is briefly addressed.

  8. Modelling radionuclide transport in fractured media with a dynamic update of Kd values

    NASA Astrophysics Data System (ADS)

    Trinchero, Paolo; Painter, Scott; Ebrahimi, Hedieh; Koskinen, Lasse; Molinero, Jorge; Selroos, Jan-Olof

    2016-01-01

    Radionuclide transport in fractured crystalline rocks is a process of interest in evaluating long term safety of potential disposal systems for radioactive wastes. Given their numerical efficiency and the absence of numerical dispersion, Lagrangian methods (e.g. particle tracking algorithms) are appealing approaches that are often used in safety assessment (SA) analyses. In these approaches, many complex geochemical retention processes are typically lumped into a single parameter: the distribution coefficient (Kd). Usually, the distribution coefficient is assumed to be constant over the time frame of interest. However, this assumption could be critical under long-term geochemical changes as it is demonstrated that the distribution coefficient depends on the background chemical conditions (e.g. pH, Eh, and major chemistry). In this work, we provide a computational framework that combines the efficiency of Lagrangian methods with a sound and explicit description of the geochemical changes of the site and their influence on the radionuclide retention properties.

  9. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  10. The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes.

    PubMed

    Roffel, A F; in't Hout, W G; de Zeeuw, R A; Zaagsma, J

    1987-05-01

    We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepine exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 microM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors. PMID:3614390

  11. A Newly-Identified Polymorphism in Rhesus Macaque Complement Factor H Modulates Binding Affinity for Meningococcal FHbp

    PubMed Central

    Konar, Monica; Beernink, Peter T.; Granoff, Dan M.

    2015-01-01

    Background Two meningococcal serogroup B vaccines contain Factor H binding protein (FHbp). Binding of Factor H (FH) to FHbp was thought to be specific for human or chimpanzee FH. However, in a previous study an amino acid polymorphism in rhesus macaque FH domain 6, tyrosine at position 352 (Y352) was associated with high binding to FHbp, whereas histidine at position 352 (H352) was associated with low binding. Methods and Results Here we report that a second FH polymorphism at position 360 also affects macaque FH binding. Of 43 macaques, 11 had high FH binding and 32 had low binding. As in our previous study, all 11 animals with high binding had Y352, and 24 with low binding had H352. However the remaining eight with low FH binding had Y352, which was predicted to yield high binding. All eight had S360 instead of P360. Thus, three allelic variants at positions 352 and 360 affect macaque FH binding to FHbp: HP (low), YS (low), and YP (high). We measured binding affinity of each FH sequence type to FHbp by surface plasmon resonance. Two animals with high binding types (YS/YP and HP/YP) had dissociation constants (KD) of 10.4 and 18.2 nM, respectively, which were similar to human FH (19.8 nM). Two macaques with low binding (HP/HP and HP/YS) had KD values approximately five-fold higher (100.3 and 99.5 nM, respectively). A third macaque with low binding (YS/YS) had a KD value too high to be measured. Conclusions Macaques have at least three allelic variants encoding FH with different affinities for FHbp (five genotypic combinations of these variants). Since in previous studies binding of FH to FHbp vaccines decreased protective antibody responses, our data will aid in selection of macaques with FH binding that is similar to humans for further investigation of FHbp vaccine immunogenicity. PMID:26285122

  12. KdV-like equations for fluid dynamics

    NASA Astrophysics Data System (ADS)

    Ruggieri, M.; Speciale, M. P.

    2014-12-01

    Main goal of the authors is to consider the generalized system of KdV equations ut+uxxx+2uux+2e1vvx+e2(uxv+uvx)+e3vxxx = 0 c1vt+vxxx+2vvx+c2vx+c3(e1(uxv+uvx)+2e2uux+e3uxxx) = 0 (1), and to construct the optimal system of one dimensional subalgebras. The reduction of the above system to ODEs through the optimal systems is performed and finally an application is shown.

  13. The Linear KdV Equation with an Interface

    NASA Astrophysics Data System (ADS)

    Deconinck, Bernard; Sheils, Natalie E.; Smith, David A.

    2016-07-01

    The interface problem for the linear Korteweg-de Vries (KdV) equation in one-dimensional piecewise homogeneous domains is examined by constructing an explicit solution in each domain. The location of the interface is known and a number of compatibility conditions at the boundary are imposed. We provide an explicit characterization of sufficient interface conditions for the construction of a solution using Fokas's Unified Transform Method. The problem and the method considered here extend that of earlier papers to problems with more than two spatial derivatives.

  14. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  15. 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)

  16. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  17. An averaging theorem for a perturbed KdV equation

    NASA Astrophysics Data System (ADS)

    Guan, Huang

    2013-06-01

    We consider a perturbed KdV equation: \\begin{equation*} \\fl \\dot{u}+u_{xxx}-6uu_x=\\epsilon f(x,u(\\cdot)),\\quad x\\in {T},\\tqs\\int_{{T}} u \\,\\rmd x=0. \\end{equation*} For any periodic function u(x), let I(u)=(I_1(u),I_2(u),\\ldots)\\in{R}_+^{\\infty} be the vector, formed by the KdV integrals of motion, calculated for the potential u(x). Assuming that the perturbation ɛf(x, u(x)) defines a smoothing mapping u(x) ↦ f(x, u(x)) (e.g. it is a smooth function ɛ f(x), independent from u), and that solutions of the perturbed equation satisfy some mild a priori assumptions, we prove that for solutions u(t, x) with typical initial data and for 0 ⩽ t ≲ ɛ-1, the vector I(u (t)) may be well approximated by a solution of the averaged equation.

  18. Efficient Modelling of Radionuclide Transport in Highly Heterogeneous Media and Under Variable Hydrochemical Conditions Using an "Intelligent Kd" Approach

    NASA Astrophysics Data System (ADS)

    Trinchero, P.; Painter, S. L.; Ebrahimi, H.; Koskinen, L.; Molinero, J.; Selroos, J. O.

    2014-12-01

    Due to the high heterogeneity of fractured media and the ubiquitous lack of a complete site characterization, deterministic simulations of radionuclide transport in fractured rocks are notoriously highly uncertain. This epistemic uncertainty is typically addressed using stochastic methods; e.g. the connectivity structure of the medium is described using one or multiple realizations of Discrete Fracture Networks (DFN), which are then combined to Time Domain Random Walk (TDRW) simulations (e.g. Painter and Cvetkovic, 2005). In these formulations, many complex geochemical retention processes are usually lumped into a single parameter: the distribution coefficient (Kd). Although this approach is mathematically robust and numerically efficient, it relies on an important assumption: the Kd value of each radionuclide is constant in time. This assumption could be critical under long-term geochemical changes as it is demonstrated that the distribution coefficient depends on the pH, redox conditions and major chemistry of the system. In this work, we present a novel methodology that combines the robustness of stochastic methods with a sound and explicit description of water-solute-rock interaction processes. The reconciliation of all these is achieved by using an "intelligent Kd" approach. The hydrogeochemical evolution of the site of study is first computed using long-term and large-scale mechanistic reactive transport simulations. The simulated hydrochemical conditions are then used to generate a complete database of Kd values, which represent the hydrochemical conditions in every position and time of the model domain. Then, TDRW simulations, based on one or multiple DFN realizations, are fed with these data and the results (e.g. radionuclide breakthrough curves) implicitly bring the signature of the underlying changes in the background geochemistry.

  19. Evaluation of protective immunity of 28KD-GST as a vaccine against experimental Schistosoma mansoni.

    PubMed

    Hassan, Mohsen M; Moustafa, Nahed E; El-Motayam, Mona H; El-Settawy, Magda A; Taha, Afaf A

    2009-08-01

    Schistosoma mansoni GST was purified from adult worm homogenates by affinity chromatography. SDS-PAGE analysis of the purified antigen revealed that SmGST has molecular weight around 28 KD was used as a vaccine in a dose of 25 and 35 microg. Eleven groups of BALB/c mice of 10 mice each were vaccinated by GST. Complete Freund's adjuvant (CFA) and BCG vaccine were used as adjuvant. Booster doses were given after 2 & 4 weeks. Two weeks after the last dose of vaccine, mice were challenged by S. mansoni cercariae. Blood samples were taken 7 weeks post infection for detection of IgGl, IgE and circulating antigens. Then mice were sacrificed for histopathological study of the liver. Highly significant (p > 0.001) increase in the mean optical density of IgGl & IgE in groups vaccinated by 35 microg GST and CFA was demonstrated. On the other hand, highly significant (P < 0.001) decrease in circulating antigen, grnuloma number and size in the same group. PMID:19795747

  20. Dual aptamer-immobilized surfaces for improved affinity through multiple target binding in potentiometric thrombin biosensing.

    PubMed

    Goda, Tatsuro; Higashi, Daiki; Matsumoto, Akira; Hoshi, Toru; Sawaguchi, Takashi; Miyahara, Yuji

    2015-11-15

    We developed a label-free and reagent-less potentiometric biosensor with improved affinity for thrombin. Two different oligomeric DNA aptamers that can recognize different epitopes in thrombin were introduced in parallel or serial manners on the sensing surface to capture the target via multiple contacts as found in many biological systems. The spacer and linker in the aptamer probes were optimized for exerting the best performance in molecular recognition. To gain the specificity of the sensor to the target, an antifouling molecule, sulfobeaine-3-undecanethiol (SB), was introduced on the sensor to form a self-assembled monolayer (SAM). Surface characterization revealed that the aptamer probe density was comparable to the distance of the two epitopes in thrombin, while the backfilling SB SAM was tightly aligned on the surface to resist nonspecific adsorption. The apparent binding parameters were obtained by thrombin sensing in potentiometry using the 1:1 Langmuir adsorption model, showing the improved dissociation constants (Kd) with the limit of detection of 5.5 nM on the dual aptamer-immobilized surfaces compared with single aptamer-immobilized ones. A fine control of spacer and linker length in the aptamer ligand was essential to realize the multivalent binding of thrombin on the sensor surface. The findings reported herein are effective for improving the sensitivity of potentiometric biosensor in an affordable way towards detection of tiny amount of biomolecules. PMID:26067329

  1. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

    PubMed Central

    Einav, Shirit; Gerber, Doron; Bryson, Paul D; Sklan, Ella H; Elazar, Menashe; Maerkl, Sebastian J; Glenn, Jeffrey S; Quake, Stephen R

    2014-01-01

    More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ~3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B’s RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development. PMID:18758449

  2. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  3. Inelastic Character of Solitons of Slowly Varying gKdV Equations

    NASA Astrophysics Data System (ADS)

    Muñoz, Claudio

    2012-09-01

    In this paper we study soliton-like solutions of the variable coefficients, the subcritical gKdV equation u_t + (u_{xx} -λ u + a(\\varepsilon x) u^m )_x =0,quad in quad {R}_t×{R}_x, quad m=2,3 { and } 4, with {λ≥ 0, a(\\cdot ) in (1,2)} a strictly increasing, positive and asymptotically flat potential, and {\\varepsilon} small enough. In previous works (Muñoz in Anal PDE 4:573-638, 2011; On the soliton dynamics under slowly varying medium for generalized KdV equations: refraction vs. reflection, SIAM J. Math. Anal. 44(1):1-60, 2012) the existence of a pure, global in time, soliton u( t) of the above equation was proved, satisfying lim_{tto -infty}\\|u(t) - Q_1(\\cdot -(1-λ)t) \\|_{H^1({R})} =0,quad 0≤ λ<1, provided {\\varepsilon} is small enough. Here R( t, x) := Q c ( x - ( c - λ) t) is the soliton of R t + ( R xx - λ R + R m ) x = 0. In addition, there exists {tilde λ in (0,1)} such that, for all 0 < λ < 1 with {λneq tilde λ} , the solution u( t) satisfies sup_{t≫ 1/\\varepsilon}\\|u(t) - kappa(λ)Q_{c_infty}(\\cdot-ρ(t)) \\|_{H^1({R})}lesssim \\varepsilon^{1/2}. Here {{ρ'(t) ˜ (c_infty(λ) -λ)}} , with {{kappa(λ)=2^{-1/(m-1)}}} and {{c_infty(λ)>λ}} in the case {0<λrsim \\varepsilon^{1 +δ}, for any {{δ>0}} fixed. This bound clarifies the existence of a dispersive tail and the difference with the standard solitons of the constant coefficients, gKdV equation.

  4. A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

    PubMed Central

    Ardeshir, F; Flint, J E; Richman, S J; Reese, R T

    1987-01-01

    Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:3556166

  5. (/sup 3/H)dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states

    SciTech Connect

    Hamblin, M.W.; Ariani, K.; Adriaenssens, P.I.; Ciaranello, R.D.

    1987-12-01

    (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.

  6. Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal.

    PubMed

    Morgado, Isabel; Melo, Eduardo P; Lundberg, Erik; Estrela, Nídia L; Sauer-Eriksson, A Elisabeth; Power, Deborah M

    2008-11-25

    Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR. PMID:18620020

  7. Modulational Instability in Equations of KdV Type

    NASA Astrophysics Data System (ADS)

    Bronski, Jared C.; Hur, Vera Mikyoung; Johnson, Mathew A.

    It is a matter of experience that nonlinear waves in a dispersive medium, propagating primarily in one direction, may appear periodic in small space and time scales, but their characteristics—the amplitude, the phase, the wave number, etc.—slowly vary in large space and time scales. In the 1960s, Whitham developed an asymptotic (WKB) method to study the effects of small "modulations" on nonlinear dispersive waves. Since then, there has been a great deal of work aiming at rigorously justifying the predictions from Whitham's formal theory. We discuss some recent advances in the mathematical understanding of the dynamics, in particular, the instability, of slowly modulated waves for equations of KdV type.

  8. On the continuous limits and integrability of a new coupled semidiscrete mKdV system

    SciTech Connect

    Zhu Zuonong; Zhao Haiqiong; Wu Xiaonan

    2011-04-15

    In this paper, we aim to get more insight on the relation between semidiscrete coupled mKdV system (where ''semidiscrete'' means that the system is discrete in the space variable and continuous in time) and the coupled mKdV equations; to this purpose, we propose a new coupled semidiscrete mKdV system. The Lax pairs, the Darboux transformation, soliton solutions and conservation laws for the coupled semidiscrete mKdV system are given. The coupled mKdV theory including the Lax pairs, the Darboux transformation, soliton solutions, and conservation laws is recovered through the continuous limits of corresponding theory for the new semidiscrete mKdV system.

  9. Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

    PubMed Central

    Pierce, E J; Davison, M D; Parton, R G; Habig, W H; Critchley, D R

    1986-01-01

    Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. Images Fig. 5. PMID:3539106

  10. New solutions for conformable fractional Boussinesq and combined KdV-mKdV equations using Jacobi elliptic function expansion method

    NASA Astrophysics Data System (ADS)

    Tasbozan, Orkun; Çenesiz, Yücel; Kurt, Ali

    2016-07-01

    In this paper, the Jacobi elliptic function expansion method is proposed for the first time to construct the exact solutions of the time conformable fractional two-dimensional Boussinesq equation and the combined KdV-mKdV equation. New exact solutions are found. This method is based on Jacobi elliptic functions. The results obtained confirm that the proposed method is an efficient technique for analytic treatment of a wide variety of nonlinear conformable time-fractional partial differential equations.

  11. Mathematical model accurately predicts protein release from an affinity-based delivery system.

    PubMed

    Vulic, Katarina; Pakulska, Malgosia M; Sonthalia, Rohit; Ramachandran, Arun; Shoichet, Molly S

    2015-01-10

    Affinity-based controlled release modulates the delivery of protein or small molecule therapeutics through transient dissociation/association. To understand which parameters can be used to tune release, we used a mathematical model based on simple binding kinetics. A comprehensive asymptotic analysis revealed three characteristic regimes for therapeutic release from affinity-based systems. These regimes can be controlled by diffusion or unbinding kinetics, and can exhibit release over either a single stage or two stages. This analysis fundamentally changes the way we think of controlling release from affinity-based systems and thereby explains some of the discrepancies in the literature on which parameters influence affinity-based release. The rate of protein release from affinity-based systems is determined by the balance of diffusion of the therapeutic agent through the hydrogel and the dissociation kinetics of the affinity pair. Equations for tuning protein release rate by altering the strength (KD) of the affinity interaction, the concentration of binding ligand in the system, the rate of dissociation (koff) of the complex, and the hydrogel size and geometry, are provided. We validated our model by collapsing the model simulations and the experimental data from a recently described affinity release system, to a single master curve. Importantly, this mathematical analysis can be applied to any single species affinity-based system to determine the parameters required for a desired release profile. PMID:25449806

  12. Restricted alpha- and beta-adrenoceptor affinity of sulfoconjugated catecholamines in human mononuclear leukocytes, platelets, and fat cells and reduction of the postreceptor mechanisms.

    PubMed

    Werle, E; Michel, G; Lenz, T; Kather, H; Schneider, B; Weicker, H

    1988-08-01

    The physiologic significance of the racemic 3-O-sulfate esters of epinephrine (EPI-3-O-S) and norepinephrine (NE-3-O-S) as well as 4-O-sulfoconjugated dopamine (DA-4-O-S) was evaluated. For this purpose these conjugated catecholamines (CA) were synthesized and investigated with respect to their alpha 2- and beta 2-adrenoceptor affinities and their biological activity in three different human cell systems: in mononuclear leukocytes (MNL), platelets, and fat cells. The unequivocal identification and the minimal degree of contamination of the synthesized sulfoconjugates with free CA was proved by 1H-NMR and by high-performance liquid chromatography with amperometric detection (HPLCA) respectively. In isolated human MNL, beta-adrenoceptor affinities of these conjugated CA were determined in competition experiments with the lipophilic nonspecific radioligand (-) 125I-cyanopindolol (ICYP) and, in addition, with the hydrophilic ligand 3H-CGP12177. With both ligands the affinity constants (KD) of the sulfoconjugated CA under investigation were about 100- to 1000-fold higher when compared with the respective free amines. Moreover, these sulfoconjugated CA per se induced no intracellular production of cyclic adenosine monophosphate (cAMP) in MNL. In comparison with the free amines, metanephrine (MN) and normetanephrine (NMN) showed a highly reduced competitive potency on the MNL beta-adrenoceptors labelled with 3H-CGP or ICYP. The KD values for MN and NMN in competition studies with ICYP were 10- and 5-fold higher than in those with 3H-CGP respectively, indicating a restricted access of MN and NMN to intracellular receptors. The adenylate cyclase system was not stimulated at all by MN or by NMN. In human platelets EPI-3-O-S and NE-3-O-S neither competed with the specific alpha 2-adrenoceptor antagonist 3H-yohimbine nor elicited any aggregation response at all. MN and NMN exhibited an about 40-fold reduced affinity for alpha 2-adrenoceptors in platelets when compared with the

  13. Soluble low affinity adenosine A/sub 2/ binding site from human placenta: reconstitution and characteristics

    SciTech Connect

    Hutchison, K.; Prasad, M.; Fox, I.H.

    1987-05-01

    The authors have developed a vesicle reconstitution technique that allows for rapid vacuum filtration assay, and have characterized the soluble A/sub 2/ site from placental membranes. The overall yield of reconstituted binding is 60%. Competition analysis of membranes and reconstituted vesicles yields identical agonist potency orders and affinities: N-ethylcarboxamidoadenosine (NECA) (Kd-330 nM)>2-chloroadenosine (Kd=1.7 ..mu..M) > L-phenylisopropyladenosine (Kd > 1 mM). Equilibrium binding to membranes and reconstituted vesicles of (/sup 3/H)-NECA, an adenosine agonist, was not reduced by guanine nulceotides. HPLC gel permeation chromatography of extracts from membranes preincubated with 5 mM MgCl/sub 2/ and 100 ..mu..M NECA revealed a peak of binding with kD of 0.07. Extracts prepared with either an antagonist or NECA and 100 ..mu..M guanylyl 5'-imidodiphosphate revealed a peak of binding with a kD of 0.09. These data suggest that the adenosine A/sub 2/ receptor retains its binding properties upon reconstitution and may couple to a guanine nucleotide regulatory protein.

  14. Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants.

    PubMed

    Telmer, Patrick G; Shilton, Brian H

    2003-09-01

    The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. PMID:12794084

  15. A Phage Display Screening Derived Peptide with Affinity for the Adeninyl Moiety

    PubMed Central

    Elmlund, Louise; Söderberg, Pernilla; Suriyanarayanan, Subramanian; Nicholls, Ian A.

    2014-01-01

    Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (KD of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions. PMID:25587414

  16. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  17. Various methods for solving time fractional KdV-Zakharov-Kuznetsov equation

    NASA Astrophysics Data System (ADS)

    Guner, Ozkan; Aksoy, Esin; Bekir, Ahmet; Cevikel, Adem C.

    2016-06-01

    This paper presents the exact analytical solution of the (3+1)-dimensional time fractional KdV-Zakharov-Kuznetsov (KdV-ZK) equation with the help of the Kudryashov method, the exp-function method and the functional variable method. The fractional derivatives are described in Jumarie's sense.

  18. Complex and singular solutions of KdV and MKdV equations

    NASA Technical Reports Server (NTRS)

    Buti, B.; Rao, N. N.; Khadkikar, S. B.

    1986-01-01

    The Korteweg-de Vries (KdV) and the modified Korteweg-de Vries (MKdV) equations are shown to have, besides the regular real solutions, exact regular complex as well as singular solutions. The singular solution for the KdV is real but for the MKdV it is pure imaginary. Implications of the complex solutions are discussed.

  19. Local well-posedness for the fifth-order KdV equations on T

    NASA Astrophysics Data System (ADS)

    Kwak, Chulkwang

    2016-05-01

    This paper is a continuation of the paper Low regularity Cauchy problem for the fifth-order modified KdV equations on T[7]. In this paper, we consider the fifth-order equation in the Korteweg-de Vries (KdV) hierarchy as following:

  20. Effects of Moisture Content and Redox Potential on in situ Kd Values for Radiodine in Soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the processes that determine the solid-liquid partitioning (Kd value) of Se are of fundamental importance in assessing the risk associated with the disposal of radioselenium-containing waste. Using a mini-column approach, in-situ Kd values for 75Se were determined over time in relation...

  1. The KdV equation under periodic boundary conditions and its perturbations

    NASA Astrophysics Data System (ADS)

    Guan, Huang; Kuksin, Sergei

    2014-09-01

    In this paper we discuss properties of the Korteweg-de Vries (KdV) equation under periodic boundary conditions, especially those which are important for studying perturbations of the equation. We then review what is known about the long-time behaviour of solutions for perturbed KdV equations.

  2. Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

    PubMed

    Rodrigues de Miranda, J F; Scheres, H M; Salden, H J; Beld, A J; Klaassen, A B; Kuijpers, W

    1985-07-31

    Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites. PMID:3840092

  3. Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems.

    PubMed

    Fielding, Lee; Rutherford, Samantha; Fletcher, Dan

    2005-06-01

    The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems. PMID:15816062

  4. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications

    PubMed Central

    Matsunaga, Ken-ichiro; Kimoto, Michiko; Hanson, Charlotte; Sanford, Michael; Young, Howard A.; Hirao, Ichiro

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ. PMID:26690672

  5. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications.

    PubMed

    Matsunaga, Ken-ichiro; Kimoto, Michiko; Hanson, Charlotte; Sanford, Michael; Young, Howard A; Hirao, Ichiro

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ. PMID:26690672

  6. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  7. Azole affinity of sterol 14α-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens.

    PubMed

    Warrilow, Andrew G; Parker, Josie E; Kelly, Diane E; Kelly, Steven L

    2013-03-01

    Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K(s), 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K(d), ~30,500 nM) and voriconazole (K(d), ~2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (K(d), ~40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC(50)) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC(50), ~150 μM) and did not significantly inhibit Δ60HsCYP51. PMID:23274672

  8. Modelling radionuclide transport in fractured media with a dynamic update of Kd values

    DOE PAGESBeta

    Trinchero, Paolo; Painter, Scott L.; Ebrahimi, Hedieh; Koskinen, Lasse; Molinero, Jorge; Selroos, Jan -Olof

    2015-10-13

    Radionuclide transport in fractured crystalline rocks is a process of interest in evaluating long term safety of potential disposal systems for radioactive wastes. Given their numerical efficiency and the absence of numerical dispersion, Lagrangian methods (e.g. particle tracking algorithms) are appealing approaches that are often used in safety assessment (SA) analyses. In these approaches, many complex geochemical retention processes are typically lumped into a single parameter: the distribution coefficient (Kd). Usually, the distribution coefficient is assumed to be constant over the time frame of interest. However, this assumption could be critical under long-term geochemical changes as it is demonstrated thatmore » the distribution coefficient depends on the background chemical conditions (e.g. pH, Eh, and major chemistry). In this study, we provide a computational framework that combines the efficiency of Lagrangian methods with a sound and explicit description of the geochemical changes of the site and their influence on the radionuclide retention properties.« less

  9. A novel single-molecule study to determine protein--protein association constants.

    PubMed

    Ratcliff, G C; Erie, D A

    2001-06-20

    Atomic force microscopy (AFM) is traditionally used as an imaging technique to gain qualitative information for a biological system. We have successfully used the imaging capabilities of the AFM to determine protein-protein association constants. We have developed a method to measure the molecular weight of a protein based on its volume determined from AFM images. Our volume determination method allows for rapid, accurate analysis of large protein populations. On the basis of the measured volume, the fraction of monomers as dimers was determined for the DNA helicase UvrD, and the dissociation constant (K(d)) for the helicase was calculated. We determined a K(d) for UvrD of 1.4 microM, which is in good agreement with published K(d) data obtained from analytical ultracentrifugation (AUC) studies. Our method provides a rapid method for determining protein-protein association constants. PMID:11403593

  10. N(ω)-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([(3)H]UR-MK299) with Extended Residence Time.

    PubMed

    Keller, Max; Weiss, Stefan; Hutzler, Christoph; Kuhn, Kilian K; Mollereau, Catherine; Dukorn, Stefanie; Schindler, Lisa; Bernhardt, Günther; König, Burkhard; Buschauer, Armin

    2015-11-25

    Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field. PMID:26466164

  11. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  12. Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

    PubMed Central

    VandenBerg, S R; Allgren, R L; Todd, R D; Ciaranello, R D

    1983-01-01

    High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists. PMID:6574495

  13. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  14. Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide

    SciTech Connect

    Haring, R.; Kloog, Y.; Harshak-Felixbrodt, N.A.; Sokolovsky, M.

    1987-01-30

    Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for (/sup 3/H)TCP, (/sup 3/H)N-(1-(2-thienyl)cyclohexyl)piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for (/sup 3/H)TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K or Na). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by (/sup 3/H)azido phencyclidine is selectively inhibited by monovalent ions.

  15. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  16. High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element.

    PubMed

    Roy, Subhadeep; Tanious, Farial A; Wilson, W David; Ly, Danith H; Armitage, Bruce A

    2007-09-18

    Guanine-rich DNA and RNA sequences are known to fold into secondary structures known as G-quadruplexes. Recent biochemical evidence along with the discovery of an increasing number of sequences in functionally important regions of the genome capable of forming G-quadruplexes strongly indicates important biological roles for these structures. Thus, molecular probes that can selectively target quadruplex-forming sequences (QFSs) are envisioned as tools to delineate biological functions of quadruplexes as well as potential therapeutic agents. Guanine-rich peptide nucleic acids have been previously shown to hybridize to homologous DNA or RNA sequences forming PNA-DNA (or RNA) quadruplexes. For this paper we studied the hybridization of an eight-mer G-rich PNA to a quadruplex-forming sequence derived from the promoter region of the MYC proto-oncogene. UV melting analysis, fluorescence assays, and surface plasmon resonance experiments reveal that this PNA binds to the MYC QFS in a 2:1 stoichiometry and with an average binding constant Ka = (2.0 +/- 0.2) x 10(8) M(-1) or Kd = 5.0 nM. In addition, experiments carried out with short DNA targets revealed a dependence of the affinity on the sequence of bases in the loop region of the DNA. A structural model for the hybrid quadruplex is proposed, and implications for gene targeting by G-rich PNAs are discussed. PMID:17718513

  17. Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.

    PubMed Central

    Graham, R M; Hess, H J; Homcy, C J

    1982-01-01

    The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization. PMID:6285370

  18. Adult human keratinocyte cultures express 40, 52, 58 and 67 KD keratins

    SciTech Connect

    Bhatnagar, R.S.; Chandrakasan, G.; Hussain, M.Z.; Enriquez, B.; Ryder, M.I.

    1986-03-01

    Keratins are complex fibrous proteins characteristic of epithelial cells. Although several different classes of keratins are known to occur in the epidermis, the expression of all keratins has not been observed in vitro. The authors have developed a procedure that allows us to culture and passage up to ten times, adult human kerationcytes, in the absence of mesenchymal substrates. EM examination of stratifying cultures showed the presence of numerous tonofilaments, desmosomes and keratohyaline granules. The expression of different classes of keratins was examined by immunofluorescence, radiolabeling, SDS-PAGE and Western blot, using mouse monoclonal antibodies. Analysis of water-insoluble proteins showed the presence of keratins of M.W. 40 kd, 50-52 kd, 56-57 kd and 65-67 kd. The expression of 40kd keratin is known to be associated with basal cells. In their culture system basal cells secrete a well-defined basement membrane on which they rest. These cells may be responsible for the 40kd keratin. The expression of 65-67kd keratins has not previously been observed in vitro. These keratins are considered to be markers for terminal differentiation of epidermal cells. These proteins are presumed to be synthesized in their cultures by sloughing layers of rough, granular cells.

  19. Statistical theory of chromatography: new outlooks for affinity chromatography.

    PubMed Central

    Denizot, F C; Delaage, M A

    1975-01-01

    We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods. PMID:1061072

  20. 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

  1. Detailed characterization of a purified type 4 phosphodiesterase, HSPDE4B2B: differentiation of high- and low-affinity (R)-rolipram binding.

    PubMed

    Rocque, W J; Holmes, W D; Patel, I R; Dougherty, R W; Ittoop, O; Overton, L; Hoffman, C R; Wisely, G B; Willard, D H; Luther, M A

    1997-03-01

    We have overexpressed in a baculovirus expression system, and purified to > 95% homogeneity, milligram quantities of a human recombinant rolipram-sensitive cAMP phosphodiesterase, HSPDE4B2B (amino acid residues 81-564). The protein expression levels were approximately 8 mg of HSPDE4B2B (81-564) per liter of Sf9 cells. The Km of the purified enzyme for cAMP was 4 microM and the Ki for the Type 4 phosphodiesterase-specific inhibitor (R)-rolipram was 0.6 microM. The specific activity of the purified protein was 40 mumol/min/mg protein. A nonequilibrium filter binding assay revealed a high-affinity (R)-rolipram binding site on the purified enzyme with a Kd of 1.5 nM and a stoichiometry of 0.05-0.3 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Equilibrium dialysis experiments revealed a single binding constant of 140 nM with a stoichiometry of 0.75 mol of (R)-rolipram per mol of HSPDE4B2B (81-564). Size exclusion chromatography and analytical ultracentrifugation experiments suggest that the protein exists in multiple association states larger than a monomer. Proteolysis experiments revealed a 43-kDa fragment that contained catalytic and rolipram-inhibitable activities, but the fragment showed no high-affinity (R)-rolipram binding. Based on the proteolytic cleavage studies a 43-kDa protein was constructed, expressed, and purified. This protein, HSPDE4B2B (152-528), had Km and Vmax similar to those of the HSPDE4B2B (81-564) protein, but did not exhibit high-affinity (R)-rolipram binding. The protein did show low-affinity (R)-rolipram binding using the equilibrium binding assay. These results show that a low-affinity binding site for (R)-rolipram is solely contained within the catalytic domain of HSPDE4B2B, whereas high-affinity (R)-rolipram binding requires residues within the catalytic domain and residues flanking N- and/or C-terminal to the catalytic region. PMID:9056484

  2. Nonlocal Symmetry Reductions, CTE Method and Exact Solutions for Higher-Order KdV Equation

    NASA Astrophysics Data System (ADS)

    Ren, Bo; Liu, Xi-Zhong; Liu, Ping

    2015-02-01

    The nonlocal symmetries for the higher-order KdV equation are obtained with the truncated Painlevé method. The nonlocal symmetries can be localized to the Lie point symmetries by introducing suitable prolonged systems. The finite symmetry transformations and similarity reductions for the prolonged systems are computed. Moreover, the consistent tanh expansion (CTE) method is applied to the higher-order KdV equation. These methods lead to some novel exact solutions of the higher-order KdV system.

  3. Distribution Coefficients (Kd Values) for Waste Resins Generated from the K and L Disassembly Basin Facilities

    SciTech Connect

    Kaplan, D.I.

    2002-12-02

    The objective of this study was to measure 14C, 129I, and 99Tc Kd values of spent resin generated from the K and L Disassembly Basin Facilities. The scope of the work was to conduct Kd measurements of resins combined in the ratio that they are disposed, 42:58 cation:anion. Because it was not known how these spent resins would be buried, it was necessary to measure the Kd values in such a manner as to simulate both trench and vault disposal. This was accomplished by using an acid-rain simulant (a standard U.S. Environmental Protection Agency protocol) and a cement leachate simulant .

  4. Deriving the New Traveling Wave Solutions for the Nonlinear Dispersive Equation, KdV-ZK Equation and Complex Coupled KdV System Using Extended Simplest Equation Method

    NASA Astrophysics Data System (ADS)

    Mohammed, K. Elboree

    2015-10-01

    In this paper, we investigate the traveling wave solutions for the nonlinear dispersive equation, Korteweg-de Vries Zakharov-Kuznetsov (KdV-ZK) equation and complex coupled KdV system by using extended simplest equation method, and then derive the hyperbolic function solutions include soliton solutions, trigonometric function solutions include periodic solutions with special values for double parameters and rational solutions. The properties of such solutions are shown by figures. The results show that this method is an effective and a powerful tool for handling the solutions of nonlinear partial differential equations (NLEEs) in mathematical physics.

  5. A Genome-Inspired DNA Ligand for Affinity Capture of Insulin and Insulin-like Growth Factor-2

    PubMed Central

    Xiao, Junfeng; Carter, Jennifer A.; Frederick, Kimberley A.; McGown, Linda B.

    2009-01-01

    The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here we report on interactions of insulin and the highly homologous insulin-like growth factor 2 (IGF-2) with ILPR variants a, h and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI mass spectrometry and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with KD~10−13 M and KD~10−7 M, which was not observed for insulin with variant h (KD~10−8 M) or IGF-2 with either variant (KD’s~10−9 D M). The results provide a basis for design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery. PMID:19391177

  6. A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

    PubMed Central

    Lee, C H; Leung, B; Lemmon, M A; Zheng, J; Cowburn, D; Kuriyan, J; Saksela, K

    1995-01-01

    We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef. Images PMID:7588629

  7. Comparision of atrazine and metolachlor affinity for bermudagrass ( Cynodon dactylon L.) and two soils.

    PubMed

    Dozier, M C; Senseman, S A; Hoffman, D W; Baumann, P A

    2002-10-01

    Given that bermudagrass is being used as one of the grasses of choice in grass filter strip plantings as an acceptable grass to reduce off-target losses of herbicides, laboratory experiments were conducted to determine and compare the relative affinity of bermudagrass, a Weswood soil, and a Houston Black soil for atrazine (6-chloro- N-ethyl- N-isopropyl-1,3,5-triazine-2,4-diamine) and metolachlor (2-chloro- N-(2-ethyl-6-methylphenyl)- N-(2-methoxy-1-methyethyl) acetamide). Experiments were also conducted to determine if the presence of one herbicide affects the relative affinity of the other compound to these sorbents. The experiments were carried out using radiolabeled atrazine and metolachlor. Results were reported in disintegrations min(-1) (dpms) and converted to K(d) to determine and compare relative affinity. Both K(d) values for relative affinity of atrazine (86.2) and metolachlor (131.5) to bermudagrass were significantly greater than those of the two soils, Weswood (atrazine, 20.0 and metolachlor, 28.4) and Houston Black (atrazine, 35.8 and metolachlor, 33.5). The two compounds were also mixed together to mimic the common practice of applying atrazine and metolachlor simultaneously as a tank mix. Relative affinity of atrazine to any of the sorbents was not affected by the presence of metolachlor. Similarly, when comparing the affinity of metolachlor alone to that of metolachlor with atrazine present in the solution, no significant differences were observed for bermudagrass or the Weswood soil. However, on the Houston Black soil, the presence of atrazine significantly increased the soil's affinity for metolachlor. PMID:12202924

  8. 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.

  9. Dissociative changes in Bmax and KD of dopamine D2/D3 receptors with aging observed in functional subdivisions of striatum: A revisit with an improved data analysis method

    PubMed Central

    Kuwabara, Hiroto; McCaul, Mary E.; Wand, Gary S.; Earley, Christopher J.; Allen, Richard P; Weerts, Elise M; Dannals, Robert F.; Wong, Dean F

    2013-01-01

    Separate measurements of Bmax, the density of available receptors and KD, the equilibrium dissociation constant in the human brain with positron emission tomography (PET) have contributed to our understandings of neuropsychiatric disorders, especially with respect to the dopamine D2/D3 receptor system. However, existing methods have limited applications to the whole striatum, putamen or caudate nucleus. Improved methods are required to examine Bmax and KD in detailed functional striatal subdivisions that are becoming widely used. Methods In response, a new method (bolus-plus-infusion transformation, BPIT) was developed. After completion of a validation study for [11C]raclopride scans involving 81 subjects, age-associated changes in Bmax and KD were examined in 47 healthy subjects ranging from 18 to 77 years old. Results The BPIT method was consistent with established reference tissue methods regarding regional binding potential (BPND). BPIT yielded time-consistent estimates of Bmax and KD when scan and infusion lengths were set equal in the analysis. In addition, BPIT was shown to be robust against PET measurement errors when compared to a widely accepted transient equilibrium method (TEM). Altogether, BPIT was supported as a method for BPND, Bmax, and KD. We demonstrated age-associated declines in Bmax in all five functional striatum subdivisions with BPIT when corrected for multiple comparisons. These age-related effects were not consistently attainable with TEM. Irrespective to methods, KD remained unchanged with age. Conclusion The BPIT approach may be useful for understanding dopamine receptor abnormalities in neuropsychiatric disorders by enabling separate measurements of Bmax and KD in functional striatum subdivisions. PMID:22492734

  10. Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon

    PubMed Central

    2015-01-01

    Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM – 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd’s for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes. PMID:25170558

  11. Recommended Partition Coefficient (Kd) Values for Nuclide Partitioning in the Presence of Cellulose Degradation Products

    SciTech Connect

    Serkiz, S.M.

    2001-02-23

    This report documents the data analysis of the results of the described laboratory studies in order to recommend Kd values for use in Performance Assessment modeling of nuclide transport in the presence of CDP.

  12. Changes in parathyroid hormone receptor binding affinity during egg laying: implications for calcium homeostasis in chicken.

    PubMed

    Yasuoka, T; Kawashima, M; Takahashi, T; Iwata, A; Oka, N; Tanaka, K

    1996-12-01

    Parathyroid hormone (PTH) receptor bindings were examined in the membrane fraction of the calvaria and the kidney of the hen by the use of [125I]PTH-related protein (PTHrP) binding assays. The binding specificity, reversibility, and saturation of the receptor were demonstrated. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were obtained by Scatchard analyses. In both calvaria and kidney, Kd and Bmax values decreased at 3 h before oviposition in egg-laying hens, but not in nonlaying hens. Administration of 17 beta-estradiol or progesterone in vivo caused a decrease in the Kd and Bmax values. Ionized calcium concentrations in the blood plasma showed a decrease at 13 h before oviposition. The results suggest that the PTH receptor binding in the calvaria and the kidney is affected by ovarian steroid hormones and may play a role in maintaining the calcium homeostasis in the egg-laying hen. PMID:8970893

  13. Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.

    PubMed

    Qin, Shanshan; Ren, Yiran; Fu, Xu; Shen, Jie; Chen, Xin; Wang, Quan; Bi, Xin; Liu, Wenjing; Li, Lixin; Liang, Guangxin; Yang, Cheng; Shui, Wenqing

    2015-07-30

    Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery. PMID:26320641

  14. Integrability properties of a coupled KdV system and its supersymmetric extension

    NASA Astrophysics Data System (ADS)

    Sotomayor, Adrián; Restuccia, Alvaro

    2016-05-01

    We discuss several integrability properties of a coupled KdV system. We obtain a new generalization of the already known static solutions for the system. We then consider the supersymmetric extension of the coupled KdV system, it is a new integrable system. We show that for particular Grassmann algebras the system is the limit of a Clifford algebra valued system with nice stability properties. We briefly discuss the hamiltonian structures of this supersymmetric integrable system.

  15. Identification of the 100-kD victorin binding protein from oats.

    PubMed Central

    Wolpert, T J; Navarre, D A; Moore, D L; Macko, V

    1994-01-01

    The fungus Cochliobolus victoriae, the causal agent of victoria blight of oats, produces the host-specific toxin victorin. Sensitivity of oats to victorin, and thus susceptibility to the fungus, is controlled by a single dominant gene. This gene is believed to also confer resistance to the crown rust pathogen Puccinia coronata. In the case of victoria blight, the gene has been hypothesized to condition susceptibility by encoding a toxin receptor. A 100-kD victorin binding protein (VBP) has been identified; it binds radiolabeled victorin derivatives in a ligand-specific manner and in a genotype-specific manner in vivo. The VBP may function as a toxin receptor. In vitro translation coupled with indirect immunoprecipitation was used to identify the mRNA for the 100-kD VBP, and fractionated mRNAs were used to prepare cDNA libraries enriched in the relative abundance of cDNA for the 100-kD VBP. A 3.4-kb cDNA clone was isolated that, when subjected to a 400-bp 5' deletion, was capable of directing the synthesis of a protein in Escherichia coli, which reacted to an antibody specific for the 100-kD VBP. Peptide mapping, by limited proteolysis, indicated that the protein directed by the cDNA is the 100-kD VBP. Nucleotide sequence analysis of the cDNA revealed extensive homology to a previously cloned cDNA for the P protein component of the multienzyme complex glycine decarboxylase. Glycine decarboxylase is a nuclear-encoded, mitochondrial enzyme complex. Protein gel blot analysis indicated that the 100-kD VBP copurifies with mitochondria. Based on analysis of in vitro translation products, nucleotide sequence homology, mitochondrial localization, and the widespread species distribution of the 100-kD VBP, we concluded that the 100-kD VBP is the P protein component of glycine decarboxylase. PMID:7919984

  16. Nonvanishing boundary condition for the mKdV hierarchy and the Gardner equation

    NASA Astrophysics Data System (ADS)

    Gomes, J. F.; França, Guilherme S.; Zimerman, A. H.

    2012-01-01

    A Kac-Moody algebra construction for the integrable hierarchy containing the Gardner equation is proposed. Solutions are systematically constructed by employing the dressing method and deformed vertex operators, which take into account the nonvanishing boundary value problem for the modified Korteweg-de Vries (mKdV) hierarchy. Explicit examples are given and besides the usual KdV-like solitons, our solutions contemplate the large amplitude table-top solitons, kinks, dark solitons, breathers and wobbles.

  17. Heterogenetiy of glucocorticoid binders: a high affinity triamcinolone acetonide binder in bovine serum.

    PubMed

    Do, Y; Feldman, D

    1980-11-01

    While investigating glucocorticoid-binding proteins in bovine tissues, a new binder was found in fresh bovine serum which exhibited high affinity for certain synthetic glucocorticoids. This serum binder was characterized using [3H]triamcinolone acetonide (TA) as the ligand. On sucrose gradients, the [3H]TA peak sedimented at 8S, which was easily distinguishable from the [3H]cortisol-transcortin peak at 4S. Unlike the tissue receptor, which showed ionically dependent transformation from 8S in equilibrium or formed from 4S, the serum binder sedimented at 8S in both hypo- and hypertonic gradients. Binding properties were evaluated employing sephadex G-50 chromatography to separate bound from free steroid. Scatchard analysis of specific [3H]TA binding data revealed a straight line. The apparent equilibrium dissociation constant (Kd) was 7.8 +/- 0.7 X 10(-8) M, and the binding capacity was 772 +/- 70 fmol/mg serum protein. Hormonal specificity was determined by a competitive binding assay and revealed the following sequence: TA (100%) > betamethasone (47%) > triamcinolone (33%) > dexamethasone (2%) = cortisol = progesterone; aldosterone, estradiol, and testosterone exhibited negligible competitive activity. The serum binder was very stable, withstanding heating to 37 C for 60 min and long term storage in the frozen state. However, binding was significantly destroyed by trypsin. The binder was absent from fresh samples of chicken, mouse, rat, rabbit, dog, monkey, and human sera and frozen horse and porcine sera, but was clearly present in commercially available frozen calf, fetal calf, and lamb sera. At this time, we are unable to define the function of this binder, although its existence in both ovine and bovine sera suggests a possible role in ruminants. However, since bovine serum is routinely employed in tissue culture studies, the presence of this glucocorticoid binder might significantly influence many experiments. PMID:6775927

  18. Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

    PubMed Central

    Daram, P; Brunner, S; Rausch, C; Steiner, C; Amrhein, N; Bucher, M

    1999-01-01

    An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function. PMID:10559441

  19. On the orbital stability of Gaussian solitary waves in the log-KdV equation

    NASA Astrophysics Data System (ADS)

    Carles, Rémi; Pelinovsky, Dmitry

    2014-12-01

    We consider the logarithmic Korteweg-de Vries (log-KdV) equation, which models solitary waves in anharmonic chains with Hertzian interaction forces. By using an approximating sequence of global solutions of the regularized generalized KdV equation in H^1({R}) with conserved L2 norm and energy, we construct a weak global solution of the log-KdV equation in a subset of H^1({R}) . This construction yields conditional orbital stability of Gaussian solitary waves of the log-KdV equation, provided that uniqueness and continuous dependence of the constructed solution holds. Furthermore, we study the linearized log-KdV equation at the Gaussian solitary wave and prove that the associated linearized operator has a purely discrete spectrum consisting of simple purely imaginary eigenvalues in addition to the double zero eigenvalue. The eigenfunctions, however, do not decay like Gaussian functions but have algebraic decay. Using numerical approximations, we show that the Gaussian initial data do not spread out but produce visible radiation at the left slope of the Gaussian-like pulse in the time evolution of the linearized log-KdV equation.

  20. Compressive and rarefactive DIA solitons beyond the KdV limit

    SciTech Connect

    Mamun, A. A.; Deeba, F.

    2012-04-15

    The modified Gardner equation (MGE), showing the existence of compressive and rarefactive dust-ion-acoustic (DIA) solitons in a nonplanar dusty plasma (containing inertial ions, Boltzmann electrons, and negatively charged stationary dust) beyond the KdV Korteweg-de Vries (KdV) limit, is derived and numerically solved. The basic features of the compressive and rarefactive cylindrical and spherical DIA solitons, which are found to exist beyond the KdV limit, i.e., exist for {mu} {approx} 2/3 (where {mu} = Z{sub n}n{sub d0}/n{sub i0}, z{sub d} is the number of electrons residing onto the dust grain surface, n{sub d0}(n{sub i0}) is the dust (ion) number density at equilibrium, and {mu} {approx} 2/3 means that {mu} is not equal to 2/3, but it is around 2/3) are identified. These solitons (which can be referred to as DIA Gardner solitons (DIA-GSs)) are completely different from the KdV solitons because {mu} = 2/3 corresponds to the vanishing of the nonlinear coefficient of the KdV equation, and {mu} {approx} 2/3 corresponds to extremely large amplitude KdV solitons for which the validity of the reductive perturbation method breaks down. It is also shown that the properties of the nonplanar (cylindrical and spherical) DIA-GSs are significantly different from those of the one dimensional planar ones.

  1. A MEMS Dielectric Affinity Glucose Biosensor.

    PubMed

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-06-20

    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. Physiological sodium concentrations enhance the iodide affinity of the Na+/I- symporter

    NASA Astrophysics Data System (ADS)

    Nicola, Juan P.; Carrasco, Nancy; Mario Amzel, L.

    2014-06-01

    The Na+/I- symporter (NIS) mediates active I- transport—the first step in thyroid hormonogenesis—with a 2Na+:1I- stoichiometry. NIS-mediated 131I- treatment of thyroid cancer post-thyroidectomy is the most effective targeted internal radiation cancer treatment available. Here to uncover mechanistic information on NIS, we use statistical thermodynamics to obtain Kds and estimate the relative populations of the different NIS species during Na+/anion binding and transport. We show that, although the affinity of NIS for I- is low (Kd=224 μM), it increases when Na+ is bound (Kd=22.4 μM). However, this Kd is still much higher than the submicromolar physiological I- concentration. To overcome this, NIS takes advantage of the extracellular Na+ concentration and the pronounced increase in its own affinity for I- and for the second Na+ elicited by binding of the first. Thus, at physiological Na+ concentrations, ~79% of NIS molecules are occupied by two Na+ ions and ready to bind and transport I-.

  3. 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. PMID:24630982

  4. 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

  5. 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.

  6. A method for estimating the diffuse attenuation coefficient (KdPAR)from paired temperature sensors

    USGS Publications Warehouse

    Read, Jordan S.; Rose, Kevin C.; Winslow, Luke A.; Read, Emily Kara

    2015-01-01

    A new method for estimating the diffuse attenuation coefficient for photosynthetically active radiation (KdPAR) from paired temperature sensors was derived. We show that during cases where the attenuation of penetrating shortwave solar radiation is the dominant source of temperature changes, time series measurements of water temperatures at multiple depths (z1 and z2) are related to one another by a linear scaling factor (a). KdPAR can then be estimated by the simple equation KdPAR ln(a)/(z2/z1). A suggested workflow is presented that outlines procedures for calculating KdPAR according to this paired temperature sensor (PTS) method. This method is best suited for conditions when radiative temperature gains are large relative to physical noise. These conditions occur frequently on water bodies with low wind and/or high KdPARs but can be used for other types of lakes during time periods of low wind and/or where spatially redundant measurements of temperatures are available. The optimal vertical placement of temperature sensors according to a priori knowledge of KdPAR is also described. This information can be used to inform the design of future sensor deployments using the PTS method or for campaigns where characterizing sub-daily changes in temperatures is important. The PTS method provides a novel method to characterize light attenuation in aquatic ecosystems without expensive radiometric equipment or the user subjectivity inherent in Secchi depth measurements. This method also can enable the estimation of KdPAR at higher frequencies than many manual monitoring programs allow.

  7. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  8. Potential misevaluation of the ground-state dissociation constant from fluorimetric titrations: application to the ion indicators SBFI, PBFI, and fura-2.

    PubMed

    Kowalczyk, A; Boens, N; Meuwis, K; Ameloot, M

    1997-02-01

    A test based on time-resolved fluorescence experiments is proposed to assess the interference of an excited-state reaction with the fluorimetric determination of the ground-state dissociation constant Kd of ion [symbol: see text] fluorescent indicator complexes. If an inflection point occurs in the plot of the fluorescence signal vs -log [ion] in the concentration range of the ion where both decay times are invariant, this inflection point can be associated with the correct Kd. In contrast, the inflection point(s) in the concentration range where the decay times vary cannot be attributed to Kd. The test is applied to the fluorescent ion indicators SBFI (for Na+), PBFI (for K+), and Fura-2 (for Ca2+). In all three cases the decay times are invariant in the physiological concentration ranges of the respective ions, indicating that the fluorimetrically determined Kd values are actually the true Kd values. PMID:9025965

  9. New analytical exact solutions of time fractional KdV–KZK equation by Kudryashov methods

    NASA Astrophysics Data System (ADS)

    S Saha, Ray

    2016-04-01

    In this paper, new exact solutions of the time fractional KdV–Khokhlov–Zabolotskaya–Kuznetsov (KdV–KZK) equation are obtained by the classical Kudryashov method and modified Kudryashov method respectively. For this purpose, the modified Riemann–Liouville derivative is used to convert the nonlinear time fractional KdV–KZK equation into the nonlinear ordinary differential equation. In the present analysis, the classical Kudryashov method and modified Kudryashov method are both used successively to compute the analytical solutions of the time fractional KdV–KZK equation. As a result, new exact solutions involving the symmetrical Fibonacci function, hyperbolic function and exponential function are obtained for the first time. The methods under consideration are reliable and efficient, and can be used as an alternative to establish new exact solutions of different types of fractional differential equations arising from mathematical physics. The obtained results are exhibited graphically in order to demonstrate the efficiencies and applicabilities of these proposed methods of solving the nonlinear time fractional KdV–KZK equation.

  10. A reliable algorithm for KdV equations arising in warm plasma

    NASA Astrophysics Data System (ADS)

    Goswami, Amit; Singh, Jagdev; Kumar, Devendra

    2016-03-01

    The aim of the present work is to propose a simple and reliable algorithm namely homotopy perturbation transform method (HPTM) for KdV equations in warm plasma. The homotopy perturbation transform method is a combined form of the Laplace transform method with the homotopy perturbation method. In this method, the solution is calculated in the form of a convergent series with an easily computable compact. To illustrate the simplicity and reliability of the method, several examples are provided. In this paper, the homotopy perturbation transform method has been applied to obtain the solution of the KdV, mKdV, K(2, 2) and K(3,3) equations. We compared our solutions with the exact solutions. Results illustrate the applicability, efficiency and accuracy of HPTM to solve nonlinear equations despite needlessness to any linearization or perturbation. It is predicted that the proposed algorithm can be widely applied to other nonlinear problems in science and engineering.

  11. On the asymptotic solutions of the KdV equation with higher-order corrections

    NASA Astrophysics Data System (ADS)

    Burde, Georgy I.

    2005-07-01

    A method for construction of new integrable PDEs, whose properties are related to an asymptotic perturbation expansion with the leading-order term given by an integrable equation, is developed. A new integrable equation is constructed by applying the properly defined Lie-Bäcklund group of transformations to the leading-order equation. The integrable equations related to the Korteweg-de Vries (KdV) equation with higher-order corrections are used to investigate the limits of applicability of the so-called asymptotic integrability concept. It is found that the solutions of the higher-order KdV equations obtained by a near identity transform from the normal form solitary waves cannot, in principle, describe some intrinsic features of the high-order KdV solitons.

  12. Affine generalization of the Komar complex of general relativity

    NASA Astrophysics Data System (ADS)

    Mielke, Eckehard W.

    2001-02-01

    On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

  13. Lax representations and integrable time discretizations of the DDKdV, DDmKdV, and DDHOKdV

    NASA Astrophysics Data System (ADS)

    Zhu, Zuonong; Huang, Hongci; Xue, Weimin

    1999-02-01

    From a proper 2 × 2 discrete isospectral problem, a new differential-difference integrable equation in Lax sense is proposed by a discrete zero curvature equation. The DDKdV (differential-difference DdV equation) proposed by Ohta and Hirota and DDCDGKS (DD Caudrey-Dodd-Gibbon-Kotera-Sawada equation) are rederived. Some other new discrete KdV equations, discrete mKdV equations and discrete high order KdV equations which converge to the corresponding continuous soliton equations in the continuum limit are obtained. Integrable time discretizations of the DDKdV, DDmKdV (differential-difference mKdV equation) and DDHOKdV (differential-difference high order KdV equations) are given.

  14. KD-tree based clustering algorithm for fast face recognition on large-scale data

    NASA Astrophysics Data System (ADS)

    Wang, Yuanyuan; Lin, Yaping; Yang, Junfeng

    2015-07-01

    This paper proposes an acceleration method for large-scale face recognition system. When dealing with a large-scale database, face recognition is time-consuming. In order to tackle this problem, we employ the k-means clustering algorithm to classify face data. Specifically, the data in each cluster are stored in the form of the kd-tree, and face feature matching is conducted with the kd-tree based nearest neighborhood search. Experiments on CAS-PEAL and self-collected database show the effectiveness of our proposed method.

  15. Soliton solutions of the KdV equation with higher-order corrections

    NASA Astrophysics Data System (ADS)

    Wazwaz, Abdul-Majid

    2010-10-01

    In this work, the Korteweg-de Vries (KdV) equation with higher-order corrections is examined. We studied the KdV equation with first-order correction and that with second-order correction that include the terms of the fifth-order Lax, Sawada-Kotera and Caudrey-Dodd-Gibbon equations. The simplified form of the bilinear method was used to show the integrability of the first-order models and therefore to obtain multiple soliton solutions for each one. The obstacles to integrability of some of the models with second-order corrections are examined as well.

  16. Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.

    PubMed

    Jönsson, Peter; Southcombe, Jennifer H; Santos, Ana Mafalda; Huo, Jiandong; Fernandes, Ricardo A; McColl, James; Lever, Melissa; Evans, Edward J; Hudson, Alexander; Chang, Veronica T; Hanke, Tomáš; Godkin, Andrew; Dunne, Paul D; Horrocks, Mathew H; Palayret, Matthieu; Screaton, Gavin R; Petersen, Jan; Rossjohn, Jamie; Fugger, Lars; Dushek, Omer; Xu, Xiao-Ning; Davis, Simon J; Klenerman, David

    2016-05-17

    The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination. PMID:27114505

  17. Compensating Enthalpic and Entropic Changes Hinder Binding Affinity Optimization

    SciTech Connect

    Lafont,V.; Armstrong, A.; Ohtaka, H.; Kiso, Y.; Amzel, L.; Freire, E.

    2007-01-01

    A common strategy to improve the potency of drug candidates is to introduce chemical functionalities, like hydrogen bond donors or acceptors, at positions where they are able to establish strong interactions with the target. However, it is often observed that the added functionalities do not necessarily improve potency even if they form strong hydrogen bonds. Here, we explore the thermodynamic and structural basis for those observations. KNI-10033 is a potent experimental HIV-1 protease inhibitor with picomolar affinity against the wild-type enzyme (Kd = 13 pm). The potency of the inhibitor is the result of favorable enthalpic (?H = -8.2 kcal/mol) and entropic (-T?S = -6.7 kcal/mol) interactions. The replacement of the thioether group in KNI-10033 by a sulfonyl group (KNI-10075) results in a strong hydrogen bond with the amide of Asp 30B of the HIV-1 protease. This additional hydrogen bond improves the binding enthalpy by 3.9 kcal/mol; however, the enthalpy gain is completely compensated by an entropy loss, resulting in no affinity change. Crystallographic and thermodynamic analysis of the inhibitor/protease complexes indicates that the entropy losses are due to a combination of conformational and solvation effects. These results provide a set of practical guidelines aimed at overcoming enthalpy/entropy compensation and improve binding potency.

  18. An Ultrahigh Affinity D-Peptide Antagonist Of MDM2

    PubMed Central

    Zhan, Changyou; Zhao, Le; Wei, Xiaoli; Wu, Xueji; Chen, Xishan; Yuan, Weirong; Lu, Wei-Yue; Pazgier, Marzena; Lu, Wuyuan

    2012-01-01

    The oncoprotein MDM2 negatively regulates the activity and stability of the p53 tumor suppressor, and is an important molecular target for anticancer therapy. Aided by mirror image phage display and native chemical ligation, we have previously discovered several proteolysis-resistant duodecimal D-peptide antagonists of MDM2, termed DPMI-α, β, γ. The prototypic D-peptide inhibitor DPMI-α binds (25-109)MDM2 at an affinity of 220 nM, and kills tumor cells in vitro and inhibits tumor growth in vivo by reactivating the p53 pathway. Herein, we report the design of a super-active D-peptide antagonist of MDM2, termed DPMI-δ, of which the binding affinity for (25-109)MDM2 has been improved over DPMI-α by three orders of magnitude (Kd = 220 pM). X-ray crystallographic studies validate DPMI-δ as an exceedingly potent inhibitor of the p53-MDM2 interaction, promising to be a highly attractive lead drug candidate for anticancer therapeutic development. PMID:22694121

  19. Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

    PubMed

    Fritzler, M J; Hamel, J C; Ochs, R L; Chan, E K

    1993-07-01

    Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins. PMID:8315394

  20. Key KdSOC1 gene expression profiles during plantlet morphogenesis under hormone, photoperiod, and drought treatments.

    PubMed

    Liu, C; Zhu, C; Zeng, H M

    2016-01-01

    Kalanchoe daigremontiana utilizes plantlet formation between its zigzag leaf margins as its method of asexual reproduction. In this study, K. daigremontiana SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (KdSOC1), a key intermediate in the transition from vegetative to asexual growth, was cloned. Furthermore, its expression profiles during plantlet formation under different environmental and hormone induction conditions were analyzed. The full-KdSOC1 cDNA sequence length was 1410 bp with 70% shared homology with Carya cathayensis SOC1. The conserved domain search of KdSOC1 showed the absence of I and C domains, which might indicate novel biological functions in K. daigremontiana. The full-KdSOC1 promoter sequence was 1401 bp long and contained multiple-hormone-responsive cis-acting elements. Hormone induction assays showed that gibberellins and salicylic acid mainly regulated KdSOC1 expression. The swift change from low to high KdSOC1 expression levels during long-day induction was accompanied by the rapid emergence of plantlets. Drought stress stimulated KdSOC1 expression in leaves both with and without plantlet formation. Together, the results suggested that KdSOC1 was closely involved in environmental stimulation signal perception and the transduction of K. daigremontiana plantlet formation. Therefore, future identification of KdSOC1 functions might reveal key information that will help elucidate the transition network between embryogenesis and organogenesis during plantlet formation. PMID:26909971

  1. Lie Symmetry Analysis and Explicit Solutions of the Time Fractional Fifth-Order KdV Equation

    PubMed Central

    Wang, Gang wei; Xu, Tian zhou; Feng, Tao

    2014-01-01

    In this paper, using the Lie group analysis method, we study the invariance properties of the time fractional fifth-order KdV equation. A systematic research to derive Lie point symmetries to time fractional fifth-order KdV equation is performed. In the sense of point symmetry, all of the vector fields and the symmetry reductions of the fractional fifth-order KdV equation are obtained. At last, by virtue of the sub-equation method, some exact solutions to the fractional fifth-order KdV equation are provided. PMID:24523885

  2. Lie symmetry analysis and explicit solutions of the time fractional fifth-order KdV equation.

    PubMed

    Wang, Gang Wei; Xu, Tian Zhou; Feng, Tao

    2014-01-01

    In this paper, using the Lie group analysis method, we study the invariance properties of the time fractional fifth-order KdV equation. A systematic research to derive Lie point symmetries to time fractional fifth-order KdV equation is performed. In the sense of point symmetry, all of the vector fields and the symmetry reductions of the fractional fifth-order KdV equation are obtained. At last, by virtue of the sub-equation method, some exact solutions to the fractional fifth-order KdV equation are provided. PMID:24523885

  3. Method of Multiple Scales and Travelling Wave Solutions for (2+1)-Dimensional KdV Type Nonlinear Evolution Equations

    NASA Astrophysics Data System (ADS)

    Ayhan, Burcu; Özer, M. Naci; Bekir, Ahmet

    2016-08-01

    In this article, we applied the method of multiple scales for Korteweg-de Vries (KdV) type equations and we derived nonlinear Schrödinger (NLS) type equations. So we get a relation between KdV type equations and NLS type equations. In addition, exact solutions were found for KdV type equations. The ( G'} over G )-expansion methods and the ( {G'} over G, {1 over G}} )-expansion methods were proposed to establish new exact solutions for KdV type differential equations. We obtained periodic and hyperbolic function solutions for these equations. These methods are very effective for getting travelling wave solutions of nonlinear evolution equations (NEEs).

  4. On the Khinchin Constant

    NASA Technical Reports Server (NTRS)

    Bailey, David H.; Borwein, Jonathan M.; Crandall, Richard E.; Craw, James M. (Technical Monitor)

    1995-01-01

    We prove known identities for the Khinchin constant and develop new identities for the more general Hoelder mean limits of continued fractions. Any of these constants can be developed as a rapidly converging series involving values of the Riemann zeta function and rational coefficients. Such identities allow for efficient numerical evaluation of the relevant constants. We present free-parameter, optimizable versions of the identities, and report numerical results.

  5. Solar constant secular changes

    NASA Technical Reports Server (NTRS)

    Schatten, Kenneth H.; Orosz, Jerome A.

    1990-01-01

    A recent model for solar constant secular changes is used to calculate a 'proxy' solar constant for: (1) the past four centuries, based upon the sunspot record, (2) the past nine centuries, based upon C-14 observations and their relation to solar activity, and (3) the next decade, based upon a dynamo theory model for the solar cycle. The proxy solar constant data is tabulated as it may be useful for climate modelers studying global climate changes.

  6. Soliton equations related to the affine Kac-Moody algebra D{4/(1)}

    NASA Astrophysics Data System (ADS)

    Gerdjikov, V. S.; Mladenov, D. M.; Stefanov, A. A.; Varbev, S. K.

    2015-06-01

    We have derived the hierarchy of soliton equations associated with the untwisted affine Kac-Moody algebra D {4/(1)} by calculating the corresponding recursion operators. The Hamiltonian formulation of the equations from the hierarchy is also considered. As an example we have explicitly presented the first non-trivial member of the hierarchy, which is an one-parameter family of mKdV equations. We have also considered the spectral properties of the Lax operator and introduced a minimal set of scattering data.

  7. 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

  8. Experimental values for 241Am and 239+240Pu Kd's in French agricultural soils.

    PubMed

    Roussel-Debet, Sylvie

    2005-01-01

    Experiments resulted in determination of K(d) values for (241)Am and (239+240)Pu in 6 soils whose characteristics are representative of agricultural soils found around French nuclear power plant sites. These experiments were conducted in stirred batch reactors and the sorption isotherms were plotted. For americium, the experimental K(d) values varied from 60 to 4000 Lkg(-1) (d.w.) and correlated well with soil pH, K(d) increased with increasing pH. As regards plutonium, the experimental K(d) values varied between 300 and 9600 Lkg(-1) and decreased with increasing total sand content. The estimation of the total residence time determined by using a particular and experimentally refined value for K(d)-with a model similar to those currently used for impact assessments-illustrates the importance of establishing values that are better suited to specific soils than generic values. Lastly, depending on the type of evaluation envisioned, it might be important to look for a specific value of K(d)-and even modelling-more suited to the specificity of the scenario studied, by performing more complex, or even in situ, experiments. PMID:15603906

  9. Singular solutions of the KdV equation and the inverse scattering method

    SciTech Connect

    Arkad'ev, V.A.; Pogrebkov, A.K.; Polivanov, M.K.

    1985-12-20

    The paper is devoted to the construction of singular solutions of the KdV equation. The presentation is based on a variant of the inverse scattering method for singular solutions of nonlinear equations developed in previous works of the authors.

  10. A Local Discontinuous Galerkin Method for the Complex Modified KdV Equation

    SciTech Connect

    Li Wenting; Jiang Kun

    2010-09-30

    In this paper, we develop a local discontinuous Galerkin(LDG) method for solving complex modified KdV(CMKdV) equation. The LDG method has the flexibility for arbitrary h and p adaptivity. We prove the L{sup 2} stability for general solutions.

  11. Analytical integrability and physical solutions of d-KdV equation

    SciTech Connect

    Karmakar, P.K.; Dwivedi, C.B.

    2006-03-15

    A new idea of electron inertia-induced ion sound wave excitation for transonic plasma equilibrium has already been reported. In such unstable plasma equilibrium, a linear source driven Korteweg-de Vries (d-KdV) equation describes the nonlinear ion sound wave propagation behavior. By numerical techniques, two distinct classes of solution (soliton and oscillatory shocklike structures) are obtained. Present contribution deals with the systematic methodological efforts to find out its (d-KdV) analytical solutions. As a first step, we apply the Painleve method to test whether the derived d-KdV equation is analytically integrable or not. We find that the derived d-KdV equation is indeed analytically integrable since it satisfies Painleve property. Hirota's bilinearization method and the modified sine-Gordon method (also termed as sine-cosine method) are used to derive the analytical results. Perturbative technique is also applied to find out quasistationary solutions. A few graphical plots are provided to offer a glimpse of the structural profiles obtained by different methods applied. It is conjectured that these solutions may open a new scope of acoustic spectroscopy in plasma hydrodynamics.

  12. Decouple a coupled KdV system of Nutku and Og˜uz

    NASA Astrophysics Data System (ADS)

    Hu, Heng Chun; Liu, Q. P.

    2002-02-01

    A coupled KdV system with a free parameter proposed by Nutku and Og˜uz is considered. It is shown that the system passes the WTC's Painlevé test for arbitrary value of the parameter. A further analysis yields that the parameter can be removed and the system can be decoupled.

  13. Fundamental Physical Constants

    National Institute of Standards and Technology Data Gateway

    SRD 121 CODATA Fundamental Physical Constants (Web, free access)   This site, developed in the Physics Laboratory at NIST, addresses three topics: fundamental physical constants, the International System of Units (SI), which is the modern metric system, and expressing the uncertainty of measurement results.

  14. 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.

  15. A {gamma} dose distribution evaluation technique using the k-d tree for nearest neighbor searching

    SciTech Connect

    Yuan Jiankui; Chen Weimin

    2010-09-15

    Purpose: The authors propose an algorithm based on the k-d tree for nearest neighbor searching to improve the {gamma} calculation time for 2D and 3D dose distributions. Methods: The {gamma} calculation method has been widely used for comparisons of dose distributions in clinical treatment plans and quality assurances. By specifying the acceptable dose and distance-to-agreement criteria, the method provides quantitative measurement of the agreement between the reference and evaluation dose distributions. The {gamma} value indicates the acceptability. In regions where {gamma}{<=}1, the predefined criterion is satisfied and thus the agreement is acceptable; otherwise, the agreement fails. Although the concept of the method is not complicated and a quick naieve implementation is straightforward, an efficient and robust implementation is not trivial. Recent algorithms based on exhaustive searching within a maximum radius, the geometric Euclidean distance, and the table lookup method have been proposed to improve the computational time for multidimensional dose distributions. Motivated by the fact that the least searching time for finding a nearest neighbor can be an O(log N) operation with a k-d tree, where N is the total number of the dose points, the authors propose an algorithm based on the k-d tree for the {gamma} evaluation in this work. Results: In the experiment, the authors found that the average k-d tree construction time per reference point is O(log N), while the nearest neighbor searching time per evaluation point is proportional to O(N{sup 1/k}), where k is between 2 and 3 for two-dimensional and three-dimensional dose distributions, respectively. Conclusions: Comparing with other algorithms such as exhaustive search and sorted list O(N), the k-d tree algorithm for {gamma} evaluation is much more efficient.

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

    SciTech Connect

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

    1989-07-05

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

  17. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    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, and 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:18429272

  18. 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

  19. Robust Affinity Standards for Cu(I) Biochemistry

    PubMed Central

    Bagchi, Pritha; Morgan, M. Thomas; Bacsa, John; Fahrni, Christoph J.

    2014-01-01

    The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. Concluding from X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations, all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10−10 and 10−17 M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate (BCS) or 2,2′-bicinchoninic acid (BCA), we further calibrated their Cu(I) stability constants against the MCL-series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (logK = 14.3±0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in E. coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small molecule ligands. PMID:24298878

  20. In-situ Kd values and geochemical behavior for inorganic and organic constituents of concern at the TNX Outfall Delta

    SciTech Connect

    Kaplan, D.I.

    2000-02-11

    A series of tests were conducted to provide site-specific Kd values for constituents of concern at the TNX Outfall Delta Operable Unit. These Kd values can be used to calculate contaminant migration within the operable unit and are, at this time considered to be the most defensible values.

  1. A Bäcklund Transformation of the Restricted mKdV Flow with a Rosochatius Deformation

    NASA Astrophysics Data System (ADS)

    Zhou, Ru-Guang

    2013-09-01

    A Bäcklund transformation of the restricted mKdV flow with a Rosochatius deformation is constructed. Its Lax representation and thus N invariants in involution are presented. Such Bäcklund transformation is a Rosochatius deformation of that of the restricted mKdV flow.

  2. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  3. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  4. Synthesis and binding affinity of an iodinated juvenile hormone

    SciTech Connect

    Prestwich, G.D.; Eng, W.S.; Robles, S.; Vogt, R.G.; Wisniewski, J.R.; Wawrzenczyk, C.

    1988-01-25

    The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural /sup 3/H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated (/sup 125/I)12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added /sup 125/I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of (/sup 125/I)12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

  5. 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.…

  6. 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…

  7. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa. PMID:8183950

  8. Differentiation of extrastriatal dopamine D2 receptor density and affinity in the human brain using PET.

    PubMed

    Olsson, Hans; Halldin, Christer; Farde, Lars

    2004-06-01

    Dopaminergic neurotransmission in extrastriatal regions may play a crucial role in the pathophysiology and treatment of neuropsychiatric disorders. The high-affinity radioligands [(11)C]FLB 457, [(123)I]epidepride, and [(18)F]fallypride are now used in clinical studies to measure these low-density receptor populations in vivo. However, a single determination of the regional binding potential (BP) does not differentiate receptor density (B(max)) from the apparent affinity (K(D)). In this positron emission tomography (PET) study, we measured extrastriatal dopamine D2 receptor density (B(max)) and apparent affinity (K(D)) in 10 healthy subjects using an in vivo saturation approach. Each subject participated in two to three PET measurements with different specific radioactivity of [(11)C]FLB 457. The commonly used simplified reference tissue model (SRTM) was used in a comparison of BP values with the B(max) values obtained from the saturation analysis. The calculated regional receptor density values were of the same magnitude (0.33-1.68 nM) and showed the same rank order as reported from postmortem studies, that is, in descending order thalamus, lateral temporal cortex, anterior cinguli, and frontal cortex. The affinity ranged from 0.27 to 0.43 nM, that is, approximately 10-20 times the value found in vitro (20 pM). The area under the cerebellar time activity curve (TAC) was slightly lower (11 +/- 8%, mean +/- SD, P = 0.004, n = 10) after injection of low as compared with high specific radioactivity, indicating sensitivity to the minute density of dopamine D2 receptors in the this region. The results of the present study support that dopamine D2 receptor density and affinity can be differentiated in low-density regions using a saturation approach. There was a significant (P < 0.001) correlation between the binding potential calculated with SRTM and the receptor density (B(max)), which supports the use of BP in clinical studies where differentiation of B(max) and K(D

  9. The cosmological constant problem

    SciTech Connect

    Dolgov, A.D.

    1989-05-01

    A review of the cosmological term problem is presented. Baby universe model and the compensating field model are discussed. The importance of more accurate data on the Hubble constant and the Universe age is stressed. 18 refs.

  10. Space Shuttle astrodynamical constants

    NASA Technical Reports Server (NTRS)

    Cockrell, B. F.; Williamson, B.

    1978-01-01

    Basic space shuttle astrodynamic constants are reported for use in mission planning and construction of ground and onboard software input loads. The data included here are provided to facilitate the use of consistent numerical values throughout the project.

  11. Constant potential pulse polarography

    USGS Publications Warehouse

    Christie, J.H.; Jackson, L.L.; Osteryoung, R.A.

    1976-01-01

    The new technique of constant potential pulse polarography, In which all pulses are to be the same potential, is presented theoretically and evaluated experimentally. The response obtained is in the form of a faradaic current wave superimposed on a constant capacitative component. Results obtained with a computer-controlled system exhibit a capillary response current similar to that observed In normal pulse polarography. Calibration curves for Pb obtained using a modified commercial pulse polarographic instrument are in good accord with theoretical predictions.

  12. Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

    PubMed Central

    Kahn, N N; Bauman, W A; Sinha, A K

    1996-01-01

    Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI. PMID:8552614

  13. GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells.

    PubMed

    Meier, E; Drejer, J; Schousboe, A

    1984-12-01

    The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors. PMID:6149269

  14. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

  15. 92-kD gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.

    PubMed Central

    Ståhle-Bäckdahl, M; Inoue, M; Guidice, G J; Parks, W C

    1994-01-01

    Eosinophils are prominent in bullous pemphigoid (BP), and proteases secreted from these and other inflammatory cells may induce disruption of the basement membrane. We used in situ hybridization and immunohistochemistry to localize the sites of 92-kD gelatinase expression in BP lesions. In all samples (20/20), a strong signal for gelatinase mRNA was detected only in eosinophils and was most pronounced where these cells accumulated at the floor of forming blisters. No other cells were positive for enzyme mRNA. Both eosinophils and neutrophils, however, contained immunoreactive 92-kD gelatinase indicating that active expression occurred only in eosinophils. Degranulated eosinophils were also seen near blisters, and as demonstrated by gelatin zymography, immunoblotting, and ELISA, 92-kD gelatinase protein was prominent in BP blister fluid. No other gelatinolytic activity was specifically detected in BP fluid, and only small amounts of 92-kD gelatinase were present in suction blister fluids. As demonstrated in vitro, 92-kD gelatinase cleaved the extracellular, collagenous domain of recombinant 180-kD BP autoantigen (BP180, BPAG2, HD4, type XVII collagen), a transmembrane molecule of the epidermal hemidesmosome. Our results suggest that production and release 92-kD gelatinase by eosinophils contributes significantly to tissue damage in BP. Images PMID:8182134

  16. Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

    PubMed

    Mavaddat, N; Mason, D W; Atkinson, P D; Evans, E J; Gilbert, R J; Stuart, D I; Fennelly, J A; Barclay, A N; Davis, S J; Brown, M H

    2000-09-01

    Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions. PMID:10831600

  17. On the problem of periodicity and hidden solitons for the KdV model.

    PubMed

    Engelbrecht, Jüri; Salupere, Andrus

    2005-03-01

    In continuum limit, the Fermi-Pasta-Ulam lattice is modeled by a Korteweg-de Vries (KdV) equation. It is shown that the long-time behavior of a KdV soliton train emerging from a harmonic excitation has a regular periodicity of right- and left-going trajectories. In a soliton train not all the solitons are visible, the solitons with smaller amplitude are hidden and their influence is seen through the changes of phase shifts of larger solitons. In the case of an external harmonic force several resonance schemes are revealed where both visible and hidden solitons have important roles. The weak, moderate, strong, and dominating fields are distinguished and the corresponding solution types presented. PMID:15836291

  18. The origin of gauge symmetries in integrable systems of the KdV type

    SciTech Connect

    Bakas, I.; Depireux, D.A. )

    1992-03-30

    Generalized systems of integrable nonlinear differential equations of the KdV type are considered from the point of view of self-dual Yang-Mills theory in space-times with signature. This paper presents a systematic method for embedding the rth flows of the SL(N) KdV hierarchy with N {ge} 2 and r {lt} N in the dimensionally reduced self-dual system using SL(N) as a gauge group. We also find that for r {gt} N the corresponding equations can be described in a similar fashion, provided that (in general) the rank of the gauge group increases accordingly. Certain connections of this formalism with W{sub N} algebras are also discussed. Finally the authors obtain a new class of nonlinear systems in two dimensions by introducing self-dual Ansatze associated with the W{sup (l)} {sub N} algebras of Bershadsky and Polyakov.

  19. The Fall and Rise of X-ray Supernova 2005kd

    NASA Astrophysics Data System (ADS)

    Dwarkadas, Vikram; Reddy, Ratuja; Bauer, Franz E.

    2015-01-01

    Supernova (SN) 2005kd belongs to the class of Type IIn SNe, which show narrow lines in their early spectra. It is one of the most luminous X-ray SNe known - in excess of Lx = 1041 ergs/s at peak. The evolution of its X-ray luminosity with time is rather atypical, with a general dimming, as expected, followed by an unexpected re-brightening. We report on a recent 30 ks Chandra observation of SN 2005kd, which extends the X-ray lightcurve of this SN from 2005 to 2013. We use the time evolution of the X-ray flux to explore the expansion of the forward and reverse shock of the SN within the surrounding medium, as well as to study the structure of the surrounding medium.

  20. Lymphocyte expression of a 90 kD brush border antigen.

    PubMed Central

    Van Leer, E H; Moullier, P; Ronco, P; Verroust, P

    1987-01-01

    This study analyses the expression by rat lymphocytes of six renal brush border (BB) antigens defined by a set of monoclonal antibodies and demonstrates that a 90 kD protein (gp 90), synthesized by BB and glomeruli is found on the surface of T and B cells. The other antigens, including the 330 kD protein involved in Heymann's nephritis--a model of epimembranous glomerulonephritis--are not detectable on the surface of lymphocytes. Immunochemical studies indicate that gp 90 and the related protein immunoprecipitated from thymocyte membranes co-migrate in SDS-PAGE. Quantitative binding analysis shows that the number of antigenic sites is in the same order of magnitude on thymocytes, spleen and bone-marrow lymphocytes as well as on two lines of pre-B and pre-T cells, but considerably lower on a highly differentiated helper T cell line. Images Fig. 4 PMID:3301100

  1. DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

    PubMed

    Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I

    2016-03-01

    Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196

  2. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

  3. The geometric property of soliton solutions for the integrable KdV6 equations

    NASA Astrophysics Data System (ADS)

    Li, Jibin; Zhang, Yi

    2010-04-01

    The geometric property of soliton solutions of the three completely integrable sixth-order nonlinear equations (KdV6) is studied by using the method of dynamical systems and the work of Wazwaz [Appl. Math. Comput. 204, 963 (2008)]. This paper proved that a solitary wave solution corresponds to a homoclinic orbit of a four-dimensional dynamical system to a equilibrium point. The orbit lies on the intersection curve of two level set passing through the same equilibrium point.

  4. KdV-Burgers equation in the modified continuum model considering anticipation effect

    NASA Astrophysics Data System (ADS)

    Liu, Huaqing; Zheng, Pengjun; Zhu, Keqiang; Ge, Hongxia

    2015-11-01

    The new continuum model mentioned in this paper is developed based on optimal velocity car-following model, which takes the drivers' anticipation effect into account. The critical condition for traffic flow is derived, and nonlinear analysis shows density waves occur in traffic flow because of the small disturbance. Near the neutral stability line, the KdV-Burgers equation is derived and one of the solutions is given. Numerical simulation is carried out to show the local cluster described by the model.

  5. Structure of the Λ (1405 ) and the K-d →π Σ n reaction

    NASA Astrophysics Data System (ADS)

    Ohnishi, Shota; Ikeda, Yoichi; Hyodo, Tetsuo; Weise, Wolfram

    2016-02-01

    The Λ (1405 ) resonance production reaction is investigated within the framework of the coupled-channels Alt-Grassberger-Sandhas (AGS) equations. We perform full three-body calculations for the K ¯N N -π Y N amplitudes on the physical real energy axis and investigate how the signature of the Λ (1405 ) appears in the cross sections of the K-d →π Σ n reactions, also in view of the planned E31 experiment at J-PARC. Two types of meson-baryon interaction models are considered: an energy-dependent interaction based on chiral S U (3 ) effective field theory, and an energy-independent version that has been used repeatedly in phenomenological approaches. These two models have different off-shell properties that imply correspondingly different behavior in the three-body system. We investigate how these features show up in differential cross sections of K-d →π Σ n reactions. Characteristic patterns distinguishing between the two models are found in the invariant mass spectrum of the final π Σ state. The K-d →π Σ n reaction, with different (π±Σ∓ and π0Σ0 ) charge combinations in the final state, is thus demonstrated to be a useful tool for investigating the subthreshold behavior of the K ¯N interaction.

  6. Compound Galactosylceramidase Gene (GALC) Heterozygosity in a Boy with Infantile Krabbe Disease (KD).

    PubMed

    Gucev, Zoran; Tasic, Velibor

    2015-01-01

    Krabbe disease (KD) (globoid cell leukodystrophy) is a degenerative, lysosomal storage disease, caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. The inheritance is autosomal recessive. KD affects the white matter of the central and peripheral nervous systems. We present a 3 year old boy in whom the disease had an 'infantile' or 'classic' presentation, with spasticity, irritability, and developmental delay. In addition the boy showed progressive severe motor and mental deterioration, difficulties in swallowing and decerebration. Molecular analysis revealed that the child is a compound heterozygote: p.Asp187Val (c.560A>T) and p.Ile250Thr (c.749T>C). The father was the carrier of p.Asp187Val (c.560A>T), while the mother was the carrier of the p.Ile250Thr (c.749T>C) in exon 6 of the GALC gene. The clinical course in this compound heterozygote is severe and the patient passed away at the age of 3 years. Genotype-phenotype relations are discussed in this Macedonian patient with KD. PMID:27442402

  7. Effect of hyperoxaluria on the inhibitory activity of a 45-kD urinary protein.

    PubMed

    Selvam, Ramasamy; Balakrishnan, Selvakumar; Kalaiselvi, Periandavan

    2002-02-01

    Proteins are thought to play a major role in stone formation and structurally abnormal proteins have been reported to be present in the urine of stone formers. This study was aimed to determine whether hyperoxaluria modifies the kinetic properties of urinary inhibitory proteins. Hyperoxaluria was induced by feeding 1% ethylene glycol to rats. Oxalate, uric acid and calcium excretion were increased progressively during hyperoxaluria, while magnesium level was decreased. Urinary proteins were separated on a DEAE-cellulose column by eluting with stepwise increasing salt concentration in 0.05 M Tris-HCl buffer (pH 7.0). Each protein fraction was studied for its crystallization inhibitory potential by the spectrophotometric method. The protein eluted in 0.3 M NaCl containing buffer had the maximal nucleation as well as inhibitory activity. The protein had a molecular weight of 45 kD. In hyperoxaluria, the urinary excretion of this protein significantly increased. In the crystal growth assay, the control rat 45-kD protein inhibited nucleation by 75% and aggregation by 100%. In contrast, it is very interesting to note that the protein derived from 28th day hyperoxaluric urine, behaved as a promoter of nucleation (-113%, percentage inhibition) and weak inhibitor of aggregation (28%). A significantly high negative correlation (r = -0.97) between oxalate excretion and the inhibitory activity of the 45-kD protein was observed suggesting a modification of the protein by oxalate. PMID:11818706

  8. Estimation of Kd of lead and (210)Po in 11 soils from India.

    PubMed

    Maity, Sukanta; Pandit, G G

    2014-12-01

    The fate of contaminant transport is often estimated using the distribution (partition) coefficient, Kd. It is a measure of sorption of contaminants to soil. As Kd is element, soil type and ground water dependent, chemical characterization of soil and ground water of the particular site is essential. In this study, soil and ground water samples from different locations around India were collected. The soil samples were physically characterized and pH, CaCO3, cation exchange capacity (CEC), organic matter and organic carbon were determined. Equilibration time for lead and (210)Po were estimated with respect to contact time and were found to be 28 and 72 h respectively. The Kd of lead varied from 6700 to 31,000 L/kg with a geometric mean of 15,200 L/kg, and for (210)Po from 1400 to 8700 L/kg with a geometric mean of 3700 L/kg. PMID:24787466

  9. Dielectric Constant of Suspensions

    NASA Astrophysics Data System (ADS)

    Mendelson, Kenneth S.; Ackmann, James J.

    1997-03-01

    We have used a finite element method to calculate the dielectric constant of a cubic array of spheres. Extensive calculations support preliminary conclusions reported previously (K. Mendelson and J. Ackmann, Bull. Am. Phys. Soc. 41), 657 (1996).. At frequencies below 100 kHz the real part of the dielectric constant (ɛ') shows oscillations as a function of the volume fraction of suspension. These oscillations disappear at low conductivities of the suspending fluid. Measurements of the dielectric constant (J. Ackmann, et al., Ann. Biomed. Eng. 24), 58 (1996). (H. Fricke and H. Curtis, J. Phys. Chem. 41), 729 (1937). are not sufficiently sensitive to show oscillations but appear to be consistent with the theoretical results.

  10. Elastic constants of calcite

    USGS Publications Warehouse

    Peselnick, L.; Robie, R.A.

    1962-01-01

    The recent measurements of the elastic constants of calcite by Reddy and Subrahmanyam (1960) disagree with the values obtained independently by Voigt (1910) and Bhimasenachar (1945). The present authors, using an ultrasonic pulse technique at 3 Mc and 25??C, determined the elastic constants of calcite using the exact equations governing the wave velocities in the single crystal. The results are C11=13.7, C33=8.11, C44=3.50, C12=4.82, C13=5.68, and C14=-2.00, in units of 1011 dyncm2. Independent checks of several of the elastic constants were made employing other directions and polarizations of the wave velocities. With the exception of C13, these values substantially agree with the data of Voigt and Bhimasenachar. ?? 1962 The American Institute of Physics.

  11. 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.

  12. Hierarchical octree and k-d tree grids for 3D radiative transfer simulations

    NASA Astrophysics Data System (ADS)

    Saftly, W.; Baes, M.; Camps, P.

    2014-01-01

    Context. A crucial ingredient for numerically solving the three-dimensional radiative transfer problem is the choice of the grid that discretizes the transfer medium. Many modern radiative transfer codes, whether using Monte Carlo or ray tracing techniques, are equipped with hierarchical octree-based grids to accommodate a wide dynamic range in densities. Aims: We critically investigate two different aspects of octree grids in the framework of Monte Carlo dust radiative transfer. Inspired by their common use in computer graphics applications, we test hierarchical k-d tree grids as an alternative for octree grids. On the other hand, we investigate which node subdivision-stopping criteria are optimal for constructing of hierarchical grids. Methods: We implemented a k-d tree grid in the 3D radiative transfer code SKIRT and compared it with the previously implemented octree grid. We also considered three different node subdivision-stopping criteria (based on mass, optical depth, and density gradient thresholds). Based on a small suite of test models, we compared the efficiency and accuracy of the different grids, according to various quality metrics. Results: For a given set of requirements, the k-d tree grids only require half the number of cells of the corresponding octree. Moreover, for the same number of grid cells, the k-d tree is characterized by higher discretization accuracy. Concerning the subdivision stopping criteria, we find that an optical depth criterion is not a useful alternative to the more standard mass threshold, since the resulting grids show a poor accuracy. Both criteria can be combined; however, in the optimal combination, for which we provide a simple approximate recipe, this can lead to a 20% reduction in the number of cells needed to reach a certain grid quality. An additional density gradient threshold criterion can be added that solves the problem of poorly resolving sharp edges and strong density gradients. Conclusions: We advocate the use

  13. Two-component coupled KdV equations and its connection with the generalized Harry Dym equations

    SciTech Connect

    Popowicz, Ziemowit

    2014-01-15

    It is shown that three different Lax operators in the Dym hierarchy produce three generalized coupled Harry Dym equations. These equations transform, via the reciprocal link, to the coupled two-component Korteweg de Vries (KdV) system. The first equation gives us known integrable two-component KdV system, while the second reduces to the known symmetrical two-component KdV equation. The last one reduces to the Drienfeld-Sokolov equation. This approach gives us new Lax representation for these equations.

  14. XrayOpticsConstants

    2005-06-20

    This application (XrayOpticsConstants) is a tool for displaying X-ray and Optical properties for a given material, x-ray photon energy, and in the case of a gas, pressure. The display includes fields such as the photo-electric absorption attenuation length, density, material composition, index of refraction, and emission properties (for scintillator materials).

  15. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  16. Multiple GPCR conformations and signalling pathways: implications for antagonist affinity estimates

    PubMed Central

    Baker, Jillian G.; Hill, Stephen J.

    2007-01-01

    Antagonist affinity measurements have traditionally been considered important in characterizing the cell-surface receptors present in a particular cell or tissue. A central assumption has been that antagonist affinity is constant for a given receptor–antagonist interaction, regardless of the agonist used to stimulate that receptor or the downstream response that is measured. As a consequence, changes in antagonist affinity values have been taken as initial evidence for the presence of novel receptor subtypes. Emerging evidence suggests, however, that receptors can possess multiple binding sites and the same receptor can show different antagonist affinity measurements under distinct experimental conditions. Here, we discuss several mechanisms by which antagonists have different affinities for the same receptor as a consequence of allosterism, coupling to different G proteins, multiple (but non-interacting) receptor sites, and signal-pathway-dependent pharmacology (where the pharmacology observed varies depending on the signalling pathway measured). PMID:17629959

  17. Affinity Peptide for Targeted Detection of Dysplasia in Barrett's Esophagus

    PubMed Central

    Li, Meng; Anastassiades, Costas P.; Joshi, Bishnu; Komarck, Chris M.; Piraka, Cyrus; Elmunzer, Badih J.; Turgeon, Danielle K.; Johnson, Timothy D.; Appelman, Henry; Beer, David G.; Wang, Thomas D.

    2012-01-01

    Background & Aims Dysplasia is a pre-malignant condition in Barrett's esophagus that is difficult to detect on screening endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease, and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies. Methods Peptide selection was performed using phage display by removing non-specific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, ELISA, flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1 mm intervals. Results The peptide sequence SNFYMPL was selected and demonstrated preferential binding to target cells on bound phage counts, ELISA, and flow cytometry. Reducing binding was observed on competition with unlabeled peptide in a dose dependent manner, an affinity of Kd = 164 nM was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n=12), the fluorescence intensity (mean±SEM) in 1 mm intervals classified histologically as squamous (n=145), intestinal metaplasia (n=83), dysplasia (n=61) and gastric mucosa (n=69) was 46.5±1.6, 62.3±5.8, 100.0±9.0, and 42.4±3.0 arb units, respectively. Conclusions The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus, and can be fluorescence-labeled to target pre-malignant mucosa on imaging. PMID:20637198

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

    PubMed

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

    2016-01-01

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

  19. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  20. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    PubMed

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. PMID:26938582

  1. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  2. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  3. Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

    PubMed

    Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-01-01

    Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry. PMID:21478615

  4. UNDERSTANDING VARIATION IN PARTITION COEFFICIENT KD, VALUES, VOLUME III: AMERICIUM, ARSENIC, CURIUM, IODINE, NEPTUNIUM, RADIUM, AND TECHNETIUM

    EPA Science Inventory

    This report describes the conceptualization, measurement, and use of the partition (or distribution) coefficient, Kd, parameter, and the geochemical aqueous solution and sorbent properties that are most important in controlling adsorption/retardation behavior of selected contamin...

  5. Immobilized metal ion affinity chromatography.

    PubMed

    Yip, T T; Hutchens, T W

    1992-01-01

    Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme

  6. Wall of fundamental constants

    SciTech Connect

    Olive, Keith A.; Peloso, Marco; Uzan, Jean-Philippe

    2011-02-15

    We consider the signatures of a domain wall produced in the spontaneous symmetry breaking involving a dilatonlike scalar field coupled to electromagnetism. Domains on either side of the wall exhibit slight differences in their respective values of the fine-structure constant, {alpha}. If such a wall is present within our Hubble volume, absorption spectra at large redshifts may or may not provide a variation in {alpha} relative to the terrestrial value, depending on our relative position with respect to the wall. This wall could resolve the contradiction between claims of a variation of {alpha} based on Keck/Hires data and of the constancy of {alpha} based on Very Large Telescope data. We derive the properties of the wall and the parameters of the underlying microscopic model required to reproduce the possible spatial variation of {alpha}. We discuss the constraints on the existence of the low-energy domain wall and describe its observational implications concerning the variation of the fundamental constants.

  7. Renormalization of Newton's constant

    NASA Astrophysics Data System (ADS)

    Falls, Kevin

    2015-12-01

    The problem of obtaining a gauge independent beta function for Newton's constant is addressed. By a specific parametrization of metric fluctuations a gauge independent functional integral is constructed for the semiclassical theory around an arbitrary Einstein space. The effective action then has the property that only physical polarizations of the graviton contribute, while all other modes cancel with the functional measure. We are then able to compute a gauge independent beta function for Newton's constant in d dimensions to one-loop order. No Landau pole is present provided Ng<18 , where Ng=d (d -3 )/2 is the number of polarizations of the graviton. While adding a large number of matter fields can change this picture, the absence of a pole persists for the particle content of the standard model in four spacetime dimensions.

  8. Estimation of the Diffuse Attenuation Coefficient KdPAR Using MERIS Satellite Reflections for European Coastal Waters

    NASA Astrophysics Data System (ADS)

    Saulquin, Bertand; Hamdi, Anouar; Populus, Jacques; Loutier, Romain; Demaria, Julien; Mangin, Antoine; D'Andon, Odile Fanton

    2010-12-01

    Accurate estimations of the diffuse attenuation coefficient is critical to understand physical processes such as the heat transfer in the upper layer of the ocean and also biological processes such as phytoplankton photosynthesis in the ocean euphotic zone. Light availability in the water column and the seabed determine the euphotic zone and constraints the type and distribution of the algae species. The EuSeaMap project's aim is to characterize at a resolution of 250m the European infralitoral benthic zone, according to biology, physic and geology criteriums and using observations and models. Satellite observations of the diffuse attenuation coefficient of the downwelling spectral irradiance at wavelength 490 nm (Kd490) or the diffuse attenuation coefficient for the downwelling photosynthetically available radiation (KdPAR) is an effective method to provide large scale maps of these parameters at high spatial and temporal resolution. Several empirical and semi-analytical models are commonly used to derive the Kd490 and KdPAR maps from ocean colour satellite sensors such as the Medium Resolution Imaging Spectrometer Instrument (MERIS), the Sea- viewing Wide Field-of-view Sensor (SeaWiFS), and the Moderate Resolution Imaging Spectroradiometer (MODIS). Most of these existing empirical or semi- analytical models have been calibrated on open ocean waters and provide good results in these areas, but tend to underestimate the attenuation of light in coastal waters, our area of interest. We propose here a new estimation of the euphotic depth and the KdPAR for coastal European waters using MERIS reflectances at the resolution of 1km and 250 m. First, a semi-analytical model is used to estimate the Kd490, and in a second step, two relationships have been developed between the KdPAR and the Kd490 for respectively clear and turbid waters. Satellite-derived fields of Kd490 and the deduced KdPAR are validated using matchups collected over the world. Distribution maps of seabed

  9. Varying constants quantum cosmology

    SciTech Connect

    Leszczyńska, Katarzyna; Balcerzak, Adam; Dabrowski, Mariusz P. E-mail: abalcerz@wmf.univ.szczecin.pl

    2015-02-01

    We discuss minisuperspace models within the framework of varying physical constants theories including Λ-term. In particular, we consider the varying speed of light (VSL) theory and varying gravitational constant theory (VG) using the specific ansätze for the variability of constants: c(a) = c{sub 0} a{sup n} and G(a)=G{sub 0} a{sup q}. We find that most of the varying c and G minisuperspace potentials are of the tunneling type which allows to use WKB approximation of quantum mechanics. Using this method we show that the probability of tunneling of the universe ''from nothing'' (a=0) to a Friedmann geometry with the scale factor a{sub t} is large for growing c models and is strongly suppressed for diminishing c models. As for G varying, the probability of tunneling is large for G diminishing, while it is small for G increasing. In general, both varying c and G change the probability of tunneling in comparison to the standard matter content (cosmological term, dust, radiation) universe models.

  10. Connecting Fundamental Constants

    SciTech Connect

    Di Mario, D.

    2008-05-29

    A model for a black hole electron is built from three basic constants only: h, c and G. The result is a description of the electron with its mass and charge. The nature of this black hole seems to fit the properties of the Planck particle and new relationships among basic constants are possible. The time dilation factor in a black hole associated with a variable gravitational field would appear to us as a charge; on the other hand the Planck time is acting as a time gap drastically limiting what we are able to measure and its dimension will appear in some quantities. This is why the Planck time is numerically very close to the gravitational/electric force ratio in an electron: its difference, disregarding a {pi}{radical}(2) factor, is only 0.2%. This is not a coincidence, it is always the same particle and the small difference is between a rotating and a non-rotating particle. The determination of its rotational speed yields accurate numbers for many quantities, including the fine structure constant and the electron magnetic moment.

  11. The Hubble Constant

    NASA Astrophysics Data System (ADS)

    Jackson, Neal

    2015-09-01

    I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. There are two broad categories of measurements. The first uses individual astrophysical objects which have some property that allows their intrinsic luminosity or size to be determined, or allows the determination of their distance by geometric means. The second category comprises the use of all-sky cosmic microwave background, or correlations between large samples of galaxies, to determine information about the geometry of the Universe and hence the Hubble constant, typically in a combination with other cosmological parameters. Many, but not all, object-based measurements give H_0 values of around 72-74 km s^-1 Mpc^-1, with typical errors of 2-3 km s^-1 Mpc^-1. This is in mild discrepancy with CMB-based measurements, in particular those from the Planck satellite, which give values of 67-68 km s^-1 Mpc^-1 and typical errors of 1-2 km s^-1 Mpc^-1. The size of the remaining systematics indicate that accuracy rather than precision is the remaining problem in a good determination of the Hubble constant. Whether a discrepancy exists, and whether new physics is needed to resolve it, depends on details of the systematics of the object-based methods, and also on the assumptions about other cosmological parameters and which datasets are combined in the case of the all-sky methods.

  12. Water Wave Solutions of the Coupled System Zakharov-Kuznetsov and Generalized Coupled KdV Equations

    PubMed Central

    Seadawy, A. R.; El-Rashidy, K.

    2014-01-01

    An analytic study was conducted on coupled partial differential equations. We formally derived new solitary wave solutions of generalized coupled system of Zakharov-Kuznetsov (ZK) and KdV equations by using modified extended tanh method. The traveling wave solutions for each generalized coupled system of ZK and KdV equations are shown in form of periodic, dark, and bright solitary wave solutions. The structures of the obtained solutions are distinct and stable. PMID:25374940

  13. Water wave solutions of the coupled system Zakharov-Kuznetsov and generalized coupled KdV equations.

    PubMed

    Seadawy, A R; El-Rashidy, K

    2014-01-01

    An analytic study was conducted on coupled partial differential equations. We formally derived new solitary wave solutions of generalized coupled system of Zakharov-Kuznetsov (ZK) and KdV equations by using modified extended tanh method. The traveling wave solutions for each generalized coupled system of ZK and KdV equations are shown in form of periodic, dark, and bright solitary wave solutions. The structures of the obtained solutions are distinct and stable. PMID:25374940

  14. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  15. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  16. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  17. Crystal structure of the 500 kD yeast acetyl-CoA carboxylase holoenzyme dimer

    PubMed Central

    Wei, Jia; Tong, Liang

    2015-01-01

    Acetyl-CoA carboxylase (ACC) has crucial roles in fatty acid metabolism and is an attractive target for drug discovery against diabetes, cancer and other diseases1–6. Saccharomyces cerevisiae ACC (ScACC) is crucial for the production of very-long-chain fatty acids and the maintenance of the nuclear envelope7,8. ACC contains biotin carboxylase (BC) and carboxyltransferase (CT) activities, and its biotin is linked covalently to the biotin carboxyl carrier protein (BCCP). Most eukaryotic ACCs are 250 kD, multi-domain enzymes and function as homo-dimers and higher oligomers. They contain a unique, 80 kD central region that shares no homology with other proteins. While the structures of the BC, CT and BCCP domains and other biotin-dependent carboxylase holoenzymes are known1,9–14, currently there is no structural information on the ACC holoenzyme. Here we report the crystal structure of the full-length, 500 kD holoenzyme dimer of ScACC. The structure is strikingly different from those of the other biotin-dependent carboxylases. The central region contains five domains and is important for positioning the BC and CT domains for catalysis. The structure unexpectedly reveals a dimer of the BC domain and extensive conformational differences compared to the structure of BC domain alone, which is a monomer. These structural changes explain why the BC domain alone is catalytically inactive and define the molecular mechanism for the inhibition of eukaryotic ACC by the natural product soraphen A15,16 and by phosphorylation of a Ser residue just prior to the BC domain core in mammalian ACC. The BC and CT active sites are separated by 80 Å, and the entire BCCP domain must translocate during catalysis. PMID:26458104

  18. Banach frames in the affine synthesis problem

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2009-10-01

    We consider the problem of representing functions f\\in L^p(\\mathbb R^d) by a series in elements of the affine system \\displaystyle \\psi_{j,k}(x)=\\lvert\\det a_j\\rvert^{1/2}\\psi(a_jx-bk), \\qquad j\\in\\mathbb N, \\quad k\\in\\mathbb Z^d. The corresponding representation theorems are established on the basis of the frame inequalities \\displaystyle A\\Vert g\\Vert _q\\le\\Vert\\{(g,\\psi_{j,k})\\}\\Vert _Y\\le B\\Vert g\\Vert _q for the Fourier coefficients \\displaystyle(g,\\psi_{j,k})=\\int_{\\mathbb R^d}g(x)\\psi_{j,k}(x)\\,dx of functions g\\in L^q(\\mathbb R^d), 1/p+1/q=1, where {\\Vert\\cdot\\Vert}_Y is the norm in some Banach space of number families \\{y_{j,k}\\} and 0 are constants. In particular, it is proved that if the integral of a function \\psi\\in L^1\\cap L^p(\\mathbb R^d), 1, is nonzero, so \\displaystyle\\int_{\\mathbb R^d}\\psi(x)\\,dx\

  19. Indian craniometric variability and affinities.

    PubMed

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with "Caucasoid" populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  20. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  1. Adler{endash}Kostant{endash}Symes construction, bi-Hamiltonian manifolds, and KdV equations

    SciTech Connect

    Guha, P. |

    1997-10-01

    This paper focuses a relation between Adler{endash}Kostant{endash}Symes (AKS) theory applied to Fordy{endash}Kulish scheme and bi-Hamiltonian manifolds. The spirit of this paper is closely related to Casati{endash}Magri{endash}Pedroni work on Hamiltonian formulation of the KP equation. Here the KdV equation is deduced via the superposition of the Fordy{endash}Kulish scheme and AKS construction on the underlying current algebra C{sup {infinity}}(S{sup 1},g{circle_times}{bold C}[[{lambda}

  2. Averaging principle for the KdV equation with a small initial value

    NASA Astrophysics Data System (ADS)

    Yuan, Xiaoping; Zhang, Jing

    2016-02-01

    The averaging principle with a small initial value is constructed for a Hamiltonian perturbed Korteweg-de Vries (KdV) equation under periodic boundary condition where the positive integer n 0 is not divisible by three and K 1 and K 2 are real analytic functions. More precisely, any action I with the small initial value \\parallel I(0){{\\parallel}{{\\tilde{\\mathop{\\ell} }s}}}≤slant \\varepsilon evolves slowly over a long time interval: where s is the index of some space.

  3. Liouville Correspondence Between the Modified KdV Hierarchy and Its Dual Integrable Hierarchy

    NASA Astrophysics Data System (ADS)

    Kang, Jing; Liu, Xiaochuan; Olver, Peter J.; Qu, Changzheng

    2016-02-01

    We study an explicit correspondence between the integrable modified KdV hierarchy and its dual integrable modified Camassa-Holm hierarchy. A Liouville transformation between the isospectral problems of the two hierarchies also relates their respective recursion operators and serves to establish the Liouville correspondence between their flows and Hamiltonian conservation laws. In addition, a novel transformation mapping the modified Camassa-Holm equation to the Camassa-Holm equation is found. Furthermore, it is shown that the Hamiltonian conservation laws in the negative direction of the modified Camassa-Holm hierarchy are both local in the field variables and homogeneous under rescaling.

  4. Chaotic motion for the generalized KdV-Burgers equation with external perturbation

    NASA Astrophysics Data System (ADS)

    Yu, Jun; Li, Jieru; Ng, Tick Wan

    2009-12-01

    The bifurcation and chaos in the generalized KdV-Burgers equation under periodic perturbation are investigated numerically in some detail. It is shown that dynamical chaos can occur when we choose appropriately systematic parameters and initial conditions. Abundant bifurcation structures and different routes to chaos such as period-doubling and inverse period-doubling cascades, intermittent bifurcation and crisis are found by using bifurcation diagrams, Poincaré maps and phase portraits. To characterize the chaotic behavior of this system, the spectrum of the Lyapunov exponent and the Lyapunov dimension of the attractor are also employed.

  5. Approximate Symmetry Reduction Approach: Infinite Series Reductions to the KdV-Burgers Equation

    NASA Astrophysics Data System (ADS)

    Jiao, Xiaoyu; Yao, Ruoxia; Zhang, Shunli; Lou, Sen Y.

    2009-11-01

    For weak dispersion and weak dissipation cases, the (1+1)-dimensional KdV-Burgers equation is investigated in terms of approximate symmetry reduction approach. The formal coherence of similarity reduction solutions and similarity reduction equations of different orders enables series reduction solutions. For the weak dissipation case, zero-order similarity solutions satisfy the Painlevé II, Painlevé I, and Jacobi elliptic function equations. For the weak dispersion case, zero-order similarity solutions are in the form of Kummer, Airy, and hyperbolic tangent functions. Higher-order similarity solutions can be obtained by solving linear variable coefficients ordinary differential equations.

  6. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    PubMed

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

  7. 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

  8. Distribution coefficients (Kd) and desorption rates of 137Cs and 241Am in Black Sea sediments.

    PubMed

    Topcuoğlu, S; Güngör, N; Kirbaşoğlu, C

    2002-12-01

    The distribution coefficients (Kd) and desorption rates of 137Cs and 241Am radionuclides in bottom sediments at different locations in the Black Sea were studied under laboratory conditions. The Kd values were found to be 500 for 137Cs and 3800 for 241Am at the steady state and described exponential curves. Rapid uptake of the radionuclides occurred during the initial period and little accumulation happened after four days. The desorption rates for 137Cs in different bottom sediments were best described by a three-component exponential model. The desorption half-times of 137Cs ranged from 26 to 50 d at the slow components. However, the desorption rate of 241Am described one component for all sediment samples and desorption half-time was found to be 75 d. In general, the results showed that the 241Am radionuclide is more effectively transferred to bottom sediment and has longer turnover time than 137Cs under Black Sea conditions. PMID:12489734

  9. A 38-kD Mycobacterium tuberculosis antigen associated with infection. Its isolation and serologic evaluation.

    PubMed Central

    Espitia, C; Cervera, I; González, R; Mancilla, R

    1989-01-01

    To identify antigens that could be specifically associated with tuberculosis infection, the antibody response to Mycobacterium tuberculosis antigens of patients with pulmonary tuberculosis and of healthy individuals were compared by immunoblot. In healthy individuals, serum antibodies were found in the majority of cases. Bands of 60 and 32-31 kilodaltons (kD) were the antigens more frequently recognized by antibodies of normal sera (55.8 and 64.7%, respectively). In patients with pulmonary tuberculosis, the number and intensity of the developed antigen bands were much higher than in normal individuals. Antigens reacting preferentially with tuberculosis sera were also identified. Furthermore, a unique disease-associated protein antigen of 38 kD was found to react with 57% of patients' sera but with none of the controls. This antigen was isolated by elution from nitrocellulose membranes and tested as an ELISA reagent in the serodiagnosis of pulmonary tuberculosis. A specificity of 0.96 and sensitivity of 0.68 were obtained. Images Fig. 1 Fig. 3 PMID:2478322

  10. 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

  11. Purification of a 53kD pI 4. 8 cytosolic phosphoprotein from HL60

    SciTech Connect

    Biser, P.S.; Spearman, T.N.; Bruzzone, M.; Durham, J.P.

    1987-05-01

    In order to study the potential role of a 53kD pI 4.8 phosphoprotein in the differentiation of HL60 using monoclonal antibodies, a partial purification has been carried out. Cytosol from cells differentiated with 1 M retinoic acid was applied to a DEAE-cellulose column and eluted with a linear NaCl gradient. Fractions were screened by in vitro phosphorylation of aliquots using /sup 32/P ATP and highly purified protein kinase C, SDS-PAGE, and autoradiography. Fraction which showed autoradiographic bands of the correct molecular weight were further analyzed using 2-D electrophoresis involving isolectric focusing over a pH range of 4-6 followed by SDS-PAGE on a 10% slab gel. Autoradiograms of these gels showed the 53 kD pI 4.8 phosphoprotein to elute with a peak at 0.24 NaCl. This 53 kD pI 4.8 protein was identified as the 53kD pI 4.8 phosphoprotein whose synthesis and phosphorylation is induced by retinoic acid by DEAE chromatography of cytosol from cells labelled in vivo with /sup 32/PO/sub 4//sup -2/ followed by 2-D electrophoresis. Fractions containing the 53 kD pI 4.8 protein were concentrated and applied to a chromatofocusing column which was eluted with a gradient from pH 6 to 4. Analysis of fractions via in vitro phosphorylation and SDS PAGE showed the 53 kD pI 4.8 protein eluting with a peak at pH 4.8 as a silver-stained band well separated from contaminating proteins. Experiments are currently in progress to produce monoclonal antibodies to the 53 kD pI 4.8 protein using the partially purified antigen.

  12. O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities.

    PubMed

    Chen, Wan-Na; Kuppan, Kekini Vahini; Lee, Michael David; Jaudzems, Kristaps; Huber, Thomas; Otting, Gottfried

    2015-04-01

    O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional (1)H NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl (1)H NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant Kd (0.2 μM) of the complex between glutamate and the Escherichia coli aspartate/glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the (1)H NMR signal of the Tby tert-butyl group allows Kd measurements using less concentrated protein solutions than usual, providing access to Kd values 1 order of magnitude lower than established NMR methods that employ direct protein detection for Kd measurements. PMID:25789794

  13. Modified gravity in three dimensional metric-affine scenarios

    NASA Astrophysics Data System (ADS)

    Bambi, Cosimo; Ghasemi-Nodehi, M.; Rubiera-Garcia, D.

    2015-08-01

    We consider metric-affine scenarios where a modified gravitational action is sourced by electrovacuum fields in a three dimensional space-time. We first study the case of f (R ) theories, finding deviations near the center as compared to the solutions of general relativity. We then consider Born-Infeld gravity, which has raised a lot of interest in the last few years regarding its applications in astrophysics and cosmology, and show that new features always arise at a finite distance from the center. Several properties of the resulting space-times, in particular in presence of a cosmological constant term, are discussed.

  14. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  15. [3H]-lifarizine, a high affinity probe for inactivated sodium channels.

    PubMed Central

    MacKinnon, A. C.; Wyatt, K. M.; McGivern, J. G.; Sheridan, R. D.; Brown, C. M.

    1995-01-01

    1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582509

  16. The Hubble constant.

    PubMed

    Tully, R B

    1993-06-01

    Five methods of estimating distances have demonstrated internal reproducibility at the level of 5-20% rms accuracy. The best of these are the cepheid (and RR Lyrae), planetary nebulae, and surface-brightness fluctuation techniques. Luminosity-line width and Dn-sigma methods are less accurate for an individual case but can be applied to large numbers of galaxies. The agreement is excellent between these five procedures. It is determined that Hubble constant H0 = 90 +/- 10 km.s-1.Mpc-1 [1 parsec (pc) = 3.09 x 10(16) m]. It is difficult to reconcile this value with the preferred world model even in the low-density case. The standard model with Omega = 1 may be excluded unless there is something totally misunderstood about the foundation of the distance scale or the ages of stars. PMID:11607391

  17. When constants are important

    SciTech Connect

    Beiu, V.

    1997-04-01

    In this paper the authors discuss several complexity aspects pertaining to neural networks, commonly known as the curse of dimensionality. The focus will be on: (1) size complexity and depth-size tradeoffs; (2) complexity of learning; and (3) precision and limited interconnectivity. Results have been obtained for each of these problems when dealt with separately, but few things are known as to the links among them. They start by presenting known results and try to establish connections between them. These show that they are facing very difficult problems--exponential growth in either space (i.e. precision and size) and/or time (i.e., learning and depth)--when resorting to neural networks for solving general problems. The paper will present a solution for lowering some constants, by playing on the depth-size tradeoff.

  18. Unitaxial constant velocity microactuator

    DOEpatents

    McIntyre, Timothy J.

    1994-01-01

    A uniaxial drive system or microactuator capable of operating in an ultra-high vacuum environment. The mechanism includes a flexible coupling having a bore therethrough, and two clamp/pusher assemblies mounted in axial ends of the coupling. The clamp/pusher assemblies are energized by voltage-operated piezoelectrics therewithin to operatively engage the shaft and coupling causing the shaft to move along its rotational axis through the bore. The microactuator is capable of repeatably positioning to sub-manometer accuracy while affording a scan range in excess of 5 centimeters. Moreover, the microactuator generates smooth, constant velocity motion profiles while producing a drive thrust of greater than 10 pounds. The system is remotely controlled and piezoelectrically driven, hence minimal thermal loading, vibrational excitation, or outgassing is introduced to the operating environment.

  19. A Constant Pressure Bomb

    NASA Technical Reports Server (NTRS)

    Stevens, F W

    1924-01-01

    This report describes a new optical method of unusual simplicity and of good accuracy suitable to study the kinetics of gaseous reactions. The device is the complement of the spherical bomb of constant volume, and extends the applicability of the relationship, pv=rt for gaseous equilibrium conditions, to the use of both factors p and v. The method substitutes for the mechanical complications of a manometer placed at some distance from the seat of reaction the possibility of allowing the radiant effects of reaction to record themselves directly upon a sensitive film. It is possible the device may be of use in the study of the photoelectric effects of radiation. The method makes possible a greater precision in the measurement of normal flame velocities than was previously possible. An approximate analysis shows that the increase of pressure and density ahead of the flame is negligible until the velocity of the flame approaches that of sound.

  20. The Hubble constant.

    PubMed Central

    Tully, R B

    1993-01-01

    Five methods of estimating distances have demonstrated internal reproducibility at the level of 5-20% rms accuracy. The best of these are the cepheid (and RR Lyrae), planetary nebulae, and surface-brightness fluctuation techniques. Luminosity-line width and Dn-sigma methods are less accurate for an individual case but can be applied to large numbers of galaxies. The agreement is excellent between these five procedures. It is determined that Hubble constant H0 = 90 +/- 10 km.s-1.Mpc-1 [1 parsec (pc) = 3.09 x 10(16) m]. It is difficult to reconcile this value with the preferred world model even in the low-density case. The standard model with Omega = 1 may be excluded unless there is something totally misunderstood about the foundation of the distance scale or the ages of stars. PMID:11607391

  1. Protein Complex Purification by Affinity Capture.

    PubMed

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

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  2. 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.

  3. 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.

  4. Compact noncontraction semigroups of affine operators

    NASA Astrophysics Data System (ADS)

    Voynov, A. S.; Protasov, V. Yu

    2015-07-01

    We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.

  5. Generation of high-affinity DNA aptamers using an expanded genetic alphabet.

    PubMed

    Kimoto, Michiko; Yamashige, Rie; Matsunaga, Ken-ichiro; Yokoyama, Shigeyuki; Hirao, Ichiro

    2013-05-01

    DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids. PMID:23563318

  6. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  7. Modulation of Perfusion and Oxygenation by Red Blood Cell Oxygen Affinity during Acute Anemia

    PubMed Central

    Cabrales, Pedro; Tsai, Amy G.; Intaglietta, Marcos

    2008-01-01

    Responses to exchange transfusion using red blood cells (RBCs) with modified hemoglobin (Hb) oxygen (O2) affinity were studied in the hamster window chamber model during acute anemia to determine its role on microvascular perfusion and tissue oxygenation. Allosteric effectors were introduced in the RBCs by electroporation. Inositol hexaphosphate (IHP) and 5-hydroxymethyl-2-furfural (5HMF) were used to decrease and increase Hb-O2 affinity. In vitro P50s (partial pressure of O2 at 50% Hb saturation) were modified to 10, 25, 45, and 50 mm Hg (normal P50 is 32 mm Hg). Allosteric effectors also decreased the Hill coefficient. Anemic condition was induced by isovolemic hemodilution exchanges using 6% dextran 70 kD to 18% hematocrit (Hct). Modified RBCs (at 18% Hct in 5% albumin solution) were infused by exchange transfusion of 35% of blood volume. Systemic parameters, microvascular perfusion, capillary perfusion (functional capillary density, FCD), and microvascular Po2 levels were measured. RBcs with P50 of 45 mm Hg increased tissue Po2 and decreased O2 delivery (Do2) and extraction (Vo2) and RBCs with P50 of 60 mmHg reduced FCD, microvascular flow, tissue Po2, Do2 and Vo2. Erythrocytes with increased Hb-O2 affinity maintained hemodynamic conditions, Do2 and decreased tissue Po2. This study shows that in an anemic condition, maximal tissue Po2 does not correspond to maximal Do2 and Vo2. PMID:17884988

  8. Development of high-affinity single chain Fv against foot-and-mouth disease virus.

    PubMed

    Jung, Joon-Goo; Jeong, Gu Min; Yim, Sung Sun; Jeong, Ki Jun

    2016-03-01

    Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV. PMID:26827774

  9. 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

  10. 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

  11. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  12. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  13. Linker peptide and affinity tag for detection and purification of single-chain Fv fragments.

    PubMed

    Küttner, Gabriele; Giessmann, Elke; Wessner, Helga; Scholz, Christa; Reinhardt, Dina; Winkler, Karsten; Marx, Uwe; Höhne, Wolfgang

    2004-05-01

    The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. PMID:15152607

  14. Bacterial cadherin domains as carbohydrate binding modules: determination of affinity constants to insoluble complex polysaccharides.

    PubMed

    Fraiberg, Milana; Borovok, Ilya; Weiner, Ronald M; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    Cadherin (CA) and cadherin-like (CADG) doublet domains from the complex polysaccharide-degrading marine bacterium, Saccharophagus degradans 2-40, demonstrated reversible calcium-dependent binding to different complex polysaccharides, which serve as growth substrates for the bacterium. Here we describe a procedure based on adsorption of CA and CADG doublet domains to different insoluble complex polysaccharides, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for visualizing and quantifying the distribution of cadherins between the bound and unbound fractions. Scatchard plots were employed to determine the kinetics of interactions of CA and CADG with several complex carbohydrates. On the basis of these binding studies, the CA and CADG doublet domains are proposed to form a new family of carbohydrate-binding module (CBM). PMID:22843394

  15. Age-related decrease in the responsiveness of rat articular chondrocytes to EGF is associated with diminished number and affinity for the ligand of cell surface binding sites.

    PubMed

    Ribault, D; Habib, M; Abdel-Majid, K; Barbara, A; Mitrovic, D

    1998-01-12

    The effect of age on the responsiveness of articular chondrocytes (AC) to epidermal growth factor (EGF) was examined. Cells were isolated by digesting cartilage fragments from the humeral and femoral heads of 21-day old, 8- and 14-month old rats with collagenase. The cells were cultured under standard conditions, as monolayers. DNA synthesis was measured by [3H]thymidine incorporation and cell proliferation by the DNA content of subconfluent cultures. [125I]EGF binding and the amounts of EGF and EGF-receptor mRNAs were determined using confluent cells. DNA synthesis was decreased with age of animals. EGF stimulated DNA synthesis in cultures in 1- and 8-month old rats at low serum concentrations (< 5%), and in cultures in 14-month old animals at high serum concentrations. It also increased 5-day DNA content of cultures compared to serum-treated controls but this effect was weak in cultures in 14-month old rats. The number of high affinity binding sites for [125I]EGF decreased from 37,800 in the 1-month old to 1950 in the 14-month old rat AC. The apparent dissociation constant (Kd) also decreased with age: 0.18 nmol/l in the 1-month old; 0.12 nmol/l in the 8-month old; and 0.07 nmol/l in the 14-month old cells. AC in older rats contained more EGF mRNA and less EGF-receptor mRNA. Incubation of the cells with EGF resulted in down regulation of the EGF- and upregulation of EGF-receptor mRNA expressions. These findings show the age-related quantitative and qualitative alterations in EGF and EGF-receptor which may account, at least in part, for the diminished responsiveness of senescent AC to EGF. PMID:9509392

  16. High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold.

    PubMed

    Dryla, Agnieszka; Hoffmann, Bernd; Gelbmann, Dieter; Giefing, Carmen; Hanner, Markus; Meinke, Andreas; Anderson, Annaliesa S; Koppensteiner, Walter; Konrat, Robert; von Gabain, Alexander; Nagy, Eszter

    2007-01-01

    Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins. PMID:17041047

  17. The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

    PubMed Central

    De La Roche, Marc A; Lee, Sheu-Fen; Côté, Graham P

    2003-01-01

    Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains. PMID:12826013

  18. Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose.

    PubMed

    Wilkens, Casper; Auger, Kyle D; Anderson, Nolan T; Meekins, David A; Raththagala, Madushi; Abou Hachem, Maher; Payne, Christina M; Gentry, Matthew S; Svensson, Birte

    2016-01-01

    The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for β-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and β-cyclodextrin. SEX4 has 50-fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long-distance mutual effects of binding at SBSs and the active site in LSF2. PMID:26763114

  19. Differential Mobility Spectrometry: Preliminary Findings on Determination of Fundamental Constants

    NASA Technical Reports Server (NTRS)

    Limero, Thomas; Cheng, Patti; Boyd, John

    2007-01-01

    The electron capture detector (ECD) has been used for 40+ years (1) to derive fundamental constants such as a compound's electron affinity. Given this historical perspective, it is not surprising that differential mobility spectrometry (DMS) might be used in a like manner. This paper will present data from a gas chromatography (GC)-DMS instrument that illustrates the potential capability of this device to derive fundamental constants for electron-capturing compounds. Potential energy curves will be used to provide possible explanation of the data.

  20. 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.

  1. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  2. Affitins for protein purification by affinity magnetic fishing.

    PubMed

    Fernandes, Cláudia S M; Dos Santos, Raquel; Ottengy, Stella; Viecinski, Aline Canani; Béhar, Ghislaine; Mouratou, Barbara; Pecorari, Frédéric; Roque, A Cecília A

    2016-07-29

    Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing. PMID:27342136

  3. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

    PubMed

    Avery, Adam W; Crain, Jonathan; Thomas, David D; Hays, Thomas S

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  4. Scaling analysis of affinity propagation.

    PubMed

    Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang

    2010-06-01

    We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N((h+2)/(h+1))) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N((2-d)/(h+1)d). Finally, under some conditions we observe that there is a value s* of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss*) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set. PMID:20866473

  5. Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms.

    PubMed Central

    Kloprogge, E; Akkerman, J W

    1986-01-01

    When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding

  6. The KdV equation on the half-line: the Dirichlet to Neumann map

    NASA Astrophysics Data System (ADS)

    >Jonatan Lenells,

    2013-08-01

    We consider initial-boundary value problems for the KdV equation ut + ux + 6uux + uxxx = 0 on the half-line x ⩾ 0. For a well-posed problem, the initial data u(x, 0) as well as one of the three boundary values {u(0, t), ux(0, t), uxx(0, t)} can be prescribed; the other two boundary values remain unknown. We provide a characterization of the unknown boundary values for the Dirichlet as well as the two Neumann problems in terms of a system of nonlinear integral equations. The characterizations are effective in the sense that the integral equations can be solved perturbatively to all orders in a well-defined recursive scheme.

  7. On Lie symmetries, exact solutions and integrability to the KdV-Sawada-Kotera-Ramani equation

    NASA Astrophysics Data System (ADS)

    Ma, Pan-Li; Tian, Shou-Fu; Zhang, Tian-Tian; Zhang, Xing-Yong

    2016-04-01

    In this paper, the KdV-Sawada-Kotera-Ramani equation is investigated, which is used to describe the resonances of solitons in one-dimensional space. By using the Lie symmetry analysis method, the vector field and optimal system of the equation are derived, respectively. The optimal system is further used to study the symmetry reductions and exact solutions. Furthermore, the exact analytic solutions of the equation can be obtained by considering the power series theory. Finally, the complete integrability of the equation is systematically presented by using binary Bell's polynomials, which includes the bilinear representation, bilinear Bäcklund transformation, Lax pair and infinite conservation laws. Based on its bilinear representation, the N-soliton solutions of the equation are also constructed with exact analytic expression.

  8. Tau Function and Virasoro Action for the n × n KdV Hierarchy

    NASA Astrophysics Data System (ADS)

    Terng, Chuu-Lian; Uhlenbeck, Karen

    2016-02-01

    This is the third in a series of papers attempting to describe a uniform geometric framework in which many integrable systems can be placed. A soliton hierarchy can be constructed from a splitting of an infinite dimensional group L as positive and negative subgroups {L_±} and a commuting sequence in the Lie algebra {{L}_+} of {L_+}. Given {fin L_-}, there is a formal inverse scattering solution u f of the hierarchy. When there is a 2 co-cycle on {{L}} that vanishes on both {{L}_+ and {L}_-}, Wilson constructed for each {fin L_-} a tau function {τ_f} for the hierarchy. In this third paper, we prove the following results for the n × n KdV hierarchy: (1) The second partials of {lnτ_f} are differential polynomials of the formal inverse scattering solution u f . Moreover, {u_f} can be recovered from the second partials of {lnτ_f}.

  9. A hybrid LDG-HWENO scheme for KdV-type equations

    NASA Astrophysics Data System (ADS)

    Luo, Dongmi; Huang, Weizhang; Qiu, Jianxian

    2016-05-01

    A hybrid LDG-HWENO scheme is proposed for the numerical solution of KdV-type partial differential equations. It evolves the cell averages of the physical solution and its moments (a feature of Hermite WENO) while discretizes high order spatial derivatives using the local DG method. The new scheme has the advantages of both LDG and HWENO methods, including the ability to deal with high order spatial derivatives and the use of a small number of global unknown variables. The latter is independent of the order of the scheme and the spatial order of the underlying differential equations. One and two dimensional numerical examples are presented to show that the scheme can attain the same formal high order accuracy as the LDG method.

  10. The Gardner category and nonlocal conservation laws for N=1 Super KdV

    SciTech Connect

    Andrea, S.; Restuccia, A.; Sotomayor, A.

    2005-10-01

    The nonlocal conserved quantities of the N=1 Super KdV are obtained using a Gardner map. A fermionic substitution semigroup and the resulting Gardner category are defined and several propositions concerning their algebraic structure are obtained. This algebraic framework makes it possible to define general transformations between different nonlinear SUSY differential equations. A SUSY ring extension is then introduced to deal with the nonlocal conserved quantities of SKdV. The algebraic version of the nonlocal conserved quantities is solved in terms of the exponential function applied to the D{sup -1} of the local conserved quantities of SKdV. Finally the same formulas are shown to work for rapidly decreasing superfields.

  11. A Haar wavelet collocation method for coupled nonlinear Schrödinger-KdV equations

    NASA Astrophysics Data System (ADS)

    Oruç, Ömer; Esen, Alaattin; Bulut, Fatih

    2016-04-01

    In this paper, to obtain accurate numerical solutions of coupled nonlinear Schrödinger-Korteweg-de Vries (KdV) equations a Haar wavelet collocation method is proposed. An explicit time stepping scheme is used for discretization of time derivatives and nonlinear terms that appeared in the equations are linearized by a linearization technique and space derivatives are discretized by Haar wavelets. In order to test the accuracy and reliability of the proposed method L2, L∞ error norms and conserved quantities are used. Also obtained results are compared with previous ones obtained by finite element method, Crank-Nicolson method and radial basis function meshless methods. Error analysis of Haar wavelets is also given.

  12. Mechanical Deformation of KD2xH2(1-x)PO4

    SciTech Connect

    Kucheyev, S; Siekhaus, W; Land, T; Demos, S

    2003-11-10

    The deformation behavior of rapidly-grown tetragonal KD{sub 2x} H{sub 1(1-x)} PO{sub 4} (KDP and DKDP) single crystals, with a deuteration degree x of 0.0, 0.3, and 0.6, is studied by nanoindentation with a 1 {micro}m radius spherical indenter. Within experimental error, the deformation behavior is found to be independent of deuterium content and different for (001) and (100) surfaces. Multiple discontinuities (so called ''pop-in'' events) in force-displacement curves are observed during indentation loading, but not during unloading. Slip is identified as the major mode of plastic deformation in DKDP, and pop-in events are attributed to the initiation of slip.

  13. Two kinds of peaked solitary waves of the KdV, BBM and Boussinesq equations

    NASA Astrophysics Data System (ADS)

    Liao, ShiJun

    2012-12-01

    It is well-known that the celebrated Camassa-Holm equation has the peaked solitary waves, which have been not reported for other mainstream models of shallow water waves. In this letter, the closed-form solutions of peaked solitary waves of the KdV equation, the BBM equation and the Boussinesq equation are given for the first time. All of them have either a peakon or an anti-peakon. Each of them exactly satisfies the corresponding Rankine-Hogoniot jump condition and could be understood as weak solution. Therefore, the peaked solitary waves might be common for most of shallow water wave models, no matter whether or not they are integrable and/or admit breaking-wave solutions.

  14. Integrability and exact solutions of the nonautonomous mixed mKdV-sinh-Gordon equation

    NASA Astrophysics Data System (ADS)

    Yong, Xuelin; Wang, Hui; Gao, Jianwei

    2014-07-01

    In this paper, a nonautonomous mixed mKdV-sinh-Gordon equation with one arbitrary time-dependent variable coefficient is discussed in detail. It is proved that the equation passes the Painlevé test in the case of positive and negative resonances, respectively. Furthermore, a dependent variable transformation is introduced to get its bilinear form. Then, soliton, negaton, positon and interaction solutions are introduced by means of the Wronskian representation. Velocities are found to depend on the time-dependent variable coefficient appearing in the equation and this leads to a wide range of interesting behaviours. The singularities and asymptotic estimate of these solutions are discussed. At last, the superposition formulae for these solutions are also constructed.

  15. 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

  16. Affine root systems and dual numbers

    NASA Astrophysics Data System (ADS)

    Kostyakov, I. V.; Gromov, N. A.; Kuratov, V. V.

    The root systems in Carroll spaces with degenerate metric are defined. It is shown that their Cartan matrices and reflection groups are affine. Due to the geometric consideration the root system structure of affine algebras is determined by a sufficiently simple algorithm.

  17. Loop realizations of quantum affine algebras

    SciTech Connect

    Cautis, Sabin; Licata, Anthony

    2012-12-15

    We give a simplified description of quantum affine algebras in their loop presentation. This description is related to Drinfeld's new realization via halves of vertex operators. We also define an idempotent version of the quantum affine algebra which is suitable for categorification.

  18. Traveling Wave Solutions of the Gardner Equation and Motion of Plane Curves Governed by the mKdV Flow

    SciTech Connect

    Vassilev, V. M.; Djondjorov, P. A.; Hadzhilazova, M. Ts.; Mladenov, I. M.

    2011-11-29

    The Gardner equation is well-known in the mathematical literature since the late sixties of 20th century. Initially, it appeared in the context of the construction of local conservation laws admitted by the KdV equation. Later on, the Gardner equation was generalized and found to be applicable in various branches of physics (solid-state and plasma physics, fluid dynamics and quantum field theory). In this paper, we examine the travelling wave solutions of the Gardner equation and derive the full set of solutions to the corresponding reduced equation in terms of Weierstrass and Jacobi elliptic functions. Then, we use the travelling wave solutions of the focusing mKdV equation and obtain in explicit analytic form exact solutions of a special type of plane curve flow, known as the mKdV flow.

  19. 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.

  20. Amyloid Precursor-like Protein 2 Increases the Endocytosis, Instability, and Turnover of the H2-Kd MHC Class I Molecule1

    PubMed Central

    Tuli, Amit; Sharma, Mahak; McIlhaney, Mary M.; Talmadge, James E.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

    2008-01-01

    The defense against the invasion of viruses and tumors relies on the presentation of viral and tumor-derived peptides to cytotoxic T lymphocytes by cell surface major histocompatibility complex (MHC) class I molecules. Previously, we showed that the ubiquitously expressed protein amyloid precursor-like protein 2 (APLP2) associates with the folded form of the MHC class I molecule Kd. In the current study, APLP2 was found to associate with folded Kd molecules following their endocytosis and to increase the amount of endocytosed Kd. In addition, increased expression of APLP2 was shown to decrease Kd surface expression and thermostability. Correspondingly, Kd thermostability and surface expression were increased by down-regulation of APLP2 expression. Overall, these data suggest that APLP2 modulates the stability and endocytosis of Kd molecules. PMID:18641335

  1. Distorted DNA structures induced by HMGB2 possess a high affinity for HMGB2.

    PubMed

    Nakamura, Yasuyuki; Shimizu, Mitsuhiro; Yoshida, Michiteru

    2002-01-01

    HMGB2 (HMG2) protein binds with DNA duplex in a sequence-nonspecific manner, then bends and unwinds the DNA. In DNA cyclization analyses for the bending activity of HMGB2, two unidentified bands, denoted alpha and beta, were observed in addition to monomer circular DNA (1C) on the gel. Re-electrophoresis and proteinase K digestion revealed that alpha and beta are complexes of circularized probe DNA (seeming 1C) with HMGB2 (K(d) approximately 10(-10) M). The DNA components of alpha and beta (alpha- and beta-DNA) showed higher affinities to HMGB2 than did the linear probe DNA (K(d) approximately 10(-7) M). The DNAs have distorted structures containing partial single-stranded regions. Nicked circular molecules presumably due to severe DNA distortion by HMGB2 were observed in alpha- and beta-DNA, in addition to closed circular double-stranded molecules. The alpha and beta bands were not formed in the presence of sole DNA binding regions which are necessary for DNA bending, indicating that the acidic C-tail in the HMGB2 molecule is necessary for inducing the peculiar distorted structures of higher affinity to HMGB2. HMGB2 binds with linker DNA and/or the entry and exit of nucleosomes fixed at both ends likewise mini-circles similar to alpha-DNA and beta-DNA. Thus, the distorted structures present in alpha-DNA and beta-DNA should be important in considering the functional mechanisms in which HMGB2 participates. PMID:11754747

  2. Affinity of aptamers binding 33-mer gliadin peptide and gluten proteins: Influence of immobilization and labeling tags.

    PubMed

    Amaya-González, Sonia; López-López, Laura; Miranda-Castro, Rebeca; de-los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo José; Lobo-Castañón, María Jesús

    2015-05-11

    Aptamers are starting to increase the reagents tool box to develop more sensitive and reliable methods for food allergens. In most of these assays, aptamers have to be modified for detection and/or immobilization purposes. To take full advantage of their affinity, which decisively influence the detectability, these modifications must be faced rationally. In this work, a recently developed aptamer for an immunotoxic peptide of gliadin associated to celiac disease is used in different configurations and modified with various markers and anchored groups to evaluate the influence of such modifications on the real affinity. The interaction in solution with the peptide is strong for a relatively small molecule (Kd = 45 ± 10 nM, 17 °C) and slightly stronger than that for the immobilized intact protein due to a cooperative binding effect. Comparatively, while only minor differences were found when the peptide or the aptamer were immobilized, labeling with a biotin resulted preferable over fluorescein (Kd = 102 ± 11 vs 208 ± 54 nM, 25 °C). These findings are of prime importance for the design of an aptamer-based analytical method for gluten quantification. PMID:25911431

  3. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    SciTech Connect

    Sherman, S.J.; Catterall, W.A.

    1982-11-01

    Specific binding of /sup 3/H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors.

  4. Beyond the Hubble Constant

    NASA Astrophysics Data System (ADS)

    1995-08-01

    about the distances to galaxies and thereby about the expansion rate of the Universe. A simple way to determine the distance to a remote galaxy is by measuring its redshift, calculate its velocity from the redshift and divide this by the Hubble constant, H0. For instance, the measured redshift of the parent galaxy of SN 1995K (0.478) yields a velocity of 116,000 km/sec, somewhat more than one-third of the speed of light (300,000 km/sec). From the universal expansion rate, described by the Hubble constant (H0 = 20 km/sec per million lightyears as found by some studies), this velocity would indicate a distance to the supernova and its parent galaxy of about 5,800 million lightyears. The explosion of the supernova would thus have taken place 5,800 million years ago, i.e. about 1,000 million years before the solar system was formed. However, such a simple calculation works only for relatively ``nearby'' objects, perhaps out to some hundred million lightyears. When we look much further into space, we also look far back in time and it is not excluded that the universal expansion rate, i.e. the Hubble constant, may have been different at earlier epochs. This means that unless we know the change of the Hubble constant with time, we cannot determine reliable distances of distant galaxies from their measured redshifts and velocities. At the same time, knowledge about such change or lack of the same will provide unique information about the time elapsed since the Universe began to expand (the ``Big Bang''), that is, the age of the Universe and also its ultimate fate. The Deceleration Parameter q0 Cosmologists are therefore eager to determine not only the current expansion rate (i.e., the Hubble constant, H0) but also its possible change with time (known as the deceleration parameter, q0). Although a highly accurate value of H0 has still not become available, increasing attention is now given to the observational determination of the second parameter, cf. also the Appendix at the

  5. PET studies with low and high affinity dopamine D2 receptor radioligands: Effects of 4-hydroxybutyrate (4HB)

    SciTech Connect

    Gatley, S.J.; Fowler, J.S.; Dewey, S.

    1994-05-01

    D2 radioligands of varying affinities have been developed as PET and SPECT radiotracers, but no consensus has been reached on the abilities of these tracers to quantify D2 receptor concentrations in vivo. Amongst other differences, competition of the radioligand with endogenous DA is expected to depend on affinity for the D2 receptor, so that changes in DA might confound estimates of Bmax. We examined the uptake and kinetics if C-11 raclopride (RAC; Kd = 1.2 nM) and C-11 N-methylspiperone (NMS); Kd = 75 pM in baboon striatum after pretreatment with 4HB (200 mg/Kg, i/v) which inhibits DA release by nigrostriatal nerve terminals. While 4HB diminished uptake (%ID/g) of NMS, it prolonged tissue retention of RAC, confirming previous observations in rodent models. Logan (for RAC) and Patlak (for NMS) plots gave changes of +24% and -20%, respectively, between control and 4HB treated animals. Since decreased competition with DA should increase uptake of NMS as well as RAC the paradoxical decrease in NMS uptake could be due to a second synaptic effect of DA, such as a decrease in agonist mediated internalization of NMS. Alternatively, it could result from an independent effect of 4HB, perhaps related to this drug`s ability to induce anesthesia and to depress cerebral glucose utilization. Although previous work in the rat suggests that 4HB does not alter brain blood flow, we found O-15 water that baboon striatal blood flow was decreased 22% and 42% at 30 and 60 minutes, respectively, after 4HB. Smaller changes were seen in cerebellar blood flow. Though a 4HB induced decrease in blood flow does not rule out a DA mediated alteration in D2 receptor Bmax or Kd for NMS, or other factor, it is unnecessary to invoke this to account for our results.

  6. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  7. A novel high affinity human monoclonal antibody to mesothelin

    PubMed Central

    Ho, Mitchell; Feng, Mingqian; Fisher, Robert J.; Rader, Christoph; Pastan, Ira

    2010-01-01

    Mesothelin is a glycosylphosphatidylinisotol-anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here we report the identification of a novel human mAb to mesothelin. HN1, a human single chain Fv specific for mesothelin, was isolated from a naïve human scFv phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain, and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody-dependent cell-mediated cytotoxicity. HN1 binds a conformation-sensitive epitope in human mesothelin with high affinity (KD = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface-associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin-expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin-expressing cancer treatment and diagnosis. PMID:20635390

  8. Protein-protein binding affinities by pulse proteolysis: application to TEM-1/BLIP protein complexes.

    PubMed

    Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M

    2010-10-01

    Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein-ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein-protein complex involving the β-lactamase TEM-1 and various β-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C(m), of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein-protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC(m) was observed. From this "calibration curve," accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ΔC(m) values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

  9. Constant Domain-regulated Antibody Catalysis*

    PubMed Central

    Sapparapu, Gopal; Planque, Stephanie; Mitsuda, Yukie; McLean, Gary; Nishiyama, Yasuhiro; Paul, Sudhir

    2012-01-01

    Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies. PMID:22948159

  10. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.

    PubMed

    Lu, Dan; Shen, Juqun; Vil, Marie D; Zhang, Haifan; Jimenez, Xenia; Bohlen, Peter; Witte, Larry; Zhu, Zhenping

    2003-10-31

    Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides

  11. Recommended Distribution Coefficients, Kd Values, for Special Analysis Risk Calculations Related to Waste Disposal and Tank Closure on the Savannah River Site

    SciTech Connect

    Kaplan, D

    2005-08-31

    The purpose of this document is to provide a technically defensible list of distribution coefficients, or Kd values, for use in performance assessment (PA) and special analysis (SA) calculations on the SRS. Only Kd values for radionuclides that have new information related to them or that have recently been recognized as being important are discussed in this report. Some 150 Kd values are provided in this report for various waste-disposal or tank-closure environments: soil, corrosion in grout, oxidizing grout waste, gravel, clay, and reducing concrete environments. Documentation and justification for the selection of each Kd value is provided.

  12. Use of quantitative affinity chromatography for characterizing high-affinity interactions: binding of heparin to antithrombin III.

    PubMed

    Hogg, P J; Jackson, C M; Winzor, D J

    1991-02-01

    The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 +/- 2.2 x 10(7), 3.4 +/- 0.3 x 10(7), and 1.0 +/- 0.2 x 10(7) M-1 were observed at 15, 25, and 35 degrees C, respectively. The standard enthalpy change of -4.2 +/- 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements. PMID:2035830

  13. Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

    PubMed

    Sengupta, Tanusree; Manoj, Narayanan

    2016-01-01

    Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process. PMID:27584039

  14. 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

  15. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

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

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  16. 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.

  17. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  18. Current concepts. I. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

    SciTech Connect

    Moody, T.W.; Carney, D.N.; Cuttitta, F.; Quattrocchi, K.; Minna, J.D.

    1985-07-15

    The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (/sup 125/I-Tyr/sup 4/)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated, using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (/sup 125/I-Tyr/sup 4/)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC. 31 references, 6 figures, 2 tables.

  19. Painlevé analysis, nonlocal symmetry and explicit interaction solutions for supersymmetric mKdVB equation

    NASA Astrophysics Data System (ADS)

    Ren, Bo

    2016-08-01

    The N = 1 supersymmetric mKdVB system is transformed to a coupled bosonic system by using the bosonization approach. By a singularity structure analysis, the bosonized supersymmetric mKdVB (BSmKdVB) equation admits the Painlevé property. Starting from the standard truncated Painlevé method, the nonlocal symmetry for the BSmKdVB equation is obtained. To solve the first Lie's principle related with the nonlocal symmetry, the nonlocal symmetry is localized to the Lie point symmetry by introducing multiple new fields. Thanks to localization processes, similarity reductions for the prolonged systems are studied by the Lie point symmetry method. The interaction solutions among solitons and other complicated waves including Painlevé II waves and periodic cnoidal waves are given through the reduction theorems. The soliton-cnoidal wave interaction solutions are explicitly given by using the mapping and deformation method. The concrete soliton-cnoidal interaction solutions are displayed both in analytical and graphical ways.

  20. Kd Values for Agricultural and Surface Soils for Use in Hanford Site Farm, Residential, and River Shoreline Scenarios

    SciTech Connect

    Serne, R. Jeffrey

    2007-08-01

    This report provides best estimate Kd values and a minimum and maximum range of Kd values to be used for agricultural soils and Columbia River bank sediments that exist today or would exist in the future when portions of the Hanford Site are released for farming, residential, and recreational use after the U. S. Department of Energy (DOE) completes clean up of defense waste on the site. The Kd values should be used to determine the fate and transport rates of contaminants and their availability for plant and animal uptake in selected non-groundwater scenarios included in Hanford Site environmental impact statements, risk assessments and specific facility performance assessments. This report describes scenarios such as a small farm where drilling of a well inadvertently goes through buried waste and brings waste to the surface, allowing the tailings to become available for direct human exposure or incorporation into garden crops and farm animals used for food by the farm family. The Kd values recommended in this report can also be used to calculate sediment-water partitioning factors used to predict plant and animal uptake from interaction with the contaminated soil.

  1. Correlation functions of the KdV hierarchy and applications to intersection numbers over M bar g , n

    NASA Astrophysics Data System (ADS)

    Bertola, Marco; Dubrovin, Boris; Yang, Di

    2016-07-01

    We derive an explicit generating function of correlation functions of an arbitrary tau-function of the KdV hierarchy. In particular applications, our formulation gives closed formulæ of a new type for the generating series of intersection numbers of ψ-classes as well as of mixed ψ- and κ-classes in full genera.

  2. Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato.

    PubMed

    Meshi, T; Motoyoshi, F; Maeda, T; Yoshiwoka, S; Watanabe, H; Okada, Y

    1989-05-01

    A resistance-breaking strain of tobacco mosaic virus (TMV), Ltb1, is able to multiply in tomatoes with the Tm-2 gene, unlike its parent strain, L. Nucleotide sequence analysis of Ltb1 RNA revealed two amino acid changes in the 30-kD protein: from Cys68 to Phe and from Glu133 to Lys (from L to Ltb1). Strains with these two changes generated in vitro multiplied in tomatoes with the Tm-2 gene and induced essentially the same symptoms as those caused by Ltb1. Strains with either one of the two changes did not overcome the resistance as efficiently as Ltb1, although increased levels of multiplication were observed compared with the L strain. Results showed that both mutations are involved in the resistance-breaking property of Ltb1. Sequence analysis indicated that another resistance-breaking strain and its parent strain had two amino acid changes in the 30-kD protein: from Glu52 to Lys and from Glu133 to Lys. The fact that the amino acid changes occurred in or near the well conserved regions in the 30-kD protein suggests that the mechanism of Tm-2 resistance may be closely related to the fundamental function of the 30-kD protein, presumably in cell-to-cell movement. PMID:2535549

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

    PubMed Central

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

    2015-01-01

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

  4. Ligand-Protein Affinity Studies Using Long-Lived States of Fluorine-19 Nuclei.

    PubMed

    Buratto, Roberto; Mammoli, Daniele; Canet, Estel; Bodenhausen, Geoffrey

    2016-03-10

    The lifetimes TLLS of long-lived states or TLLC of long-lived coherences can be used for the accurate determination of dissociation constants of weak protein-ligand complexes. The remarkable contrast between signals derived from LLS or LLC in free and bound ligands can be exploited to search for weak binders with large dissociation constants KD > 1 mM that are important for fragment-based drug discovery but may escape detection by other screening techniques. Alternatively, the high sensitivity of the proposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumption of precious proteins. The detection of (19)F-(19)F long-lived states in suitably designed fluorinated spy molecules allows one to perform competition binding experiments with high sensitivity while avoiding signal overlap that tends to hamper the interpretation of proton spectra of mixtures. PMID:26800391

  5. New Quasar Studies Keep Fundamental Physical Constant Constant

    NASA Astrophysics Data System (ADS)

    2004-03-01

    Very Large Telescope sets stringent limit on possible variation of the fine-structure constant over cosmological time Summary Detecting or constraining the possible time variations of fundamental physical constants is an important step toward a complete understanding of basic physics and hence the world in which we live. A step in which astrophysics proves most useful. Previous astronomical measurements of the fine structure constant - the dimensionless number that determines the strength of interactions between charged particles and electromagnetic fields - suggested that this particular constant is increasing very slightly with time. If confirmed, this would have very profound implications for our understanding of fundamental physics. New studies, conducted using the UVES spectrograph on Kueyen, one of the 8.2-m telescopes of ESO's Very Large Telescope array at Paranal (Chile), secured new data with unprecedented quality. These data, combined with a very careful analysis, have provided the strongest astronomical constraints to date on the possible variation of the fine structure constant. They show that, contrary to previous claims, no evidence exist for assuming a time variation of this fundamental constant. PR Photo 07/04: Relative Changes with Redshift of the Fine Structure Constant (VLT/UVES) A fine constant To explain the Universe and to represent it mathematically, scientists rely on so-called fundamental constants or fixed numbers. The fundamental laws of physics, as we presently understand them, depend on about 25 such constants. Well-known examples are the gravitational constant, which defines the strength of the force acting between two bodies, such as the Earth and the Moon, and the speed of light. One of these constants is the so-called "fine structure constant", alpha = 1/137.03599958, a combination of electrical charge of the electron, the Planck constant and the speed of light. The fine structure constant describes how electromagnetic forces hold

  6. Transforming growth factor-beta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.

    PubMed Central

    Marti, H. P.; Lee, L.; Kashgarian, M.; Lovett, D. H.

    1994-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is generally considered to exert positive effects on the accumulation of extracellular matrices. These occur as the net result of enhanced matrix protein synthesis, diminished matrix metalloproteinase (MMP) synthesis, and augmented production of specific inhibitors, including the tissue inhibitor of metalloproteinases (TIMP-1). Given that glomerular TGF-beta 1 synthesis is induced by inflammation, the effects of this cytokine on synthesis of the 72-kd type IV collagenase and TIMP-1 by cultured human mesangial cells were evaluated. Concentrations of TGF-beta 1 of 5 ng/ml and above specifically stimulated the synthesis of the 72-kd type IV collagenase. This effect was independent of the stimulatory effect of TGF-beta 1 on TIMP-1 synthesis, which was maximal in a lower concentration range (0.1 to 1 ng/ml). Most significantly, the net effect at the higher concentrations of TGF-beta 1 was an excess of enzyme over the TIMP-1 inhibitor. Northern blot analysis of TGF-beta 1-stimulated human mesangial cells demonstrated a specific increase in the abundance of the 3.1 kb mRNA transcript encoding the 72-kd type IV collagenase, presumably mediated by a direct stimulation of 72-kd type IV collagenase mRNA transcription observed as early as 3 hours after exposure to TGF-beta 1. These studies were extended to an analysis of the expression of TGF-beta 1 and 72-kd type IV collagenase mRNAs in normal and nephritic rats. In normal animals, basal TGF-beta 1 and 72-kd type IV collagenase mRNA expression was observed in a strictly mesangial distribution. After induction of acute immune complex-mediated glomerulonephritis, there was a major increase in TGF-beta 1 and 72-kd type IV collagenase mRNA expression, which was strictly limited to the expanded, hypercellular mesangial compartment. Enhanced synthesis of the mesangial type IV collagenase in response to TGF-beta 1 released during glomerular inflammatory processes could have an important

  7. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    SciTech Connect

    Tiberi, M.; Magnan, J. )

    1990-05-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).

  8. 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...

  9. Minimal information to determine affine shape equivalence.

    PubMed

    Wagemans, J; Van Gool, L; Lamote, C; Foster, D H

    2000-04-01

    Participants judged the affine equivalence of 2 simultaneously presented 4-point patterns. Performance level (d') varied between 1.5 and 2.7, depending on the information available for solving the correspondence problem (insufficient in Experiment 1a, superfluous in Experiment 1b, and minimal in Experiments 1c, 2a, 2b) and on the exposure time (unlimited in Experiments 1 and 2a and 500 ms in Experiment 2b), but it did not vary much with the complexity of the affine transformation (rotation and slant in Experiment 1 and same plus tilt in Experiment 2). Performance in Experiment 3 was lower with 3-point patterns than with 4-point patterns, whereas blocking the trials according to the affine transformation parameters had little effect. Determining affine shape equivalence with minimal-information displays is based on a fast assessment of qualitatively or quasi-invariant properties such as convexity/ concavity, parallelism, and collinearity. PMID:10811156

  10. 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

  11. Visualizing antibody affinity maturation in germinal centers.

    PubMed

    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

    2016-03-01

    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 nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  12. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.

    PubMed

    Estep, Patricia; Reid, Felicia; Nauman, Claire; Liu, Yuqi; Sun, Tingwan; Sun, Joanne; Xu, Yingda

    2013-01-01

    Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods. PMID:23575269

  13. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed Central

    Zhang, S; Klessig, D F

    1997-01-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses. PMID:9165755

  14. Cluster Segmentation of Thermal Image Sequences Using kd-Tree Structure

    NASA Astrophysics Data System (ADS)

    Świta, R.; Suszyński, Z.

    2014-12-01

    This paper presents optimization methods for the K-means segmentation algorithm for a sequence of thermal images. Images of the sample response in the frequency domain to the thermal stimulation with a known spectrum were subjected to cluster segmentation, grouping pixels with similar frequency characteristics. Compared were all pixel characteristics in the function of the frame number and grouped using the minimal sum of deviations of the pixels from their segment mean for all the frames of the processed image sequence. A new initialization method for the K-means algorithm, using density information, was used. A K-means algorithm with a kd-tree structure C# implementation was tested for speed and accuracy. This algorithm divides the set of pixels to the subspaces in the hierarchy of a binary tree. This allows skipping the calculation of distances of pixels to some centroids and pruning a set of centroid clusters through the hierarchy tree. Results of the segmentation were compared with the K-means and FCM algorithm MATLAB implementations.

  15. Deceleration of the small solitons in the soliton lattice: KdV-type framework

    NASA Astrophysics Data System (ADS)

    Shurgalina, Ekaterina; Gorshkov, Konstantin; Talipova, Tatiana; Pelinovsky, Efim

    2016-04-01

    As it is known the solitary waves (solitons) in the KdV-systems move with speed which exceeds the speed of propagation of long linear waves (sound speed). Due to interaction between them, solitons do not lose their individuality (elastic interaction). Binary interaction of neigborough solitons is the major contribution in the dynamics of soliton gas. Taking into account the integrability of the classic and modified Korteweg-de Vries equations the process of the soliton interaction can be analyzed in the framework of the rigorous analytical two-soliton solutions. Main physical conclusion from this solution is the phase shift which is positive for large solitons and negative for small solitons. This fact influences the average velocity of individual soliton in the soliton lattice or soliton gas. We demonstrate that soliton of relative small amplitude moves in soliton gas in average in opposite (negative) direction, meanwhile a free soliton moves always in the right direction. Approximated analytical theory is created for the soliton motion in the periodic lattice of big solitons of the same amplitudes, and the critical amplitude of the small soliton changed its averaged speed is found. Numerical simulation is conducted for a statistical assembly of solitons with random amplitudes and phases. The application of developed theory to the long surface and internal waves is discussed.

  16. Stability of discontinuity structures described by a generalized KdV-Burgers equation

    NASA Astrophysics Data System (ADS)

    Chugainova, A. P.; Shargatov, V. A.

    2016-02-01

    The stability of discontinuities representing solutions of a model generalized KdV-Burgers equation with a nonmonotone potential of the form φ( u) = u 4- u 2 is analyzed. Among these solutions, there are ones corresponding to special discontinuities. A discontinuity is called special if its structure represents a heteroclinic phase curve joining two saddle-type special points (of which one is the state ahead of the discontinuity and the other is the state behind the discontinuity).The spectral (linear) stability of the structure of special discontinuities was previously studied. It was shown that only a special discontinuity with a monotone structure is stable, whereas special discontinuities with a nonmonotone structure are unstable. In this paper, the spectral stability of nonspecial discontinuities is investigated. The structure of a nonspecial discontinuity represents a phase curve joining two special points: a saddle (the state ahead of the discontinuity) and a focus or node (the state behind the discontinuity). The set of nonspecial discontinuities is examined depending on the dispersion and dissipation parameters. A set of stable nonspecial discontinuities is found.

  17. KD loop for increasing arch perimeter in cleft and noncleft cases.

    PubMed

    Dholakia, Kartik D; Bhat, Shweta R

    2012-01-01

    One of the many indications for dental arch expansion in treating malocclusion is to achieve arch compatibility, especially in surgical cases with severe Bolton discrepancies or collapsed arches due to congenitally missing anterior teeth. These cases usually require expansion in both the sagittal and transverse plane to achieve normal arch compatibility. Arch compatibility can be achieved by either dentoalveolar or skeletal expansion or both. Orthodontically, dentoalveolar expansion can be achieved by means of expanded arch form, vertical loops, or an added assembly such as a quad helix or Ni-Ti expander from the palatal or lingual aspect of the arch. However, these modalities normally provide expansion along transverse plane. If any expansion along sagittal plane is required, then additional appliances such as TransForce or modifications in the appliance system (eg, a quad helix with extension on anterior teeth) are necessary. Vertical loops do overcome these drawbacks to a certain extent; however, at the expense of generating moments during preactivation, which may lead to tipping of segments adjacent to the loop and precludes its use for larger changes of arch dimension. This article describes a new loop design--the KD loop--that increases the arch perimeter by sagittal and transverse expansion without generating significant moments along vertical plane. PMID:22567644

  18. Fast focal zooming scheme for direct drive fusion implemented by inserting KD2PO4 crystal

    NASA Astrophysics Data System (ADS)

    Zhong, Zheqiang; Hu, Xiaochuan; Zhang, Bin

    2016-06-01

    The highly required uniformity of target in direct-drive fusion is difficult to achieve and maintain during the entire laser fusion implosion. To mitigate the increasing nonuniformity, the fast focal zooming scheme implemented by inserting an electro-optic (EO) crystal in the front end of beamline has been proposed. Functioning as a phase plate, the specifically designed EO crystal may add the induced spherical wavefront to the laser beam and alter its focusing characteristics. However, in order to zoom out the focal spot by half, the required voltage for KD2PO4 (DKDP) with single pair of electrodes is relatively high. In order to decrease the voltage while maintaining the zooming performance, the DKDP cylinder with multi pairs of electrodes has been presented. The continuous phase plate (CPP) is designed according to both the injected beam and the output field. However, the conventional CPP is designed based on the assumption of an injected beam without wavefront distortion, which would zoom in the focal spot variation in the focal zooming scheme. In order to zoom out the focal spot, a redesigned CPP has been proposed by adding a spherical wavefront to the phase variation of the conventional CPP and further optimizing. On the basis, the focusing characteristics of laser beam during the fast focal zooming process have been analyzed. Results indicate that the focal spot size decreases with the increasing voltage on DKDP crystal, meanwhile the uniformity maintains high during the focal zooming process.

  19. Atomic layer deposited titanium dioxide coatings on KD-II silicon carbide fibers and their characterization

    NASA Astrophysics Data System (ADS)

    Cao, Shiyi; Wang, Jun; Wang, Hao

    2016-03-01

    To provide oxidation protection and/or to act as an interfacial coating, titanium oxide (TiO2) coatings were deposited on KD-II SiC fibers by employing atomic layer deposition (ALD) technique with tetrakis(dimethylamido)titanium (TDMAT) and water (H2O) as precursors. The average deposition rate was about 0.08 nm per cycle, and the prepared coatings were smooth, uniform and conformal, shielding the fibers entirely. The as-deposited coatings were amorphous regardless of the coating thickness, and changed to anatase and rutile crystal phase after annealing at 600 °C and 1000 °C, respectively. The oxidation measurement suggests that the TiO2 coating enhanced the oxidation resistance of SiC fibers obviously. SiC fibers coated with a 70-nm-thick TiO2 layer retained a relatively high tensile strength of 1.66 GPa even after exposition to air at 1400 °C for 1 h, and thick silica layer was not observed. In contrast, uncoated SiC fibers were oxidized dramatically through the same oxidation treatment, covered with a macro-cracked thick silica film, and the tensile strength was not measurable due to interfilament adhesion. The above results indicate that TiO2 films deposited by ALD are a promising oxidation resistance coating for SiC fibers.

  20. The tomato 66.3-kD polyphenoloxidase gene: molecular identification and developmental expression.

    PubMed Central

    Shahar, T; Hennig, N; Gutfinger, T; Hareven, D; Lifschitz, E

    1992-01-01

    A gene coding for a polypeptide abundant in tomato floral meristems was isolated and shown to represent a tomato 66.3-kD polyphenoloxidase. Analysis of cDNA clones and a corresponding intronless genomic clone indicated that the plastid-bound 587-residue-long polypeptide, designated P2, contains two conserved copper-binding domains, similar to those found in fungal and mammalian tyrosinases. P2 transcripts and polypeptides are accumulated in the arrested floral primordia of the anantha mutant inflorescences and are equally abundant in primordia of wild-type flowers; the gene continues to be expressed at high levels in developing floral organs. In young expanding leaves, P2 protein is concentrated in palisade cells and in epidermal trichomes. Expression patterns of P2 in plant meristems permit molecular distinction between floral and vegetative primordia, and, in a companion study, comparison with dUTPase suggests that the two genes mark two alternative complementary developmental programs in the floral and vegetative meristems of the tomato plants. PMID:1633491

  1. A 72 kD trophoblast glycoprotein defined by a monoclonal antibody.

    PubMed

    Hole, N; Stern, P L

    1988-03-01

    A novel trophoblast cell surface antigen has been defined by a monoclonal antibody 5T4, raised following immunisation with wheat germ agglutinin (WGA) purified glycoproteins from deoxycholate (DOC) solubilised human syncytiotrophoblast plasma membrane (StMPM). The distribution of the antigen was determined by indirect immunoperoxidase staining of sections of normal organ and placental tissues as well as immunofluorescence and radiobinding assays with a wide variety of cell lines representing differing normal and tumour cell types. In frozen sections of normal full term placenta, 5T4 is strongly expressed only by the syncytiotrophoblast, some extravillous cytotrophoblast and the amniotic epithelium. The 5T4 antigen is apparently not expressed by any maternal component of the placenta nor is it detected in adult liver, lung, bronchus, heart, testis, ovary, brain, or muscle. The antigen is apparently expressed by several specialised epithelia. Immunoprecipitation of radiolabelled StMPM indicated that 5T4 molecules are glycoproteins of mol. wt of approximately 72 kD on SDS-PAGE. 5T4 antigen is selectively expressed by diverse tumour cell lines, including those of developmental origin. The molecular characteristics, relatively restricted normal tissue distribution and expression by certain tumour cell types make this antigen worthy of future study for use as a diagnostic marker of malignancy. PMID:3355761

  2. The 13-kD FK506 Binding Protein, FKBP13, Interacts with a Novel Homologue of the Erythrocyte Membrane Cytoskeletal Protein 4.1

    PubMed Central

    Walensky, Loren D.; Gascard, Philippe; Field, Michael E.; Blackshaw, Seth; Conboy, John G.; Mohandas, Narla; Snyder, Solomon H.

    1998-01-01

    We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G–CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677– 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705–708). We report the specific association of FKBP13 with 4.1G–CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G–CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G–CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G–CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds. PMID:9531554

  3. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

    PubMed Central

    1987-01-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N- ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin- membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  4. High affinity FRβ-specific CAR T cells eradicate AML and normal yeloid lineage without HSC toxicity

    PubMed Central

    Lynn, Rachel C; Feng, Yang; Schutsky, Keith; Poussin, Mathilde; Kalota, Anna; Dimitrov, Dimiter S; Powell, Daniel J

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  5. Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands.

    PubMed

    Clune, A; Lee, K S; Ferenci, T

    1984-05-31

    Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein. PMID:6375667

  6. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  7. EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

    PubMed

    Tanner, John J; Frey, Benjamin B; Pemberton, Travis; Henzl, Michael T

    2016-09-13

    The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5. PMID:27541325

  8. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  9. Formulas for determining rotational constants

    NASA Astrophysics Data System (ADS)

    Guelachvili, G.

    This document is part of Subvolume B `Linear Triatomic Molecules', Part 9, of Volume 20 `Molecular Constants mostly from Infrared Spectroscopy' of Landolt-Börnstein Group II `Molecules and Radicals'. Part of the introduction, it states formulas for determining rotational constants, band center, band origin, and quadrupole coupling. Specific comments relate to BHO (HBO) and COS (OCS).

  10. QCD coupling constants and VDM

    SciTech Connect

    Erkol, G.; Ozpineci, A.; Zamiralov, V. S.

    2012-10-23

    QCD sum rules for coupling constants of vector mesons with baryons are constructed. The corresponding QCD sum rules for electric charges and magnetic moments are also derived and with the use of vector-meson-dominance model related to the coupling constants. The VDM role as the criterium of reciprocal validity of the sum rules is considered.

  11. Notch ligand delta-like1: X-ray crystal structure and binding affinity.

    PubMed

    Kershaw, Nadia J; Church, Nicole L; Griffin, Michael D W; Luo, Cindy S; Adams, Timothy E; Burgess, Antony W

    2015-05-15

    The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 μM for this interaction. PMID:25715738

  12. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    PubMed

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

  13. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  14. Identification of high affinity folate binding proteins in human erythrocyte membranes.

    PubMed

    Antony, A C; Kincade, R S; Verma, R S; Krishnan, S R

    1987-09-01

    Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging. PMID:3624486

  15. Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels*

    PubMed Central

    Thompson, Christopher H.; Olivetti, Pedro R.; Fuller, Matthew D.; Freeman, Cody S.; McMaster, Denis; French, Robert J.; Pohl, Jan; Kubanek, Julia; McCarty, Nael A.

    2009-01-01

    The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function. PMID:19574231

  16. Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction.

    PubMed

    Papadakos, Grigorios; Sharma, Amit; Lancaster, Lorna E; Bowen, Rebecca; Kaminska, Renata; Leech, Andrew P; Walker, Daniel; Redfield, Christina; Kleanthous, Colin

    2015-04-29

    The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost. PMID:25856265

  17. Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity

    PubMed Central

    Dickey, Seth W.; Baker, Rosanna P.; Cho, Sangwoo; Urban, Siniša

    2013-01-01

    SUMMARY Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in over a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (Kd ~190 μM, or 0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in kcat values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications. PMID:24315097

  18. Head-on collision and overtaking collision between an envelope solitary wave and a KdV solitary wave in a dusty plasma

    NASA Astrophysics Data System (ADS)

    Zhang, Heng; Duan, Wen-Shan; Qi, Xin; Yang, Lei

    2016-02-01

    Head-on collision and overtaking collision between a KdV solitary wave and an envelope solitary wave are first studied in present paper by using Particle-in-cell (PIC) method in a dusty plasma. There are phase shifts of the KdV solitary wave in both head-on collision and the overtaking collision, while no phase shift is found for the envelop solitary wave in any cases. The remarkable difference between head-on collision and the overtaking collision is that the phase shift of KdV solitary wave increases as amplitude of KdV solitary wave increases in head-on collision, while it decreases as amplitude of the KdV solitary wave increases in the overtaking collision. It is found that the maximum amplitude during the collision process is less than sum of two amplitudes of both solitary waves, but is larger than either of the amplitude.

  19. Head-on collision and overtaking collision between an envelope solitary wave and a KdV solitary wave in a dusty plasma.

    PubMed

    Zhang, Heng; Duan, Wen-Shan; Qi, Xin; Yang, Lei

    2016-01-01

    Head-on collision and overtaking collision between a KdV solitary wave and an envelope solitary wave are first studied in present paper by using Particle-in-cell (PIC) method in a dusty plasma. There are phase shifts of the KdV solitary wave in both head-on collision and the overtaking collision, while no phase shift is found for the envelop solitary wave in any cases. The remarkable difference between head-on collision and the overtaking collision is that the phase shift of KdV solitary wave increases as amplitude of KdV solitary wave increases in head-on collision, while it decreases as amplitude of the KdV solitary wave increases in the overtaking collision. It is found that the maximum amplitude during the collision process is less than sum of two amplitudes of both solitary waves, but is larger than either of the amplitude. PMID:26868526

  20. Head-on collision and overtaking collision between an envelope solitary wave and a KdV solitary wave in a dusty plasma

    PubMed Central

    Zhang, Heng; Duan, Wen-Shan; Qi, Xin; Yang, Lei

    2016-01-01

    Head-on collision and overtaking collision between a KdV solitary wave and an envelope solitary wave are first studied in present paper by using Particle-in-cell (PIC) method in a dusty plasma. There are phase shifts of the KdV solitary wave in both head-on collision and the overtaking collision, while no phase shift is found for the envelop solitary wave in any cases. The remarkable difference between head-on collision and the overtaking collision is that the phase shift of KdV solitary wave increases as amplitude of KdV solitary wave increases in head-on collision, while it decreases as amplitude of the KdV solitary wave increases in the overtaking collision. It is found that the maximum amplitude during the collision process is less than sum of two amplitudes of both solitary waves, but is larger than either of the amplitude. PMID:26868526

  1. Affinity purification and characterization of (2'-5')oligo(adenylate)-dependent RNase from mouse spleen.

    PubMed

    Bayard, B; Bette-Bobillo, P; Aliau, S

    1994-07-15

    Murine (2'-5')An-dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2'-5')An. Since the ribonuclease has high affinity to (2'-5')An, optimal non-denaturing conditions were obtained to disrupt the (2'-5')An-nuclease complex. Low-pH buffers in the presence of 0.1% Triton X-100 removed almost 80% of the enzyme from the (2'-5')An-agarose, preserving its (2'-5')An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent crosslinking method and denaturing-renaturing affinity blotting assay. The purified enzyme was a 160-kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver-stained SDS gels. Both a 160-kDa dimer and 78-kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2'-5')An to (2'-5')An-dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4 degrees C. 2-Mercaptoethanol was required to obtain (2-'5')An-binding activity but, interestingly, in the absence of this reducing agent, (2'-5')An-binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2'-5')An. Furthermore, when activated by low amounts of (2'-5')An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2'-5')An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2'-5')An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2'-5')An-binding properties to those identified in spleen extracts. PMID:8055909

  2. Automated evaluation of protein binding affinity of anti-inflammatory choline based ionic liquids.

    PubMed

    Ribeiro, Rosa; Pinto, Paula C A G; Azevedo, Ana M O; Bica, Katharina; Ressmann, Anna K; Reis, Salette; Saraiva, M Lúcia M F S

    2016-04-01

    In this work, an automated system for the study of the interaction of drugs with human serum albumin (HSA) was developed. The methodology was based on the quenching of the intrinsic fluorescence of HSA by binding of the drug to one of its binding sites. The fluorescence quenching assay was implemented in a sequential injection analysis (SIA) system and the optimized assay was applied to ionic liquids based on the association of non-steroidal anti-inflammatory drugs with choline (IL-API). In each cycle, 100 µL of HSA and 100 µL of IL-API (variable concentration) were aspirated at a flow rate of 1 mL min(-1) and then sent through the reaction coil to the detector where the fluorescence intensity was measured. In the optimized conditions the effect of increasing concentrations of choline ketoprofenate and choline naproxenate (and respective starting materials: ketoprofen and naproxen) on the intrinsic fluorescence of HSA was studied and the dissociation constants (Kd) were calculated by means of models of drug-protein binding in the equilibrium. The calculated Kd showed that all the compounds bind strongly to HSA (Kd<100 µmol L(-1)) and that the use of the drugs in the IL format does not affect or can even improve their HSA binding. The obtained results were compared with those provided by a conventional batch assay and the relative errors were lower than 4.5%. The developed SIA methodology showed to be robust and exhibited good repeatability in all the assay conditions (rsd<6.5%). PMID:26838377

  3. Characterization of methacrylate chromatographic monoliths bearing affinity ligands.

    PubMed

    Černigoj, Urh; Vidic, Urška; Nemec, Blaž; Gašperšič, Jernej; Vidič, Jana; Lendero Krajnc, Nika; Štrancar, Aleš; Podgornik, Aleš

    2016-09-16

    We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. PMID:27554023

  4. Pulse length dependence of laser conditioning and bulk damage in KD2PO4

    SciTech Connect

    Adams, J J; Weiland, T L; Stanley, J R; Sell, W D; Luthi, R L; Vickers, J L; Carr, C W; Feit, M D; Rubenchik, A M; Spaeth, M L; Hackel, R P

    2004-11-10

    An experimental technique has been developed to measure the damage density {rho}({phi}) variation with fluence from scatter maps of bulk damage sites in plates of KD{sub 2}PO{sub 4} (DKDP) crystals combined with calibrated images of the damaging beam's spatial profile. Unconditioned bulk damage in tripler-cut DKDP crystals has been studied using 351 nm (3 {omega}) light at pulse lengths of 0.055, 0.091, 0.30, 0.86, 2.6, and 10 ns. It is found that there is less scatter due to damage at fixed fluence for longer pulse lengths. The results also show that for all the pulse lengths the scatter due to damage is a strong function of the damaging fluence. It is determined that the pulse length scaling for bulk damage scatter in unconditioned DKDP material varies as {tau}{sup 0.24 {+-} 0.05} over two orders of magnitude of pulse lengths. The effectiveness of 3 {omega} laser conditioning at pulse lengths of 0.055, 0.096, 0.30, 0.86, 3.5, and 23 ns is analyzed in term of damage density {rho}({phi}) at 3 {omega}, 2.6 ns. The 860 ps conditioning to a peak irradiance of 7 GW/cm{sup 2} had the best performance under 3 {omega}, 2.6 ns testing. It is shown that the optimal conditioning pulse length appears to lies in the range from 0.3 to 1 ns with a low sensitivity of 0.5 J/cm{sup 2}/ns to the exact pulse length.

  5. 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

  6. 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

  7. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future. PMID:26032605

  8. Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity.

    PubMed

    Santos, Adriano; Carvalho, Fernanda C; Roque-Barreira, Maria-Cristina; Bueno, Paulo R

    2014-12-15

    Characterization of lectin-carbohydrate binding using label-free methods such as impedance-derived electrochemical capacitance spectroscopy (ECS) is desirable to evaluate specific interactions, for example, ArtinM lectin and horseradish peroxidase (HRP) glycoprotein, used here as a model for protein-carbohydrate binding affinity. An electroactive molecular film comprising alkyl ferrocene as a redox probe and ArtinM as a carbohydrate receptive center to target HRP was successfully used to determine the binding affinity between ArtinM and HRP. The redox capacitance, a transducer signal associated with the alkyl ferrocene centers, was obtained by ECS and used in the Langmuir adsorption model to obtain the affinity constant (1.6±0.6)×10(8) L mol(-1). The results shown herein suggest the feasibility of ECS application for lectin glycoarray characterization. PMID:24994505

  9. One-step surface modification of polyurethane using affinity binding peptides for enhanced fouling resistance.

    PubMed

    Wang, Yibing; Yu, Yong; Zhang, Liting; Qin, Peng; Wang, Ping

    2015-01-01

    Affinity binding peptides were examined for surface fabrication of synthetic polymeric materials. Peptides possessing strong binding affinities toward polyurethane (PU) were discovered via biopanning of M13 phage peptide library. The apparent binding constant (K(app)) was as high as 2.68 × 10(9) M(-1) with surface peptide density exceeded 1.8 μg/cm(2). Structural analysis showed that the ideal peptide had a high content (75%) of H-donor amino acid residues, and that intensified hydrogen bond interaction was the key driving force for the highly stable binding of peptides on PU. PU treated with such affinity peptides promises applications as low-fouling materials, as peptides increased its wettability and substantially reduced protein adsorption and cell adhesion. These results demonstrated a facile but highly efficient one-step strategy for surface property modification of polymeric materials for biotechnological applications. PMID:25732121

  10. 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,…

  11. Development of a large field-of-view KD potassium di-deuterium phosphate modulator: Center Director's Discretionary Fund

    NASA Astrophysics Data System (ADS)

    West, E. A.

    1993-06-01

    Magnetographs, which measure polarized light, allow solar astronomers to infer the magnetic field intensity on the Sun. The Marshall Space Flight Center (MSFC) Vector Magnetograph is such an imaging instrument. The instrument requires rapid modulation between polarization states to minimize seeing effects. The accuracy of those polarization measurements is dependent on stable modulators with small field-of-view errors. Although these devices are very important in ground-based telescopes, extending the field of view of electro-optical crystals such as KD*Ps (potassium di-deuterium phosphate) could encourage the development of these devices for other imaging applications. The work that was done at MSFC as part of the Center Director's Discretionary Fund (CDDF) to reduce the field-of-view errors of instruments that use KD*P modulators in their polarimeters is described.

  12. Time-fractional KdV equation for plasma of two different temperature electrons and stationary ion

    SciTech Connect

    El-Wakil, S. A.; Abulwafa, Essam M.; El-Shewy, E. K.; Mahmoud, Abeer A.

    2011-09-15

    Using the time-fractional KdV equation, the nonlinear properties of small but finite amplitude electron-acoustic solitary waves are studied in a homogeneous system of unmagnetized collisionless plasma. This plasma consists of cold electrons fluid, non-thermal hot electrons, and stationary ions. Employing the reductive perturbation technique and the Euler-Lagrange equation, the time-fractional KdV equation is derived and it is solved using variational method. It is found that the time-fractional parameter significantly changes the soliton amplitude of the electron-acoustic solitary waves. The results are compared with the structures of the broadband electrostatic noise observed in the dayside auroral zone.

  13. A well-separated pairs decomposition algorithm for k-d trees implemented on multi-core architectures

    NASA Astrophysics Data System (ADS)

    Lopes, Raul H. C.; Reid, Ivan D.; Hobson, Peter R.

    2014-06-01

    Variations of k-d trees represent a fundamental data structure used in Computational Geometry with numerous applications in science. For example particle track fitting in the software of the LHC experiments, and in simulations of N-body systems in the study of dynamics of interacting galaxies, particle beam physics, and molecular dynamics in biochemistry. The many-body tree methods devised by Barnes and Hutt in the 1980s and the Fast Multipole Method introduced in 1987 by Greengard and Rokhlin use variants of k-d trees to reduce the computation time upper bounds to O(n log n) and even O(n) from O(n2). We present an algorithm that uses the principle of well-separated pairs decomposition to always produce compressed trees in O(n log n) work. We present and evaluate parallel implementations for the algorithm that can take advantage of multi-core architectures.

  14. Development of a large field-of-view KD potassium di-deuterium phosphate modulator: Center Director's Discretionary Fund

    NASA Technical Reports Server (NTRS)

    West, E. A.

    1993-01-01

    Magnetographs, which measure polarized light, allow solar astronomers to infer the magnetic field intensity on the Sun. The Marshall Space Flight Center (MSFC) Vector Magnetograph is such an imaging instrument. The instrument requires rapid modulation between polarization states to minimize seeing effects. The accuracy of those polarization measurements is dependent on stable modulators with small field-of-view errors. Although these devices are very important in ground-based telescopes, extending the field of view of electro-optical crystals such as KD*Ps (potassium di-deuterium phosphate) could encourage the development of these devices for other imaging applications. The work that was done at MSFC as part of the Center Director's Discretionary Fund (CDDF) to reduce the field-of-view errors of instruments that use KD*P modulators in their polarimeters is described.

  15. 150-kD von Willebrand factor binding protein extracted from human vascular subendothelium is type VI collagen.

    PubMed Central

    Rand, J H; Patel, N D; Schwartz, E; Zhou, S L; Potter, B J

    1991-01-01

    We have previously shown that von Willebrand factor (vWF), a glycoprotein which plays a critical role in the adhesion of platelets to injured blood vessels, is present within vascular subendothelium. We investigated the identity of the subendothelial binding site(s) for vWF by examining vWF binding to subendothelial constituents and solubilized a 150-kD protein with SDS-urea that bound vWF. This protein had an amino-acid composition similar to that of the type VI collagen alpha-1/alpha-2 chains, was recognized by specific polyclonal antibodies against type VI collagen, and had a similar acidic isoelectric point. Furthermore, we found that purified type VI collagen also bound vWF. Thus, we have identified the extracted 150-kD protein as type VI collagen. This protein may play a significant role in the binding of vWF to vascular subendothelium in vivo. Images PMID:2056120

  16. On Affine Fusion and the Phase Model

    NASA Astrophysics Data System (ADS)

    Walton, Mark A.

    2012-11-01

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

  17. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  18. Poisson structure and stability analysis of a coupled system arising from the supersymmetric breaking of Super KdV

    NASA Astrophysics Data System (ADS)

    Sotomayor, Adrián; Restuccia, Alvaro

    2013-11-01

    The Poisson structure of a coupled system arising from a supersymmetric breaking of N=1 Super KdV equations is obtained. The supersymmetric breaking is implemented by introducing a Clifford algebra instead of a Grassmann algebra. The Poisson structure follows from the Dirac brackets obtained by the constraint analysis of the hamiltonian of the system. The coupled system has multisolitonic solutions. We show that the one soliton solutions are Liapunov stable.

  19. A pseudo-spectral method for a non-local KdV-Burgers equation posed on R

    NASA Astrophysics Data System (ADS)

    de la Hoz, Francisco; Cuesta, Carlota M.

    2016-04-01

    In this paper, we present a new pseudo-spectral method to solve the initial value problem associated to a non-local KdV-Burgers equation involving a Caputo-type fractional derivative. The basic idea is, using an algebraic map, to transform the whole real line into a bounded interval where we can apply a Fourier expansion. Special attention is given to the correct computation of the fractional derivative in this setting.

  20. GENERAL Pseudopotentials, Lax Pairs and Bäcklund Transformations for Generalized Fifth-Order KdV Equation

    NASA Astrophysics Data System (ADS)

    Yang, Yun-Qing; Chen, Yong

    2011-01-01

    Based on the method developed by Nucci, the pseudopotentials, Lax pairs and the singularity manifold equations of the generalized fifth-order KdV equation are derived. By choosing different coefficient, the corresponding results and the Bäcklund transformations can be obtained on three conditioners which include Caudrey—Dodd—Gibbon—Sawada—Kotera equation, the Lax equation and the Kaup-kupershmidt equation.

  1. REVISED GUIDELINES FOR USING CELLULOSE DEGRADATION PRODUCT-IMPACTED KD VALUES FOR PERFORMANCE ASSESSMENTS AND COMPOSITE ANALYSES

    SciTech Connect

    Kaplan, D.

    2012-05-14

    Cellulosic materials include wood, paper, rags, and cardboard products. These materials are co-disposed with radiological waste at the Savannah River Site's (SRS) E-Area Low-Level Waste Facility (ELLWF). Cellulosic materials readily degrade in the environment to form cellulose degradation products (CDP) that will partition to the sediment or remain mobile in the groundwater. Savannah River National Lab (SRNL) has conducted studies to estimate the impact of CDP on radionuclide sorption to SRS sediments (Kd values). It was found that CDP impact on radionuclide sorption varies with radionuclide and CDP concentration. Furthermore, it was found that the amount of carbon (C) in the system could increase or decrease Kd values with respect to the base case of when no CDP was added. Throughout the expected pH range of the ELLWF, a low concentration of CDP in the system would increase Kd values (because C would sorb to the sediment and provide more exchange sites for radionuclides to sorb), whereas greater concentrations of CDP ({ge}20 mg/L C) would decrease Kd values (because C would remain in solution and complex the radionuclide and not permit the radionuclide to sorb to the sediment). A review of >230 dissolved organic carbon (DOC) groundwater concentrations in the Old Radioactive Waste Burial Ground (ORWBG) at the SRS indicated that the average DOC concentration, a gross measure of CDP, was 5 mg/L C. At approximately this DOC concentration, the laboratory studies demonstrated that no anions (Tc, I, or Se) or cations (Ni, Sr, Ce, Eu, Zr, or Th) have decreased sorption in the presence of carbon (an analogue for CDP).

  2. Yeast 3',5'-bisphosphate nucleotidase: an affinity tag for protein purification.

    PubMed

    Yang, Yang; Ma, Jianhui; Yang, Yilin; Zhang, Xiao; Wang, Yanxing; Yang, Ling; Sun, Meihao

    2014-05-01

    Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3μM and ∼11s(-)(1), respectively. Kd for PAP was 0.008μM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification. PMID:24613729

  3. Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

    SciTech Connect

    Hass, P.E.; Hotchkiss, A.; Mohler, M.; Aggarwal, B.B.

    1985-10-05

    Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

  4. 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

  5. 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

  6. Constant Communities in Complex Networks

    NASA Astrophysics Data System (ADS)

    Chakraborty, Tanmoy; Srinivasan, Sriram; Ganguly, Niloy; Bhowmick, Sanjukta; Mukherjee, Animesh

    2013-05-01

    Identifying community structure is a fundamental problem in network analysis. Most community detection algorithms are based on optimizing a combinatorial parameter, for example modularity. This optimization is generally NP-hard, thus merely changing the vertex order can alter their assignments to the community. However, there has been less study on how vertex ordering influences the results of the community detection algorithms. Here we identify and study the properties of invariant groups of vertices (constant communities) whose assignment to communities are, quite remarkably, not affected by vertex ordering. The percentage of constant communities can vary across different applications and based on empirical results we propose metrics to evaluate these communities. Using constant communities as a pre-processing step, one can significantly reduce the variation of the results. Finally, we present a case study on phoneme network and illustrate that constant communities, quite strikingly, form the core functional units of the larger communities.

  7. Estimating the Underwater Diffuse Attenuation Coefficient with a Low-Cost Instrument: The KdUINO DIY Buoy

    PubMed Central

    Bardaji, Raul; Sánchez, Albert-Miquel; Simon, Carine; Wernand, Marcel R.; Piera, Jaume

    2016-01-01

    A critical parameter to assess the environmental status of water bodies is the transparency of the water, as it is strongly affected by different water quality related components (such as the presence of phytoplankton, organic matter and sediment concentrations). One parameter to assess the water transparency is the diffuse attenuation coefficient. However, the number of subsurface irradiance measurements obtained with conventional instrumentation is relatively low, due to instrument costs and the logistic requirements to provide regular and autonomous observations. In recent years, the citizen science concept has increased the number of environmental observations, both in time and space. The recent technological advances in embedded systems and sensors also enable volunteers (citizens) to create their own devices (known as Do-It-Yourself or DIY technologies). In this paper, a DIY instrument to measure irradiance at different depths and automatically calculate the diffuse attenuation Kd coefficient is presented. The instrument, named KdUINO, is based on an encapsulated low-cost photonic sensor and Arduino (an open-hardware platform for the data acquisition). The whole instrument has been successfully operated and the data validated comparing the KdUINO measurements with the commercial instruments. Workshops have been organized with high school students to validate its feasibility. PMID:26999132

  8. Estimating the Underwater Diffuse Attenuation Coefficient with a Low-Cost Instrument: The KdUINO DIY Buoy.

    PubMed

    Bardaji, Raul; Sánchez, Albert-Miquel; Simon, Carine; Wernand, Marcel R; Piera, Jaume

    2016-01-01

    A critical parameter to assess the environmental status of water bodies is the transparency of the water, as it is strongly affected by different water quality related components (such as the presence of phytoplankton, organic matter and sediment concentrations). One parameter to assess the water transparency is the diffuse attenuation coefficient. However, the number of subsurface irradiance measurements obtained with conventional instrumentation is relatively low, due to instrument costs and the logistic requirements to provide regular and autonomous observations. In recent years, the citizen science concept has increased the number of environmental observations, both in time and space. The recent technological advances in embedded systems and sensors also enable volunteers (citizens) to create their own devices (known as Do-It-Yourself or DIY technologies). In this paper, a DIY instrument to measure irradiance at different depths and automatically calculate the diffuse attenuation Kd coefficient is presented. The instrument, named KdUINO, is based on an encapsulated low-cost photonic sensor and Arduino (an open-hardware platform for the data acquisition). The whole instrument has been successfully operated and the data validated comparing the KdUINO measurements with the commercial instruments. Workshops have been organized with high school students to validate its feasibility. PMID:26999132

  9. 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.

  10. 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.

  11. 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-}.

  12. The SOCS box encodes a hierarchy of affinities for Cullin5: implications for ubiquitin ligase formation and cytokine signalling suppression

    PubMed Central

    Babon, Jeffrey J.; Sabo, Jennifer K.; Zhang, Jian-Guo; Nicola, Nicos A.; Norton, Raymond S.

    2009-01-01

    Summary The SOCS (Suppressors of Cytokine Signalling) family of proteins inhibit the cytokine induced signalling cascade, in part by promoting the ubiquitination of signalling intermediates that are then targeted for proteasomal degradation. This activity relies upon an interaction between the SOCS box domain, the adapter complex elonginBC and a member of the Cullin family, the scaffold protein of an E3 ubiquitin ligase. Here we dissect this interaction, in vitro, using purified components. We show that all eight SOCS proteins bound Cullin5, but required prior recruitment of elonginBC. Neither SOCS nor elonginBC bound Cullin5 when in isolation. Interestingly, the affinity of each SOCS/elonginBC complex for Cullin5 varied by two orders of magnitude across the SOCS family. Unexpectedly, the most potent suppressors of signalling, SOCS-1 and SOCS-3, bound most weakly to the E3 ligase scaffold, with affinities 100- and 10-fold lower, respectively, than than the rest of the family. The remaining six SOCS proteins all bound Cullin5 with high affinity (KD ∼ 10 nM), due to a slower off-rate, and hence a longer half-life of the complex. This difference in affinity may reflect a difference in mode-of-action, as only SOCS-1 and -3 have been shown to suppress signalling using both SOCS box dependent and independent mechanisms. This is not the case with the other six SOCS proteins and our data imply the existence of two distinct sub-classes of SOCS proteins, with a high affinity for Cullin5, the E3 ligase scaffold, possibly reflecting complete dependence upon ubiquitination for suppression of cytokine signalling. PMID:19385048

  13. Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

    SciTech Connect

    Sidhu, A.

    1988-11-29

    The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist /sup 125/I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SKandF R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.

  14. When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

    NASA Astrophysics Data System (ADS)

    Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

    2012-11-01

    Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

  15. Screening of high-affinity scFvs from a ribosome displayed library using BIAcore biosensor.

    PubMed

    Yuan, Qing; Wang, Zhongkang; Nian, Siji; Yin, Youping; Chen, Gang; Xia, Yuxian

    2009-02-01

    An experimental protocol was developed to screen high-affinity single-chain Fv antibody fragments (scFvs) from a Xanthomonas axonopodis pv. citri (Xac) immunized ribosome display library using BIAcore biosensor. The screening methods involved immobilizing antigen [lipopolysaccharides (LPS) of Xac] on sensor chip HPA and then unpurified expression products of scFvs flowing over the immobilized sensor chip. The affinity-improved scFvs were selected based on dissociation rate constants (k (d)). Thirty-five enzyme-linked immunosorbent assay-positive scFvs were analyzed by BIAcore, and three of those (scFv A1, B2, and C5) with lower k (d) were screened. To demonstrate the accuracy of the screening method, the three scFvs were expressed in Escherichia coli HB2151 and purified. The purified scFvs were subsequently further identified according to association rate and affinity constants. The results showed that the three scFvs (A1, B2, and C5) had high affinity for LPS of Xac (3.51 x 10(-11), 1.13 x 10(-10), 5.06 x 10(-10) M, respectively). Furthermore, the scFv B2 was highly specific for LPS of Xac and had no any cross-reactions with bovine serum albumin and LPS from Xac-related bacteria. This provided evidence that the information from the BIAcore screening assay could be accurate. PMID:18574567

  16. Development of p-benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) derivatives and affinity photolabeling of the bovine NPR-A receptor.

    PubMed

    Coupal, M; De Léan, A; McNicoll, N; Fournier, A

    1999-04-29

    Two Nalpha-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd = 20 pM) with Kd of 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2 or Leu18 can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling. PMID:10222239

  17. Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.

    PubMed

    Slutzki, Michal; Barak, Yoav; Reshef, Dan; Schueler-Furman, Ora; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions. PMID:22608739

  18. Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

    PubMed

    Henry, Pauline C; Kanelis, Voula; O'Brien, M Christine; Kim, Brian; Gautschi, Ivan; Forman-Kay, Julie; Schild, Laurent; Rotin, Daniela

    2003-05-30

    The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression

  19. Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca2+ Binding Affinity

    PubMed Central

    Mazumder, Mohit; Padhan, Narendra; Bhattacharya, Alok; Gourinath, Samudrala

    2014-01-01

    The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca2+ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca2+-binding loops and non Ca2+-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca2+-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca2+-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca2+-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca2+-binding affinity constants of EF-hand proteins. The web server is freely available at http://202.41.10.46/calb/index.html. PMID:24760183

  20. Substituted pentacyclic carbazolones as novel muscarinic allosteric agents: synthesis and structure-affinity and cooperativity relationships.

    PubMed

    Gharagozloo, Parviz; Lazareno, Sebastian; Miyauchi, Masao; Popham, Angela; Birdsall, Nigel J M

    2002-03-14

    Two series of pentacyclic carbazolones, 22 and 23, have been synthesized utilizing a facile intramolecular Dielsminus signAlder reaction and are allosteric modulators at muscarinic acetylcholine receptors. Their affinities and cooperativities with acetylcholine and the antagonist N-methylscopolamine (NMS) at M(1)minus signM(4) receptors have been analyzed and compared. All of the synthesized compounds are negatively cooperative with acetylcholine. In contrast, the majority of the compounds exhibit positive cooperativity with NMS, particularly at M(2) and M(4) receptors. The subtype selectivity, in terms of affinity, was in general M(2) > M(1) > M(4) > M(3). The largest increases in affinity produced by a single substitution of the core structure were given by the 1-OMe (22b) and 1-Cl (22d) derivatives. The position of the N in the ring did not appear to be important for binding affinity or cooperativity. Two compounds 22y and 23i, both trisubstituted analogues, were the most potent compounds synthesized, with dissociation constants of 30minus sign100 nM for the M(2) NMS-liganded and unliganded receptor, respectively. The results indicate that the allosteric site, like the primary binding site, is capable of high-affinity interactions with molecules of relatively low molecular weight. PMID:11881995

  1. Modulating the affinity and the selectivity of engineered calmodulin EF-Hand peptides for lanthanides.

    PubMed

    Clainche, Loïc Le; Figuet, Mélanie; Montjardet-Bas, Véronique; Blanchard, Sébastien; Vita, Claudio

    2006-09-01

    A set of engineered peptides (33 amino acids long) corresponding to the helix-turn-helix (EF-Hand) motif of the metal-binding site I of the protein calmodulin from paramecium tetraurelia have been synthesized. A disulfide bridge has been introduced in the native sequence in order to stabilize a native-like conformation. The calcium-binding carboxylate residues in positions 20, 22, 24, and 31 were mutated into other amino acids and the influence of such mutations on the binding affinity of the peptides for calcium and lanthanides have been studied. It was shown that the binding affinity for terbium ions can be modulated with dissociation constants ranging from 40 nmolar to 40 mmolar. The study of the influence of the mutations on the terbium affinity showed that the residue in position 24 played a key role on the capability of the peptides to bind lanthanides and that the affinity could be enhanced by mutations on non-coordinating positions. Such peptides with high affinity for lanthanides may facilitate the development of new highly sensitive biosensors to monitor the metal pollution in the environment. PMID:16637059

  2. Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins.

    PubMed Central

    Srisawat, C; Engelke, D R

    2001-01-01

    RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs. PMID:11345441

  3. The expression of the 27-kd heat shock protein in keratinization disorders: an immunohistological study.

    PubMed

    Jonak, Constanze; Metze, Dieter; Traupe, Heiko; Happle, Rudolf; König, Arne; Trautinger, Franz

    2005-06-01

    In human skin, the 27-kd heat shock protein (hsp27), a member of the small hsp family, is expressed mainly in the upper epidermal layers. Hsp27 functions as a molecular chaperone and is involved in the regulation of cell growth and differentiation. According to experimental evidence, epidermal hsp27, through its chaperone function, might play a role in the assembly of keratin filaments and the cornified cell envelope. This study was conducted to assess the expression pattern of hsp27 in a panel of different ichthyoses. Twelve hereditary and acquired skin diseases associated with an ichthyotic phenotype and 2 corresponding mouse models were investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue, using a monoclonal antibody specific for hsp27. In ichthyosis vulgaris, lamellar ichthyosis, Sjögren-Larsson syndrome, Netherton syndrome, and acquired ichthyosiform skin condition, the pattern of hsp27 expression resembles healthy human skin. Hsp27 expression was reduced in bullous ichthyosiform erythroderma and annular epidermolytic ichthyosis, and absent in X-linked recessive ichthyosis (1/3 patients) and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome (1/1). In X-linked dominant chondrodysplasia, 3 small samples are completely negative and 2 larger samples show a pattern resembling random X inactivation. In the mouse models, tattered and bare patches, representing the murine analogues to X-linked dominant chondrodysplasia and congenital hemidysplasia with ichthyosiform nevus and limb defects syndrome, expression of hsp25 (the murine homologue of hsp27) also showed lyonization, demonstrating a clear-cut link between hsp27 expression and underlying molecular pathology. Our results show that loss of hsp27 is a rare event in human epidermis that is associated with specific genetic defects. Among the cases described here, these defects are either in suprabasal keratins or in enzymes involved in cholesterol biosynthesis

  4. DISTRIBUTION COEFICIENTS (KD) GENERATED FROM A CORE SAMPLE COLLECTED FROM THE SALTSTONE DISPOSAL FACILITY

    SciTech Connect

    Almond, P.; Kaplan, D.

    2011-04-25

    Core samples originating from Vault 4, Cell E of the Saltstone Disposal Facility (SDF) were collected in September of 2008 (Hansen and Crawford 2009, Smith 2008) and sent to SRNL to measure chemical and physical properties of the material including visual uniformity, mineralogy, microstructure, density, porosity, distribution coefficients (K{sub d}), and chemical composition. Some data from these experiments have been reported (Cozzi and Duncan 2010). In this study, leaching experiments were conducted with a single core sample under conditions that are representative of saltstone performance. In separate experiments, reducing and oxidizing environments were targeted to obtain solubility and Kd values from the measurable species identified in the solid and aqueous leachate. This study was designed to provide insight into how readily species immobilized in saltstone will leach from the saltstone under oxidizing conditions simulating the edge of a saltstone monolith and under reducing conditions, targeting conditions within the saltstone monolith. Core samples were taken from saltstone poured in December of 2007 giving a cure time of nine months in the cell and a total of thirty months before leaching experiments began in June 2010. The saltstone from Vault 4, Cell E is comprised of blast furnace slag, class F fly ash, portland cement, and Deliquification, Dissolution, and Adjustment (DDA) Batch 2 salt solution. The salt solution was previously analyzed from a sample of Tank 50 salt solution and characterized in the 4QCY07 Waste Acceptance Criteria (WAC) report (Zeigler and Bibler 2009). Subsequent to Tank 50 analysis, additional solution was added to the tank solution from the Effluent Treatment Project as well as from inleakage from Tank 50 pump bearings (Cozzi and Duncan 2010). Core samples were taken from three locations and at three depths at each location using a two-inch diameter concrete coring bit (1-1, 1-2, 1-3; 2-1, 2-2, 2-3; 3-1, 3-2, 3-3) (Hansen and

  5. Effective cosmological constant induced by stochastic fluctuations of Newton's constant

    NASA Astrophysics Data System (ADS)

    de Cesare, Marco; Lizzi, Fedele; Sakellariadou, Mairi

    2016-09-01

    We consider implications of the microscopic dynamics of spacetime for the evolution of cosmological models. We argue that quantum geometry effects may lead to stochastic fluctuations of the gravitational constant, which is thus considered as a macroscopic effective dynamical quantity. Consistency with Riemannian geometry entails the presence of a time-dependent dark energy term in the modified field equations, which can be expressed in terms of the dynamical gravitational constant. We suggest that the late-time accelerated expansion of the Universe may be ascribed to quantum fluctuations in the geometry of spacetime rather than the vacuum energy from the matter sector.

  6. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.

    PubMed

    Huang, Renhua; Gorman, Kevin T; Vinci, Chris R; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  7. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    PubMed Central

    Huang, Renhua; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K.

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  8. High affinity of lead for fetal haemoglobin.

    PubMed Central

    Ong, C N; Lee, W R

    1980-01-01

    In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis. PMID:6158989

  9. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  10. 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…

  11. Optical constants of solid methane

    NASA Technical Reports Server (NTRS)

    Khare, Bishun N.; Thompson, W. R.; Sagan, C.; Arakawa, E. T.; Bruel, C.; Judish, J. P.; Khanna, R. K.; Pollack, J. B.

    1989-01-01

    Methane is the most abundant simple organic molecule in the outer solar system bodies. In addition to being a gaseous constituent of the atmospheres of the Jovian planets and Titan, it is present in the solid form as a constituent of icy surfaces such as those of Triton and Pluto, and as cloud condensate in the atmospheres of Titan, Uranus, and Neptune. It is expected in the liquid form as a constituent of the ocean of Titan. Cometary ices also contain solid methane. The optical constants for both solid and liquid phases of CH4 for a wide temperature range are needed for radiative transfer calculations, for studies of reflection from surfaces, and for modeling of emission in the far infrared and microwave regions. The astronomically important visual to near infrared measurements of solid methane optical constants are conspicuously absent from the literature. Preliminary results are presented of the optical constants of solid methane for the 0.4 to 2.6 micron region. K is reported for both the amorphous and the crystalline (annealed) states. Using the previously measured values of the real part of the refractive index, n, of liquid methane at 110 K n is computed for solid methane using the Lorentz-Lorentz relationship. Work is in progress to extend the measurements of optical constants n and k for liquid and solid to both shorter and longer wavelengths, eventually providing a complete optical constants database for condensed CH4.

  12. How fundamental are fundamental constants?

    NASA Astrophysics Data System (ADS)

    Duff, M. J.

    2015-01-01

    I argue that the laws of physics should be independent of one's choice of units or measuring apparatus. This is the case if they are framed in terms of dimensionless numbers such as the fine structure constant, ?. For example, the standard model of particle physics has 19 such dimensionless parameters whose values all observers can agree on, irrespective of what clock, rulers or scales? they use to measure them. Dimensional constants, on the other hand, such as ?, c, G, e and k ?, are merely human constructs whose number and values differ from one choice of units to the next. In this sense, only dimensionless constants are 'fundamental'. Similarly, the possible time variation of dimensionless fundamental 'constants' of nature is operationally well defined and a legitimate subject of physical enquiry. By contrast, the time variation of dimensional constants such as ? or ? on which a good many (in my opinion, confusing) papers have been written, is a unit-dependent phenomenon on which different observers might disagree depending on their apparatus. All these confusions disappear if one asks only unit-independent questions. We provide a selection of opposing opinions in the literature and respond accordingly.

  13. Microcantilever-Based Label-Free Characterization of Temperature-Dependent Biomolecular Affinity Binding

    PubMed Central

    Wang, Bin; Huang, Fengliang; Nguyen, ThaiHuu; Xu, Yong; Lin, Qiao

    2014-01-01

    This paper presents label-free characterization of temperature-dependent biomolecular affinity binding on solid surfaces using a microcantilever-based device. The device consists of a Parylene cantilever one side of which is coated with a gold film and functionalized with molecules as an affinity receptor to a target analyte. The cantilever is located in a poly(dimethylsiloxane) (PDMS) microfluidic chamber that is integrated with a transparent indium tin oxide (ITO) resistive temperature sensor on the underlying substrate. The ITO sensor allows for real-time measurements of the chamber temperature, as well as unobstructed optical access for reflection-based optical detection of the cantilever deflection. To test the temperature-dependent binding between the target and receptor, the temperature of the chamber is maintained at a constant setpoint, while a solution of unlabeled analyte molecules is continuously infused through the chamber. The measured cantilever deflection is used to determine the target-receptor binding characteristics. We demonstrate label-free characterization of temperature-dependent binding kinetics of the platelet-derived growth factor (PDGF) protein with an aptamer receptor. Affinity binding properties including the association and dissociation rate constants as well as equilibrium dissociation constant are obtained, and shown to exhibit significant dependencies on temperature. PMID:24723743

  14. Predicting Adsorption Affinities of Small Molecules on Carbon Nanotubes Using Molecular Dynamics Simulation.

    PubMed

    Comer, Jeffrey; Chen, Ran; Poblete, Horacio; Vergara-Jaque, Ariela; Riviere, Jim E

    2015-12-22

    Computational techniques have the potential to accelerate the design and optimization of nanomaterials for applications such as drug delivery and contaminant removal; however, the success of such techniques requires reliable models of nanomaterial surfaces as well as accurate descriptions of their interactions with relevant solutes. In the present work, we evaluate the ability of selected models of naked and hydroxylated carbon nanotubes to predict adsorption equilibrium constants for about 30 small aromatic compounds with a variety of functional groups. The equilibrium constants determined using molecular dynamics coupled with free-energy calculation techniques are directly compared to those derived from experimental measurements. The calculations are highly predictive of the relative adsorption affinities of the compounds, with excellent correlation (r ≥ 0.9) between calculated and measured values of the logarithm of the adsorption equilibrium constant. Moreover, the agreement in absolute terms is also reasonable, with average errors of less than one decade. We also explore possible effects of surface loading, although we demonstrate that they are negligible for the experimental conditions considered. Given the degree of reliability demonstrated, we move on to employing the in silico techniques in the design of nanomaterials, using the optimization of adsorption affinity for the herbacide atrazine as an example. Our simulations suggest that, compared to other modifications of graphenic carbon, polyvinylpyrrolidone conjugation gives the highest affinity for atrazine-substantially greater than that of graphenic carbon alone-and may be useful as a nanomaterial for delivery or sequestration of atrazine. PMID:26506132

  15. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  16. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    PubMed

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments. PMID:9807828

  17. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor.

    PubMed

    Palmer, Wylie S; Poncet-Montange, Guillaume; Liu, Gang; Petrocchi, Alessia; Reyna, Naphtali; Subramanian, Govindan; Theroff, Jay; Yau, Anne; Kost-Alimova, Maria; Bardenhagen, Jennifer P; Leo, Elisabetta; Shepard, Hannah E; Tieu, Trang N; Shi, Xi; Zhan, Yanai; Zhao, Shuping; Barton, Michelle C; Draetta, Giulio; Toniatti, Carlo; Jones, Philip; Geck Do, Mary; Andersen, Jannik N

    2016-02-25

    The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo. PMID:26061247

  18. Optical constants of solid methane

    NASA Technical Reports Server (NTRS)

    Khare, Bishun N.; Thompson, W. R.; Sagan, C.; Arakawa, E. T.; Bruel, C.; Judish, J. P.; Khanna, R. K.; Pollack, J. B.

    1990-01-01

    Methane is the most abundant simple organic molecule in the outer solar system bodies. In addition to being a gaseous constituent of the atmospheres of the Jovian planets and Titan, it is present in the solid form as a constituent of icy surfaces such as those of Triton and Pluto, and as cloud condensate in the atmospheres of Titan, Uranus, and Neptune. It is expected in the liquid form as a constituent of the ocean of Titan. Cometary ices also contain solid methane. The optical constants for both solid and liquid phases of CH4 for a wide temperature range are needed for radiative transfer calculations, for studies of reflection from surfaces, and for modeling of emission in the far infrared and microwave regions. The astronomically important visual to near infrared measurements of solid methane optical constants are conspicuously absent from the literature. Preliminary results are presented on the optical constants of solid methane for the 0.4 to 2.6 micrometer region. Deposition onto a substrate at 10 K produces glassy (semi-amorphous) material. Annealing this material at approximately 33 K for approximately 1 hour results in a crystalline material as seen by sharper, more structured bands and negligible background extinction due to scattering. The constant k is reported for both the amorphous and the crystalline (annealed) states. Typical values (at absorption maxima) are in the .001 to .0001 range. Below lambda = 1.1 micrometers the bands are too weak to be detected by transmission through the films less than or equal to 215 micrometers in thickness, employed in the studies to date. Using previously measured values of the real part of the refractive index, n, of liquid methane at 110 K, n is computed for solid methane using the Lorentz-Lorenz relationship. Work is in progress to extend the measurements of optical constants n and k for liquid and solid to both shorter and longer wavelengths, eventually providing a complete optical constants database for

  19. Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Slamnoiu, Stefan; Vlad, Camelia; Stumbaum, Mihaela; Moise, Adrian; Lindner, Kathrin; Engel, Nicole; Vilanova, Mar; Diaz, Mireia; Karreman, Christiaan; Leist, Marcel; Ciossek, Thomas; Hengerer, Bastian; Vilaseca, Marta; Przybylski, Michael

    2014-08-01

    Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS- acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (KD) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

  20. The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain.

    PubMed

    de Paulis, Tomas

    2003-01-01

    , Berkeley, and Nashville. Evaluation of seven [(125)I]iodine substituted analogs of raclopride, including IBZM, revealed the unusual high striatum-to-cerebellum ratio of 234 of [(125)I]epidepride in the rat. Subsequent SPECT images with [(123)I]epidepride demonstrated its ability to identify extrastriatal dopamine D(2) receptors in the human brain. Exploration of the structure of epidepride confirmed its exceptional properties, to be exceeded only by its N-allyl homolog, [(125)I]nalepride. The design by others of a series of potent 5-(3-[(18)F]fluoropropyl) substituted analogs of epidepride for PET imaging, lead to the discovery of [(18)F]fallypride. By elucidating the role of lipophilicity in the substituted benzamides, the excellent imaging characteristics of [(11)C]/[(123)I]epidepride, [(11)C]/[(76)Br]isoremoxipride and [(18)F]fallypride, could not only be explained but predicted with remarkable accuracy. By using the inverse product of the receptor affinity (K(D)), and the apparent partition constant of the radioligand (P((7.4))), estimates of maximal binding potential of any radioligand for imaging of any neurotransmitter receptor or transporter site seem possible. PMID:12570798

  1. Cosmologies with variable gravitational constant

    NASA Astrophysics Data System (ADS)

    Narlikar, J. V.

    1983-03-01

    In 1937 Dirac presented an argument, based on the socalled large dimensionless numbers, which led him to the conclusion that the Newtonian gravitational constant G changes with epoch. Towards the end of the last century Ernst Mach had given plausible arguments to link the property of inertia of matter to the large scale structure of the universe. Mach's principle also leads to cosmological models with a variable gravitational constant. Three cosmologies which predict a variable G are discussed in this paper both from theoretical and observational points of view.

  2. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  3. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

    PubMed

    Orlowski, S; Champeil, P

    1991-01-15

    We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium

  4. The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation.

    PubMed Central

    Trefzer, U; Brockhaus, M; Lötscher, H; Parlow, F; Budnik, A; Grewe, M; Christoph, H; Kapp, A; Schöpf, E; Luger, T A

    1993-01-01

    In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha. Images PMID:8392091

  5. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

    PubMed Central

    He, Xiao; Garza, Denisse; Nigam, Sanjay K.; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M-1s-1 and an even slower dissociation rate (kd) in the range of 10−4 s-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not

  6. High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead

    SciTech Connect

    Mistry, P.; Lucier, G.W.; Fowler, B.A.

    1985-02-01

    The PbII binding characteristics of the previously reported PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of /sup 203/Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two /sup 203/Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of /sup 203/Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound /sup 203/Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of /sup 203/Pb by PbII, CdII and ZnII ions but not CaII ions.

  7. Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

    PubMed

    Peters, Daniel T; Fung, Herman K H; Levdikov, Vladimir M; Irmscher, Tobias; Warrander, Fiona C; Greive, Sandra J; Kovalevskiy, Oleg; Isaacs, Harry V; Coles, Mark; Antson, Alfred A

    2016-09-13

    Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions. PMID:27559824

  8. Equivalence of the Open KdV and the Open Virasoro Equations for the Moduli Space of Riemann Surfaces with Boundary

    NASA Astrophysics Data System (ADS)

    Buryak, Alexandr

    2015-10-01

    In a recent paper, Pandharipande, Solomon and Tessler initiated a study of the intersection theory on the moduli space of Riemann surfaces with boundary. The authors conjectured KdV and Virasoro type equations that completely determine all intersection numbers. In this paper, we study these equations in detail. In particular, we prove that the KdV and the Virasoro type equations for the intersection numbers on the moduli space of Riemann surfaces with boundary are equivalent.

  9. Constant-amplitude RC oscillator

    NASA Technical Reports Server (NTRS)

    Kerwin, W. J.; Westbrook, R. M.

    1970-01-01

    Sinusoidal oscillator has a frequency determined by resistance-capacitance /RC/ values of two charge control devices and a constant-amplitude voltage independent of frequency and RC values. RC elements provide either voltage-control, resistance-control, or capacitance-control of the frequency.

  10. Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate

    PubMed Central

    Olivieri, Lilian; Gardebien, Fabrice

    2014-01-01

    With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant of this intermediate should be decreased, resulting in an increase of the affinity constant . The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state. PMID:25502559

  11. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    PubMed

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  12. The 1% concordance Hubble constant

    SciTech Connect

    Bennett, C. L.; Larson, D.; Weiland, J. L.; Hinshaw, G.

    2014-10-20

    The determination of the Hubble constant has been a central goal in observational astrophysics for nearly a hundred years. Extraordinary progress has occurred in recent years on two fronts: the cosmic distance ladder measurements at low redshift and cosmic microwave background (CMB) measurements at high redshift. The CMB is used to predict the current expansion rate through a best-fit cosmological model. Complementary progress has been made with baryon acoustic oscillation (BAO) measurements at relatively low redshifts. While BAO data do not independently determine a Hubble constant, they are important for constraints on possible solutions and checks on cosmic consistency. A precise determination of the Hubble constant is of great value, but it is more important to compare the high and low redshift measurements to test our cosmological model. Significant tension would suggest either uncertainties not accounted for in the experimental estimates or the discovery of new physics beyond the standard model of cosmology. In this paper we examine in detail the tension between the CMB, BAO, and cosmic distance ladder data sets. We find that these measurements are consistent within reasonable statistical expectations and we combine them to determine a best-fit Hubble constant of 69.6 ± 0.7 km s{sup –1} Mpc{sup –1}. This value is based upon WMAP9+SPT+ACT+6dFGS+BOSS/DR11+H {sub 0}/Riess; we explore alternate data combinations in the text. The combined data constrain the Hubble constant to 1%, with no compelling evidence for new physics.

  13. Optical constants of solid methane

    SciTech Connect

    Khare, B.N.; Thompson, W.R.; Sagan, C. . Lab. for Planetary Studies); Arakawa, E.T.; Bruel, C.; Judish, J.P. ); Khanna, R.K. . Dept. of Chemistry and Biochemistry); Pollack, J.B. . Ames Research Center)

    1989-01-01

    Methane is the most abundant simple organic molecule in the outer solar system bodies. In addition to being a gaseous constituent of the atmospheres of the Jovian planets and Titan, it is present in the solid form as a constituent of icy surfaces such as those of Triton and Pluto, and as cloud condensate in the atmospheres of Titan, Uranus, and Neptune. It is expected in the liquid form as a constituent of the ocean of Titan. Cometary ices also contain solid methane. The optical constants for both solid and liquid phases of CH{sub 4} for a wide temperature range are needed for radiative transfer calculations, for studies of reflection from surfaces, and for modeling of emission in the far infrared and microwave regions. The astronomically important visual to near infrared measurements of solid methane optical constants are conspicuously absent from the literature. We present preliminary results of the optical constants of solid methane for the 0.4 {mu}m to 2.6 {mu}m region. We report k for both the amorphous and the crystalline (annealed) states. Using our previously measured values of the real part of the refractive index, n, of liquid methane at 110{degree}K (Bull. Am. Phys. Soc.31, 700 (1986)) we compute n for solid methane using the Lorentz-Lorentz relationship. Work is in progress to extend the measurements of optical constants n and k for liquid and solid to both shorter and longer wavelengths, eventually providing a complete optical constants database for condensed CH{sub 4}. 33 refs., 6 figs., 2 tabs.

  14. Modal affinities of endplate acetylcholine receptors caused by loop C mutations

    PubMed Central

    Vij, Ridhima; Purohit, Prasad

    2015-01-01

    The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity. PMID:26503719

  15. Rogue waves in electronegative space plasmas: The link between the family of the KdV equations and the nonlinear Schrödinger equation

    NASA Astrophysics Data System (ADS)

    El-Tantawy, S. A.

    2016-05-01

    We examine the likelihood of the ion-acoustic rogue waves propagation in a non-Maxwellian electronegative plasma in the framework of the family of the Korteweg-de Vries (KdV) equations (KdV/modified KdV/Extended KdV equation). For this purpose, we use the reductive perturbation technique to carry out this study. It is known that the family of the KdV equations have solutions of distinct structures such as solitons, shocks, kinks, cnoidal waves, etc. However, the dynamics of the nonlinear rogue waves is governed by the nonlinear Schrödinger equation (NLSE). Thus, the family of the KdV equations is transformed to their corresponding NLSE developing a weakly nonlinear wave packets. We show the possible region for the existence of the rogue waves and define it precisely for typical parameters of space plasmas. We investigate numerically the effects of relevant physical parameters, namely, the negative ion relative concentration, the nonthermal parameter, and the mass ratio on the propagation of the rogue waves profile. The present study should be helpful in understanding the salient features of the nonlinear structures such as, ion-acoustic solitary waves, shock waves, and rogue waves in space and in laboratory plasma where two distinct groups of ions, i.e. positive and negative ions, and non-Maxwellian (nonthermal) electrons are present.

  16. New Quasar Studies Keep Fundamental Physical Constant Constant

    NASA Astrophysics Data System (ADS)

    2004-03-01

    Very Large Telescope sets stringent limit on possible variation of the fine-structure constant over cosmological time Summary Detecting or constraining the possible time variations of fundamental physical constants is an important step toward a complete understanding of basic physics and hence the world in which we live. A step in which astrophysics proves most useful. Previous astronomical measurements of the fine structure constant - the dimensionless number that determines the strength of interactions between charged particles and electromagnetic fields - suggested that this particular constant is increasing very slightly with time. If confirmed, this would have very profound implications for our understanding of fundamental physics. New studies, conducted using the UVES spectrograph on Kueyen, one of the 8.2-m telescopes of ESO's Very Large Telescope array at Paranal (Chile), secured new data with unprecedented quality. These data, combined with a very careful analysis, have provided the strongest astronomical constraints to date on the possible variation of the fine structure constant. They show that, contrary to previous claims, no evidence exist for assuming a time variation of this fundamental constant. PR Photo 07/04: Relative Changes with Redshift of the Fine Structure Constant (VLT/UVES) A fine constant To explain the Universe and to represent it mathematically, scientists rely on so-called fundamental constants or fixed numbers. The fundamental laws of physics, as we presently understand them, depend on about 25 such constants. Well-known examples are the gravitational constant, which defines the strength of the force acting between two bodies, such as the Earth and the Moon, and the speed of light. One of these constants is the so-called "fine structure constant", alpha = 1/137.03599958, a combination of electrical charge of the electron, the Planck constant and the speed of light. The fine structure constant describes how electromagnetic forces hold

  17. Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.

    PubMed Central

    Breiteneder-Geleff, S.; Matsui, K.; Soleiman, A.; Meraner, P.; Poczewski, H.; Kalt, R.; Schaffner, G.; Kerjaschki, D.

    1997-01-01

    Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 10 Figure 12 Figure 13 Figure 14 Figure 15 PMID:9327748

  18. 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.

  19. Improved native affinity purification of RNA.

    PubMed

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  20. Protein affinity map of chemical space.

    PubMed

    Kauvar, L M; Villar, H O; Sportsman, J R; Higgins, D L; Schmidt, D E

    1998-09-11

    Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification. PMID:9792501