Affinity learning with diffusion on tensor product graph.

PubMed

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

2013-01-01

In many applications, we are given a finite set of data points sampled from a data manifold and represented as a graph with edge weights determined by pairwise similarities of the samples. Often the pairwise similarities (which are also called affinities) are unreliable due to noise or due to intrinsic difficulties in estimating similarity values of the samples. As observed in several recent approaches, more reliable similarities can be obtained if the original similarities are diffused in the context of other data points, where the context of each point is a set of points most similar to it. Compared to the existing methods, our approach differs in two main aspects. First, instead of diffusing the similarity information on the original graph, we propose to utilize the tensor product graph (TPG) obtained by the tensor product of the original graph with itself. Since TPG takes into account higher order information, it is not a surprise that we obtain more reliable similarities. However, it comes at the price of higher order computational complexity and storage requirement. The key contribution of the proposed approach is that the information propagation on TPG can be computed with the same computational complexity and the same amount of storage as the propagation on the original graph. We prove that a graph diffusion process on TPG is equivalent to a novel iterative algorithm on the original graph, which is guaranteed to converge. After its convergence we obtain new edge weights that can be interpreted as new, learned affinities. We stress that the affinities are learned in an unsupervised setting. We illustrate the benefits of the proposed approach for data manifolds composed of shapes, images, and image patches on two very different tasks of image retrieval and image segmentation. With learned affinities, we achieve the bull's eye retrieval score of 99.99 percent on the MPEG-7 shape dataset, which is much higher than the state-of-the-art algorithms. When the data- points are image patches, the NCut with the learned affinities not only significantly outperforms the NCut with the original affinities, but it also outperforms state-of-the-art image segmentation methods.

Is There Consistency between the Binding Affinity and Inhibitory Potential of Natural Polyphenols as α-amylase Inhibitors?

PubMed

Xu, Wei; Shao, Rong; Xiao, Jianbo

2016-07-26

The inhibitory potential of natural polyphenols for α-amylases has attracted great interests among researchers. The structure-affinity properties of natural polyphenols binding to α-amylase and the structure-activity relationship of dietary polyphenols inhibiting α-amylase were deeply investigated. There is a lack of consistency between the structure-affinity relationship and the structure-activity relationship of natural polyphenols as α-amylase inhibitors. Is it consistent between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors? It was found that the consistency between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors is not equivocal. For example, there is no consistency between the binding affinity and the inhibitory potential of quercetin and its glycosides as α-amylase inhibitors. However, catechins with higher α-amylase inhibitory potential exhibited higher affinity with α-amylase.

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Reversibly extracellular pH controlled cellular uptake and photothermal therapy by PEGylated mixed-charge gold nanostars.

PubMed

Wang, Shouju; Teng, Zhaogang; Huang, Peng; Liu, Dingbin; Liu, Ying; Tian, Ying; Sun, Jing; Li, Yanjun; Ju, Huangxian; Chen, Xiaoyuan; Lu, Guangming

2015-04-17

Shielding nanoparticles from nonspecific interactions with normal cells/tissues before they reach and after they leave tumors is crucial for the selective delivery of NPs into tumor cells. By utilizing the reversible protonation of weak electrolytic groups to pH changes, long-chain amine/carboxyl-terminated polyethylene glycol (PEG) decorated gold nanostars (GNSs) are designed, exhibiting reversible, significant, and sensitive response in cell affinity and therapeutic efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors. This smart nanosystem shows good dispersity and unimpaired photothermal efficacy in complex bioenvironment at pH 6.4 and 7.4 even when their surface charge is neutral. One PEGylated mixed-charge GNSs with certain surface composition, GNS-N/C 4, exhibits high cell affinity and therapeutic efficacy at pH 6.4, and low affinity and almost "zero" damage to cells at pH 7.4. Remarkably, this significant and sensitive response in cell affinity and therapeutic efficacy is reversible as local pH alternated. In vivo, GNS-N/C 4 shows higher accumulation in tumors and improved photothermal therapeutic efficacy than pH-insensitive GNSs. This newly developed smart nanosystem, whose cell affinity reversibly transforms in response to pHe gradient with unimpaired biostability, provides a novel effective means of tumor-selective therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of molecularly imprinted polymers to selective removal of clofibric acid from water.

PubMed

Dai, Chaomeng; Zhang, Juan; Zhang, Yalei; Zhou, Xuefei; Liu, Shuguang

2013-01-01

A new molecularly imprinted polymer (MIP) adsorbent for clofibric acid (CA) was prepared by a non-covalent protocol. Characterization of the obtained MIP was achieved by scanning electron microscopy (SEM) and nitrogen sorption. Sorption experimental results showed that the MIP had excellent binding affinity for CA and the adsorption of CA by MIP was well described by pseudo-second-order model. Scatchard plot analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 7.52 ± 0.46 mg L(-1) and 114 ± 4.2 mg L(-1), respectively. The selectivity of MIP demonstrated higher affinity for CA over competitive compound than that of non-imprinted polymers (NIP). The MIP synthesized was used to remove CA from spiked surface water and exhibited significant binding affinity towards CA in the presence of total dissolved solids (TDS). In addition, MIP reusability was demonstrated for at least 12 repeated cycles without significant loss in performance.

Application of Molecularly Imprinted Polymers to Selective Removal of Clofibric Acid from Water

PubMed Central

Dai, Chaomeng; Zhang, Juan; Zhang, Yalei; Zhou, Xuefei; Liu, Shuguang

2013-01-01

A new molecularly imprinted polymer (MIP) adsorbent for clofibric acid (CA) was prepared by a non-covalent protocol. Characterization of the obtained MIP was achieved by scanning electron microscopy (SEM) and nitrogen sorption. Sorption experimental results showed that the MIP had excellent binding affinity for CA and the adsorption of CA by MIP was well described by pseudo-second-order model. Scatchard plot analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 7.52±0.46 mg L−1 and 114±4.2 mg L−1, respectively. The selectivity of MIP demonstrated higher affinity for CA over competitive compound than that of non-imprinted polymers (NIP). The MIP synthesized was used to remove CA from spiked surface water and exhibited significant binding affinity towards CA in the presence of total dissolved solids (TDS). In addition, MIP reusability was demonstrated for at least 12 repeated cycles without significant loss in performance. PMID:24205143

Study of human salivary proline-rich proteins interaction with food tannins.

PubMed

Soares, Susana; García-Estévez, Ignacio; Ferrer-Galego, Raúl; Brás, Natércia F; Brandão, Elsa; Silva, Mafalda; Teixeira, Natércia; Fonseca, Fátima; Sousa, Sérgio F; Ferreira-da-Silva, Frederico; Mateus, Nuno; de Freitas, Victor

2018-03-15

In this work, saturation transfer difference-NMR, isothermal microcalorimetry and molecular dynamics simulations have been used to study the individual interactions between basic, glycosylated and acidic proline-rich proteins (bPRPS, gPRPs, aPRPs) and P-B peptide with some representative food tannins [procyanidin B2, procyanidin B2 3'-O-gallate (B2g) and procyanidin trimer (catechin-4-8-catechin-4-8-catechin)]. Results showed that P-B peptide was in general the salivary protein (SP) with higher affinity whereas aPRPs showed lower affinity to the studied procyanidins. Moreover, B2g was the procyanidin with higher affinity for all SP. Hydrophobic and hydrogen bonds were present in all interactions but the major driving force depended on the procyanidin-SP pair. Furthermore, proline clusters or residues in their vicinity were identified as the probable sites of proteins for interaction with procyanidins. For bPRP and aPRP a significant change to less extended conformations was observed, while P-B peptide did not display any structural rearrangement upon procyanidins binding. Copyright © 2017. Published by Elsevier Ltd.

Segregation of human peripheral blood lymphocytes according to their affinity for insolubilized histamine. Principal differences between males and females.

PubMed Central

Tartakovsky, B; Segal, S; Shani, A; Hellerstein, S; Weinstein, Y; Bentwich, Z

1979-01-01

An attempt was made to investigate the possible existence of differences in the composition of peripheral blood lymphocytes between males and females. Using affinity chromatography of human peripheral mononuclear cells on insolubilized histamine together with staining by fluoresceinated histamine-rabbit serum albumin (HRSA) we revealed that males possess a significantly higher proportion of mononuclear cells which bind to HRSA. These results are also reflected in sex-related differences in proliferative responses of the HRSA-non-adherent mononuclear cell population to T cell-dependent mitogens antigens and allogeneic mononuclear cells. PMID:160849

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaberdiel, Matthias R.; Gopakumar, Rajesh; Li, Wei

The relation between the bosonic higher spin W∞[λ]W∞[λ] algebra, the affine Yangian of gl 1, and the SH c algebra is established in detail. For generic λ we find explicit expressions for the low-lying W∞[λ] modes in terms of the affine Yangian generators, and deduce from this the precise identification between λ and the parameters of the affine Yangian. Furthermore, for the free field cases corresponding to λ = 0 and λ = 1 we give closed-form expressions for the affine Yangian generators in terms of the free fields. Interestingly, the relation between the W∞ modes and those of themore » affine Yangian is a non-local one, in general. We also establish the explicit dictionary between the affine Yangian and the SH c generators. Lastly, given that Yangian algebras are the hallmark of integrability, these identifications should pave the way towards uncovering the relation between the integrable and the higher spin symmetries.« less

Influence of corrosion on lipopolysaccharide affinity for two different titanium materials.

PubMed

Barão, Valentim Adelino Ricardo; Mathew, Mathew T; Yuan, Judy Chia-Chun; Knoernschild, Kent L; Assunção, Wirley Gonçalves; Wimmer, Markus A; Sukotjo, Cortino

2013-12-01

Titanium is subject to corrosion in the oral cavity, which could contribute to periimplantitis. However, the effect of corrosion on the lipopolysaccharide affinity for titanium remains unknown. This study evaluated the role of corrosion (in artificial saliva at pHs 3, 6.5, and 9) on the lipopolysaccharide (LPS) affinity for commercially pure titanium (cp-Ti) and Ti-6Al-4V alloy. Seventy-two titanium disks were anodically polarized in a controlled environment (n=9). Control specimens were not corroded. Deionized water with different concentrations of LPS (1.5, 15, and 150 μg/mL) were used to treat the disks for 24 hours to investigate LPS adherence (n=3). Then specimens were immersed in LPS-free water to evaluate LPS elution at 24, 48, and 72 hours. Data were analyzed by the 2-way, 3-way, and 3-way repeated measures ANOVA, t tests, and the Tukey honestly significant difference (HSD) tests (α=.05). A greater corrosion rate of cp-Ti and Ti-6Al-4V alloy and a higher LPS adherence to titanium surfaces (P<.05) were noted at acidic pH. The LPS affinity was higher for the Ti-6Al-4V alloy than for cp-Ti (P<.05). More LPS was eluted from titanium surfaces after a 24-hour interval. Lipopolysaccharide affinity for cp-Ti and Ti-6Al-4V alloy is influenced by the corrosion process. Copyright © 2013 Editorial Council for the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

PubMed

Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

2016-09-06

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Alternating carrier models of asymmetric glucose transport violate the energy conservation laws.

PubMed

Naftalin, Richard J

2008-11-01

Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric "carrier" (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (proportional, variant 1/KD(in)) and slower unliganded "free" carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, DeltaGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (KD(in)/KD(out)), where R is the universal gas constant (8.314 Joules/M/K degrees), and T is the temperature, assumed here to be 300 K degrees , sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport.

Assisted Design of Antibody and Protein Therapeutics (ADAPT)

PubMed Central

Vivcharuk, Victor; Baardsnes, Jason; Deprez, Christophe; Sulea, Traian; Jaramillo, Maria; Corbeil, Christopher R.; Mullick, Alaka; Magoon, Joanne; Marcil, Anne; Durocher, Yves; O’Connor-McCourt, Maureen D.

2017-01-01

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20–30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above. PMID:28750054

Low uptake affinity cultivars with biochar to tackle Cd-tainted rice--A field study over four rice seasons in Hunan, China.

PubMed

Chen, De; Guo, Hu; Li, Ruiyue; Li, Lianqing; Pan, Genxing; Chang, Andrew; Joseph, Stephen

2016-01-15

Biochar is becoming an environmentally friendly material for remediation of heavy metal contaminated soils and improving food safety. A field trial over four rice seasons was conducted to investigate the use of biochar and low Cd accumulating cultivars on Cd uptake in a heavy metal contaminated soil. Wheat straw derived biochar was applied at 0, 20 and 40 t ha(-1). Two rice cultivars with differing Cd accumulation abilities were selected in each season. The results showed that both biochar and low Cd affinity cultivars significantly reduced rice grain Cd accumulation. Biochar had no significant effect the first season but thereafter consistently reduced rice grain Cd by a maximum of 61, 86 and 57% over the next three seasons. Zn accumulation in the rice grains was not decreased by biochar application, although available soil Zn was sharply reduced (35-91%). Indica conventional rice cultivars had much lower Cd, but higher Zn and lower Cd/Zn ratios in the grain than indica hybrid cultivars. Biochar was more effective for mitigating grain Cd accumulation in low Cd affinity cultivars than in high affinity cultivars. Soil pH was sustainably increased (up to nearly 1 unit) while available Cd significantly decreased by a maximum of 85% after biochar addition. The translocation of Cd from rice roots to shoots was reduced from 20 to 80% by biochar. Low uptake affinity cultivars combined with biochar reduced late rice grain Cd concentration and Cd/Zn ratios by 69-80% and 72-80%, respectively. It indicated that the management of combining biochar and low Cd affinity cultivars should be an efficient way to remediate Cd contaminated rice paddies and reduce health risk associated with consuming rice from these soils. Copyright © 2015 Elsevier B.V. All rights reserved.

Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

NASA Astrophysics Data System (ADS)

Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2014-05-01

Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

No major role for binding by salivary proteins as a defense against dietary tannins in Mediterranean goats.

PubMed

Hanovice-Ziony, Michal; Gollop, Nathan; Landau, Serge Yan; Ungar, Eugene David; Muklada, Hussein; Glasser, Tzach Aharon; Perevolotsky, Avi; Walker, John Withers

2010-07-01

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.

Ripening-induced changes in grape skin proanthocyanidins modify their interaction with cell walls.

PubMed

Bindon, Keren A; Kennedy, James A

2011-03-23

Proanthocyanidins were isolated from the skins of Cabernet Sauvignon grapes at different stages of grape development in order to study the effect of proanthocyanidin modification on the interaction with grape cell wall material. After veraison, the degree of proanthocyanidin polymerization increased, and thereafter was variable between 24 and 33 subunits as ripening progressed. Affinity of skin cell wall material for proanthocyanidin decreased with proanthocyanidin ripeness following veraison. A significant negative relationship (R2=0.93) was found for average proanthocyanidin molecular mass and the proportion of high molecular mass proanthocyanidin adsorbed by skin cell wall material. This indicated that as proanthocyanidin polymerization increased, the affinity of a component of high molecular mass proanthocyanidins for skin cell wall material declined. This phenomenon was only associated with skin proanthocyanidins from colored grapes, as high molecular mass proanthocyanidins of equivalent subunit composition from colorless mutant Cabernet Sauvignon grapes had a higher affinity for skin cell wall material.

Affinity for Poetry and Aesthetic Appreciation of Joyful and Sad Poems

PubMed Central

Kraxenberger, Maria; Menninghaus, Winfried

2017-01-01

Artworks with sad and affectively negative content have repeatedly been reported to elicit positive aesthetic appreciation. This topic has received much attention both in the history of poetics and aesthetics as well as in recent studies on sad films and sad music. However, poetry and aesthetic evaluations of joyful and sad poetry have received only little attention in empirical studies to date. We collected beauty and liking ratings for 24 sad and 24 joyful poems from 128 participants. Following previous studies, we computed an integrated measure for overall aesthetic appreciation based on the beauty and liking ratings to test for differences in appreciation between joyful and sad poems. Further, we tested whether readers' judgments are related to their affinity for poetry. Results show that sad poems are rated significantly higher for aesthetic appreciation than joyful poems, and that aesthetic appreciation is influenced by the participants' affinity for poetry. PMID:28119649

Affinity for Poetry and Aesthetic Appreciation of Joyful and Sad Poems.

PubMed

Kraxenberger, Maria; Menninghaus, Winfried

2016-01-01

Artworks with sad and affectively negative content have repeatedly been reported to elicit positive aesthetic appreciation. This topic has received much attention both in the history of poetics and aesthetics as well as in recent studies on sad films and sad music. However, poetry and aesthetic evaluations of joyful and sad poetry have received only little attention in empirical studies to date. We collected beauty and liking ratings for 24 sad and 24 joyful poems from 128 participants. Following previous studies, we computed an integrated measure for overall aesthetic appreciation based on the beauty and liking ratings to test for differences in appreciation between joyful and sad poems. Further, we tested whether readers' judgments are related to their affinity for poetry. Results show that sad poems are rated significantly higher for aesthetic appreciation than joyful poems, and that aesthetic appreciation is influenced by the participants' affinity for poetry.

Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

PubMed

Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

2015-12-01

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

PubMed

Langel, U; Land, T; Bartfai, T

1992-06-01

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

PubMed Central

Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

2013-01-01

Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

Advancement in Higher Education: The Role of Marketing in Building Philanthropic Giving

ERIC Educational Resources Information Center

McAlexander, James H.; Koenig, Harold F.; DuFault, Beth

2014-01-01

This paper empirically explores ways in which marketers of higher education can contribute to the important task of cultivating alumni philanthropy. Advancement professionals understand that philanthropy is influenced by wealth and affinity. As marketers, we anticipate that our contribution resides with investments in building affinity. Using…

Magnetic Affinity Enzyme-Linked Immunoassay for Diagnosis of Schistosomiasis Japonicum in Persons with Low-Intensity Infection

PubMed Central

Yu, Qin; Yang, Hai; Feng, Youmei; Zhu, Yanhong; Yang, Xiangliang

2012-01-01

Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections. PMID:22869635

Contextual correlates of intensity of smoking in northeast India.

PubMed

Ladusingh, Laishram; Singh, Akansha

2015-03-01

This study assessed the significance of socio-demographic and contextual factors on the number of daily cigarette and bidi smoking in northeast India. This study is based on the data from the Global Adult Tobacco Survey-India (2009-2010). Community asset is measured as the totality of all households' durables and community affinity to smoking as the proportion of households which allowed smoking in the house. High daily cigarette and bidi smokers constitute 20 and 30 % of the respective smokers and they smoke 35.8 cigarettes and 14.6 bidis daily, respectively, on the average. The higher is the community affinity to smoking, the higher is the intensity of smoking among the high daily cigarette smokers. Advancing age and educational attainment have significant deterrent effect on the intensity of cigarette and bidi smoking. Contextual factors are found to be important for regional tobacco control programmes. The need for reaching out to communities and the importance of promotion of public-private partnership under the provision of corporate social responsibility for effectiveness of tobacco control programme is recommended.

Mutation-linked, excessively tight interaction between the calmodulin binding domain and the C-terminal domain of the cardiac ryanodine receptor as a novel cause of catecholaminergic polymorphic ventricular tachycardia.

PubMed

Nishimura, Shigehiko; Yamamoto, Takeshi; Nakamura, Yoshihide; Kohno, Michiaki; Hamada, Yoriomi; Sufu, Yoko; Fukui, Go; Nanno, Takuma; Ishiguchi, Hironori; Kato, Takayoshi; Xu, Xiaojuan; Ono, Makoto; Oda, Tetsuro; Okuda, Shinichi; Kobayashi, Shigeki; Yano, Masafumi

2018-06-01

Ryanodine receptor (RyR2) is known to be a causal gene of catecholaminergic polymorphic ventricular tachycardia (CPVT), an important inherited disease. Some of the human CPVT-associated mutations have been found in a domain (4026-4172) that has EF hand motifs, the so-called calmodulin (CaM)-like domain (CaMLD). The purpose of this study was to investigate the underlying mechanism by which CPVT is induced by a mutation at CaMLD. A new N4103K/+ knock-in (KI) mice model was generated. Sustained ventricular tachycardia was frequently observed after infusion of caffeine plus epinephrine in KI mice. Endogenous CaM bound to RyR2 decreased even at baseline in isolated KI cardiomyocytes. Ca 2+ spark frequency (CaSpF) was much higher in KI cells than in wild-type cells. Addition of GSH-CaM (higher affinity CaM to RyR2) significantly decreased CaSpF. In response to isoproterenol, spontaneous Ca 2+ transient (SCaT) was frequently observed in intact KI cells. Incorporation of GSH-CaM into intact KI cells using a protein delivery kit decreased SCaT significantly. An assay using a quartz crystal microbalance technique revealed that mutated CaMLD peptide showed higher binding affinity to CaM binding domain (CaMBD) peptide. In the N4103K mutant, CaM binding affinity to RyR2 was significantly reduced regardless of beta-adrenergic stimulation. We found that this was caused by an abnormally tight interaction between CaMBD and mutated CaM-like domain (N4103K-CaMBD). Thus, CaMBD-CaMLD interaction may be a novel therapeutic target for treatment of lethal arrhythmia. Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Human cyclophilin has a significantly higher affinity for HIV-1 recombinant p55 than p24.

PubMed

Bristow, R; Byrne, J; Squirell, J; Trencher, H; Carter, T; Rodgers, B; Saman, E; Duncan, J

1999-04-01

The ability of cyclophilin to bind a panel of recombinant HIV-gag proteins was assessed using sensitive, quantitative, sandwich enzyme-linked immunosorbant assays (ELISAs). Significantly higher binding to cyclophilin was observed when recombinants contained at least 12 carboxy-terminal amino acids of p17 in addition to p24 sequences. These results indicate that the carboxy-terminus of p17 is important for optimal binding of cyclophilin to p24 and support the theory that cyclophilin acts on the uncleaved HIV-1 gag (p17-p24) precursor.

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, S.Q.; Ren, Y.F.; Alam, B.S.

1987-05-01

The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mgmore » protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.« less

Pre-ingestive selection capacity and endoscopic analysis in the sympatric bivalves Mulinia edulis and Mytilus chilensis exposed to diets containing toxic and non-toxic dinoflagellates

PubMed Central

Widdows, John; Chaparro, Oscar R.; Ortíz, Alejandro; Mellado, Carla; Villanueva, Paola A.

2018-01-01

This study investigates the effects of toxic and non-toxic dinoflatellates on two sympatric bivalves, the clam Mulinia edulis and the mussel Mytilus chilensis. Groups of bivalves were fed one of three diets: (i) the toxic paralytic shellfish producing (PSP) Alexandrium catenella + Isochrysis galbana; (ii) the non-toxic Alexandrium affine + Isochrysis galbana and (iii) the control diet of Isochrysis galbana. Several physiological traits were measured, such as, clearance rate, pre-ingestive selection efficiency and particle transport velocity in the gill. The clearance rates of both M. chilensis and M. edulis showed a significant reduction when fed a mixed toxic diet of 50% Alexandrium catenella: 50% Isochrysis galbana. Similarly, when both species of bivalves were fed with the non-toxic diet (50% A. affine: 50% I. galbana), clearance rate was significantly lower compared with a diet of 100% I. galbana. Under all the experimental diets, M. chilensis showed higher clearance rate values, slightly more than double that of M. edulis. M. edulis and M. chilensis have the ability to select particles at the pre-ingestive level, thus eliminating a larger proportion of the toxic dinoflagellate A. catenella as well as the non-toxic A. affine in the form of pseudofaeces. Higher values of selection efficiency were registered in M. edulis than in M. chilensis when exposed to the toxic diet. Similar results were observed when these two species were exposed to the diet containing the non-toxic dinoflagellate, explained by the fact that the infaunal Mulinia edulis is adapted to dealing with larger particle sizes and higher particle densities (Navarro et al., 1993). The lower transport particle velocity observed in the present work for both species, is related to the reduced clearance rate, the higher particle concentration, and the presence of larger, toxic dinoflagellates. In addition, the species differ in their feeding responses to diets, with and without A. catenella or A. affine, largely reflecting their adaptations to different environmental conditions. The results suggest that the presence of a dinoflagellate bloom, whether toxic or non-toxic spp in Yaldad Bay, is likely to have a greater impact on the Mytilus chilensis than the infaunal Mulinia edulis, based on the combined effects on clearance rate, selection efficiency and particle transport velocity. PMID:29474467

Pre-ingestive selection capacity and endoscopic analysis in the sympatric bivalves Mulinia edulis and Mytilus chilensis exposed to diets containing toxic and non-toxic dinoflagellates.

PubMed

Navarro, Jorge M; Widdows, John; Chaparro, Oscar R; Ortíz, Alejandro; Mellado, Carla; Villanueva, Paola A

2018-01-01

This study investigates the effects of toxic and non-toxic dinoflatellates on two sympatric bivalves, the clam Mulinia edulis and the mussel Mytilus chilensis. Groups of bivalves were fed one of three diets: (i) the toxic paralytic shellfish producing (PSP) Alexandrium catenella + Isochrysis galbana; (ii) the non-toxic Alexandrium affine + Isochrysis galbana and (iii) the control diet of Isochrysis galbana. Several physiological traits were measured, such as, clearance rate, pre-ingestive selection efficiency and particle transport velocity in the gill. The clearance rates of both M. chilensis and M. edulis showed a significant reduction when fed a mixed toxic diet of 50% Alexandrium catenella: 50% Isochrysis galbana. Similarly, when both species of bivalves were fed with the non-toxic diet (50% A. affine: 50% I. galbana), clearance rate was significantly lower compared with a diet of 100% I. galbana. Under all the experimental diets, M. chilensis showed higher clearance rate values, slightly more than double that of M. edulis. M. edulis and M. chilensis have the ability to select particles at the pre-ingestive level, thus eliminating a larger proportion of the toxic dinoflagellate A. catenella as well as the non-toxic A. affine in the form of pseudofaeces. Higher values of selection efficiency were registered in M. edulis than in M. chilensis when exposed to the toxic diet. Similar results were observed when these two species were exposed to the diet containing the non-toxic dinoflagellate, explained by the fact that the infaunal Mulinia edulis is adapted to dealing with larger particle sizes and higher particle densities (Navarro et al., 1993). The lower transport particle velocity observed in the present work for both species, is related to the reduced clearance rate, the higher particle concentration, and the presence of larger, toxic dinoflagellates. In addition, the species differ in their feeding responses to diets, with and without A. catenella or A. affine, largely reflecting their adaptations to different environmental conditions. The results suggest that the presence of a dinoflagellate bloom, whether toxic or non-toxic spp in Yaldad Bay, is likely to have a greater impact on the Mytilus chilensis than the infaunal Mulinia edulis, based on the combined effects on clearance rate, selection efficiency and particle transport velocity.

Bacterial endotoxin adhesion to different types of orthodontic adhesives

PubMed Central

ROMUALDO, Priscilla Coutinho; GUERRA, Thaís Rodrigues; ROMANO, Fábio Lourenço; da SILVA, Raquel Assed Bezerra; BRANDÃO, Izaíra Tincani; SILVA, Célio Lopes; da SILVA, Lea Assed Bezerra; NELSON-FILHO, Paulo

2017-01-01

Abstract Bacterial endotoxin (LPS) adhesion to orthodontic brackets is a known contributing factor to inflammation of the adjacent gingival tissues. Objective The aim of this study was to assess whether LPS adheres to orthodontic adhesive systems, comparing two commercial brands. Material and Methods Forty specimens were fabricated from Transbond XT and Light Bond composite and bonding agent components (n=10/component), then contaminated by immersion in a bacterial endotoxin solution. Contaminated and non-contaminated acrylic resin samples were used as positive and negative control groups, respectively. LPS quantification was performed by the Limulus Amebocyte Lysate QCL-1000™ test. Data obtained were scored and subjected to the Chi-square test using a significance level of 5%. Results There was endotoxin adhesion to all materials (p<0.05). No statistically significant difference was found between composites/bonding agents and acrylic resin (p>0.05). There was no significant difference (p>0.05) among commercial brands. Affinity of endotoxin was significantly greater for the bonding agents (p=0.0025). Conclusions LPS adhered to both orthodontic adhesive systems. Regardless of the brand, the endotoxin had higher affinity for the bonding agents than for the composites. There is no previous study assessing the affinity of LPS for orthodontic adhesive systems. This study revealed that LPS adheres to orthodontic adhesive systems. Therefore, additional care is recommended to orthodontic applications of these materials. PMID:28877283

The Whereabouts of Flower Visitors: Contrasting Land-Use Preferences Revealed by a Country-Wide Survey Based on Citizen Science

PubMed Central

Deguines, Nicolas; Julliard, Romain; de Flores, Mathieu; Fontaine, Colin

2012-01-01

Background In the past decade, accumulating evidence of pollinator decline has raised concerns regarding the functioning of terrestrial ecosystems and the sustainability of crop production. Although land-use changes have been advanced as the major causes, the affinities of most wild pollinators with the main land-use types remain unknown. Filling this gap in our knowledge is a prerequisite to improving conservation and management programmes. Methodology/Principal Findings We estimated the affinity of flower visitors with urban, agricultural and natural land-uses using data from a country-wide scale monitoring scheme based on citizen science (Spipoll). We tested whether the affinities differed among insect orders and according to insect frequency (frequent or infrequent). Our results indicate that the affinities with the three land-use types differed among insect orders. Apart from Hymenopterans, which appeared tolerant to the different land-uses, all flower visitors presented a negative affinity with urban areas and a positive affinity with agricultural and natural areas. Additionally, infrequent taxa displayed a lower affinity with urban areas and a higher affinity with natural areas than did frequent taxa. Within frequent taxa, Hymenoptera and Coleoptera included specialists of the three land-use types whereas Diptera and Lepidoptera contained specialists of all but urban areas. Conclusions/Significance Our approach allowed the first standardised evaluation of the affinity of flower visitors with the main land-use types across a broad taxonomical range and a wide geographic scope. Our results suggest that the most detrimental land-use change for flower visitor communities is urbanisation. Moreover, our findings highlight the fact that agricultural areas have the potential to host highly diverse pollinator communities. We suggest that policy makers should, therefore, focus on the implementation of pollinator-friendly practices in agricultural lands. This may be a win-win strategy, as both biodiversity and crop production may benefit from healthier communities of flower visitors in these areas. PMID:23029262

Non-affine deformations in polymer hydrogels

PubMed Central

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

2012-01-01

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

The position of an arginine residue influences substrate affinity and K+ coupling in the human glutamate transporter, EAAT1.

PubMed

Ryan, Renae M; Kortt, Nicholas C; Sirivanta, Tan; Vandenberg, Robert J

2010-07-01

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and extracellular glutamate levels are controlled by a family of transporters known as excitatory amino acid transporters (EAATs). The EAATs transport glutamate and aspartate with similar micromolar affinities and this transport is coupled to the movement of Na(+), K(+), and H(+). The crystal structure of a prokaryotic homologue of the EAATs, aspartate transporter from Pyrococcus horokoshii (Glt(Ph)), has yielded important insights into the architecture of this transporter family. Glt(Ph) is a Na(+)-dependent transporter that has significantly higher affinity for aspartate over glutamate and is not coupled to H(+) or K(+). The highly conserved carboxy-terminal domains of the EAATs and Glt(Ph) contain the substrate and ion binding sites, however, there are a couple of striking differences in this region that we have investigated to better understand the transport mechanism. An arginine residue is in close proximity to the substrate binding site of both Glt(Ph) and the EAATs, but is located in transmembrane domain (TM) 8 in the EAATs and hairpin loop 1 (HP1) of Glt(Ph). Here we report that the position of this arginine residue can explain some of the functional differences observed between the EAATs and Glt(Ph). Moving the arginine residue from TM8 to HP1 in EAAT1 results in a transporter that has significantly increased affinity for both glutamate and aspartate and is K(+) independent. Conversely, moving the arginine residue from HP1 to TM8 in Glt(Ph) results in a transporter that has reduced affinity for aspartate.

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

CD73 and AMPD3 deficiency enhance metabolic performance via erythrocyte ATP that decreases hemoglobin oxygen affinity.

PubMed

O'Brien, William G; Berka, Vladimir; Tsai, Ah-Lim; Zhao, Zhaoyang; Lee, Cheng Chi

2015-08-07

Erythrocytes are the key target in 5'-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3(-/-) mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism.

Kinetics and equilibrium modelling of lead uptake by algae Gelidium and algal waste from agar extraction industry.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2007-05-08

Pb(II) biosorption onto algae Gelidium, algal waste from agar extraction industry and a composite material was studied. Discrete and continuous site distribution models were used to describe the biosorption equilibrium at different pH (5.3, 4 and 3), considering competition among Pb(II) ions and protons. The affinity distribution function of Pb(II) on the active sites was calculated by the Sips distribution. The Langmuir equilibrium constant was compared with the apparent affinity calculated by the discrete model, showing higher affinity for lead ions at higher pH values. Kinetic experiments were conducted at initial Pb(II) concentrations of 29-104 mgl(-1) and data fitted to pseudo-first Lagergren and second-order models. The adsorptive behaviour of biosorbent particles was modelled using a batch mass transfer kinetic model, which successfully predicts Pb(II) concentration profiles at different initial lead concentration and pH, and provides significant insights on the biosorbents performance. Average values of homogeneous diffusivity, D(h), are 3.6 x 10(-8); 6.1 x 10(-8) and 2.4 x 10(-8)cm(2)s(-1), respectively, for Gelidium, algal waste and composite material. The concentration of lead inside biosorbent particles follows a parabolic profile that becomes linear near equilibrium.

Synthesis and Structure–Activity Relationships of N-Benzyl Phenethylamines as 5-HT2A/2C Agonists

PubMed Central

2014-01-01

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor. PMID:24397362

Synthesis and structure-activity relationships of N-benzyl phenethylamines as 5-HT2A/2C agonists.

PubMed

Hansen, Martin; Phonekeo, Karina; Paine, James S; Leth-Petersen, Sebastian; Begtrup, Mikael; Bräuner-Osborne, Hans; Kristensen, Jesper L

2014-03-19

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor.

Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

NASA Astrophysics Data System (ADS)

Shu, Chang; Ding, Li; Zhong, Wenying

2014-10-01

In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

Water-Hydrogel Binding Affinity Modulates Freeze-Drying-Induced Micropore Architecture and Skeletal Myotube Formation.

PubMed

Rich, Max H; Lee, Min Kyung; Marshall, Nicholas; Clay, Nicholas; Chen, Jinrong; Mahmassani, Ziad; Boppart, Marni; Kong, Hyunjoon

2015-08-10

Freeze-dried hydrogels are increasingly used to create 3D interconnected micropores that facilitate biomolecular and cellular transports. However, freeze-drying is often plagued by variance in micropore architecture based on polymer choice. We hypothesized that water-polymer binding affinity plays a significant role in sizes and numbers of micropores formed through freeze-drying, influencing cell-derived tissue quality. Poly(ethylene glycol)diacrylate (PEGDA) hydrogels with alginate methacrylate (AM) were used due to AM's higher binding affinity for water than PEGDA. PEGDA-AM hydrogels with larger AM concentrations resulted in larger sizes and numbers of micropores than pure PEGDA hydrogels, attributed to the increased mass of water binding to the PEGDA-AM gel. Skeletal myoblasts loaded in microporous PEGDA-AM hydrogels were active to produce 3D muscle-like tissue, while those loaded in pure PEGDA gels were localized on the gel surface. We propose that this study will be broadly useful in designing and improving the performance of various microporous gels.

Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

PubMed Central

Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

2018-01-01

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

Activation of the mu-opiate receptor by Vitex agnus-castus methanol extracts: implication for its use in PMS.

PubMed

Webster, D E; Lu, J; Chen, S-N; Farnsworth, N R; Wang, Z Jim

2006-06-30

The dried ripe fruit of Vitex agnus-castus L. (VAC) is widely used for the treatment of premenstrual syndrome (PMS). A previous study reported that extracts of VAC showed affinity to opiate receptors; however, functional activity was not determined. We tested two different VAC extracts in receptor binding and functional assays. Our objectives were: (1) to confirm the opiate affinity; (2) to rule out interference by free fatty acids (FFA); (3) to determine the mode of action of VAC at the mu-opiate receptor. Methanol extracts of VAC were prepared either before (VAC-M1) or after (VAC-M2) extraction with petroleum ether to remove fatty acids. Both extracts showed significant affinities to the mu-opiate receptor, as indicated by the concentration-dependent displacement of [3H]DAMGO binding in Chinese hamster ovary (CHO)-human mu-opiate receptor (hMOR) cells. The IC50 values were estimated to be 159.8 microg/ml (VAC-M1) and 69.5 microg/ml (VAC-M2). Since the defatted extract not only retained, but exhibited a higher affinity (p<0.001), it argued against significant interference by fatty acids. In an assay to determine receptor activation, VAC-M1 and VAC-M2 stimulated [35S]GTPgammaS binding by 41 and 61% (p<0.001), respectively. These results suggested for the first time that VAC acted as an agonist at the mu-opiate receptor, supporting its beneficial action in PMS.

Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats.

PubMed

Bohin, Maxime C; Vincken, Jean-Paul; Westphal, Adrie H; Tripp, Annelise M; Dekker, Peter; van der Hijden, Harry T W M; Gruppen, Harry

2014-09-01

Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with α-casein and β-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to α-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to α-casein, β-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic-casein interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

PubMed Central

2015-01-01

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sloan, J.W.

1984-01-01

These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) havemore » been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.« less

Supramolecular approach to enantioselective DNA recognition using enantiomerically resolved cationic 4-amino-1,8-naphthalimide-based Tröger's bases.

PubMed

Banerjee, Swagata; Bright, Sandra A; Smith, Jayden A; Burgeat, Jeremy; Martinez-Calvo, Miguel; Williams, D Clive; Kelly, John M; Gunnlaugsson, Thorfinnur

2014-10-03

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters.

PubMed

Xu, Rong; Lord, Sarah A; Peterson, Ryan M; Fergason-Cantrell, Emily A; Lever, John R; Lever, Susan Z

2015-01-01

Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) than the parent SA4503 (DAT Ki=12650 nM; SERT Ki=760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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.

T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

PubMed

Sanecka, Anna; Yoshida, Nagisa; Kolawole, Elizabeth Motunrayo; Patel, Harshil; Evavold, Brian D; Frickel, Eva-Maria

2018-01-01

T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii- infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103 + ) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model

PubMed Central

Samy, KP; Anderson, DA; Lo, DJ; Mulvihill, MS; Song, M; Farris, AB; Parker, BS; MacDonald, AL; Lu, C; Springer, TA; Kachlany, SC; Reimann, KA; How, T; Leopardi, FV; Franke, KS; Williams, KD; Collins, BH; Kirk, AD

2016-01-01

Costimulation blockade (CoB) via belatacept is a lower morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept, and increased adhesion molecule expression. One such molecule is Leukocyte Function Associated Antigen (LFA)-1. LFA-1 exists in two forms, a commonly expressed, low-affinity form, and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 irrespective of its configuration are effective in eliminating memory T cells, but at the cost of impaired protective immunity. Here we test two novel agents, Leukotoxin A and AL-579, each of which targets the high affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity prior to efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB resistant rejection. PMID:27888551

Antenna-predominant and male-biased CSP19 of Sesamia inferens is able to bind the female sex pheromones and host plant volatiles.

PubMed

Zhang, Ya-Nan; Ye, Zhan-Feng; Yang, Ke; Dong, Shuang-Lin

2014-02-25

Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni-ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12-8.75 μM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49-1.78 μM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles. Copyright © 2013 Elsevier B.V. All rights reserved.

The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

PubMed

Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

2016-11-01

The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

PubMed

Revsbech, Inge G; Malte, Hans; Fröbert, Ole; Evans, Alina; Blanc, Stéphane; Josefsson, Johan; Fago, Angela

2013-01-01

During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

Population pharmacokinetics and penetration into prostatic, seminal, and vaginal fluid for ciprofloxacin, levofloxacin, and their combination.

PubMed

Bulitta, Jurgen B; Kinzig, Martina; Naber, Christoph K; Wagenlehner, Florian M E; Sauber, Christian; Landersdorfer, Cornelia B; Sörgel, Fritz; Naber, Kurt G

2011-01-01

Our objectives were to assess the pharmacokinetic interaction and body fluid penetration of ciprofloxacin and levofloxacin. This study was a single-dose open randomized three-way crossover in 15 healthy volunteers receiving 500 mg oral levofloxacin, 500 mg oral ciprofloxacin, or 250 mg levofloxacin and 250 mg ciprofloxacin co-administered. Serum, urine, and body fluid concentrations were determined by high-performance liquid chromatography and analyzed via population pharmacokinetic modeling. Modeling indicated that ciprofloxacin inhibited the renal reabsorption of levofloxacin. Ciprofloxacin increased the net renal clearance of levofloxacin by 13%, as its estimated affinity for a putative tubular reabsorption transporter was 12-fold higher (Km: 568 μM) compared to levofloxacin (Km: 6,830 μM). Levofloxacin increased the bioavailability of ciprofloxacin by 12% and achieved significantly (p < 0.05) higher concentrations at 3 h in ejaculate, prostatic, seminal, and vaginal fluid compared to ciprofloxacin. Modeling suggested that ciprofloxacin inhibited the tubular reabsorption of levofloxacin due to a 12-fold higher affinity for a putative tubular reabsorption transporter compared to levofloxacin. This pharmacokinetic interaction was not clinically relevant. Copyright © 2011 S. Karger AG, Basel.

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

PubMed

Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

1982-03-04

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

Enrichment and Analysis of Non-enzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron Transfer Dissociation Mass Spectrometry

PubMed Central

Zhang, Qibin; Tang, Ning; Brock, Jonathan W. C.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John W.; Smith, Richard D.; Metz, Thomas O.

2008-01-01

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus. PMID:17488106

The protein kinase promiscuities in the cancer-preventive mechanisms of NSAIDs

PubMed Central

Norvaisas, Povilas; Chan, Diana; Yokoi, Kenji; Dave, Bhuvanesh

2016-01-01

NSAIDs have been observed to have cancer-preventive properties, but the actual mechanism is elusive. We hypothesize that NSAIDs might have an effect through common pathways and targets of anticancer drugs by exploiting promiscuities of anticancer drug targets. Here, we have explored NSAIDs by their structural and pharmacophoric similarities with small anticancer molecules. In-silico analyses have shown a strong similarity between NSAIDs and protein kinase (PK) inhibitors. The calculated affinities of NSAIDs were found to be lower than the affinities of anticancer drugs, but higher than the affinities of compounds that are not specific to PKs. The competitive inhibition model suggests that PK might be inhibited by around 10%, which was confirmed by biochemical screening of some NSAIDs against PKs. NSAIDs did not affect all PKs universally, but had specificities for certain sets of PKs, which differed according to the NSAID. The study revealed potentially new features and mechanisms of NSAIDs that are useful in explaining their role in cancer prevention, which might lead to clinically significant breakthroughs in the future. PMID:25714784

Role of Pro-637 and Gln-642 in human glucocorticoid receptors and Ser-843 and Leu-848 in mineralocorticoid receptors in their differential responses to cortisol and aldosterone.

PubMed

Mani, Orlando; Nashev, Lyubomir G; Livelo, Christopher; Baker, Michael E; Odermatt, Alex

2016-05-01

Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are descended from a common ancestral corticoid receptor. The basis for specificities of human MR for aldosterone and human GR for glucocorticoids, such as cortisol, bearing 17α-hydroxyl-groups, is incompletely understood. Differences in MR at S843 and L848 and GR at the corresponding P637 and Q642 have been proposed as important in their different responses to glucocorticoids with 17α-hydroxyl-groups. We investigated the impact of these residues on binding affinity (Ki) and transcriptional activation (EC50) of mutants MR-S843P, MR-L848Q and MR-S843P/L848Q and mutants GR-P637S, GR-Q642L and GR-P637S/Q642L in the presence of different corticosteroids. Aldosterone, cortisol and corticosterone had similar affinities for wild-type MR and all mutants, while dexamethasone had increased affinity for the three mutants. However, transactivation of MR-S843P and MR-S843P/L848Q by all four steroids was significantly lower than for wild-type MR. In contrast, transactivation of MR-L848Q tended to be 3-fold higher for cortisol and corticosterone and increased 7-fold for dexamethasone, indicating that MR-L848Q has an increased response to glucocorticoids, while retaining a strong response to aldosterone. Compared to wild-type GR, GR-P637S and GR-Q642L had increased affinities and significantly increased transcriptional activity with aldosterone and corticosterone, and GR-P637S had similar transcriptional activity with cortisol and dexamethasone, while GR-Q642L and GR-P637S/Q642L had a significant decrease in transcriptional activity with cortisol and dexamethasone. 3D-models of these MR and GR mutants revealed that dexamethasone and aldosterone, respectively, fit nicely into the steroid-binding pocket, consistent with the affinity of dexamethasone for MR mutants and aldosterone for GR mutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

PubMed Central

Gao, Rong

2015-01-01

ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

[2,3 diphosphoglycerate in preterm newborns].

PubMed

Scopesi, F; Canini, S; Mazzella, M; Arioni, C; Lantieri, P; Serra, G

2000-01-01

It has been largely shown that during the first month of life, in the preterm neonate Hb levels and Hct percentages rapidly decrease, high HbF concentration persists and a high oxygen affinity occurs. Data are needed to establish the level at which 2,3 dyphosphoglycerate (2,3 DPG) interacts with the regulation of oxygen affinity. 24 samples, from eight uncomplicated preterm newborns (34.1 +/- 1.83 GW, 1869 +/- +/- 291 BW) obtained at the same time as those required for the clinical management of the infants, were collected on the 2nd, 7th and 14th day of life. Blood gases, total hemoglobin and hematocrit were obtained from 0.3 ml arterialised capillary blood. Assays of 2,3 DPG were made separately on 0.4 ml venous blood. As expected tHb concentration and Hct percentages significantly decreased from day 2 to day 14 in all eight cases. On the contrary 2,3 DPG and p50 values remained stable. Subsequently throughout the study period all neonates had an increased 2,3 DPG/Hb ratio that was significantly related with p50 at standard conditions (p < 0.05). Stable 2,3 DPG concentrations during all study period have been detected. The subsequent significant increased 2.3 DPG/Hb, ratio related to increased p50 values, could have a key role in a physiological mechanism aimed to ensure adequate oxygen delivery to the tissues and to counteract the higher oxygen affinity of fetal hemoglobin. A wider sample is needed to validate this hypothesis.

The actions of some esters of 4-hydroxyquinuclidine on guinea-pig ileum, atria and rat fundus strip.

PubMed

Barlow, R B; Kitchen, R

1982-11-01

1 The acetyl, phenylacetyl, and diphenylacetyl esters of 4-hydroxyquinuclidine and their methiodides have been prepared.2 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide) but it is less selective. At 30 degrees C its affinity for receptors in ileum is about 5 times that for receptors in atria, a difference similar to that found with diphenylacetoxytrophine methiodide. With 4-DAMP methiodide affinity for receptors in the ileum is over 10 times that for receptors in atria.3 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 3-diphenylacetoxyquinuclidine hydrochloride or its methiodide.4 4-Acetoxyquinuclidine hydrochloride has less than one-hundredth of the activity of 3-acetoxyquinuclidine hydrochloride (acecyclidine) on guinea-pig ileum, atria, and rat fundus: however, 4-acetoxyquinuclidine methiodide is consistently more active than its 3-isomer, though it is only about 1/25 times as active as acecyclidine.5 4-Acetoxyquinuclidine hydrochloride is only a poor substrate for electric eel acetylcholinesterase: its affinity is similar to that of acecyclidine but it is greatly reduced by methylation.6 The relations between the structure and activity of the agonists are very different from the relations between the structure and affinity of the antagonists, which supports the view that agonists and antagonists bind to different conformations of the muscarinic receptor.

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics.

PubMed

Seeman, P; Ko, F; Tallerico, T

2005-09-01

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2High, their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2High with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a Ki of 313 nM, as labeled by [3H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2High with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. Dizocilpine had a Ki of 0.3 nM at D2High, but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2 nM at D2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis.

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

PubMed Central

Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

2013-01-01

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy.

PubMed

Singh, Surinder M; Bandi, Swati; Jones, David N M; Mallela, Krishna M G

2017-12-01

We examined how polysorbate 20 (PS20; Tween 20) and polysorbate 80 (PS80; Tween 80) affect the higher-order structure of a monoclonal antibody (mAb) and its antigen-binding (Fab) and crystallizable (Fc) fragments, using near-UV circular dichroism and 2D nuclear magnetic resonance (NMR). Both polysorbates bind to the mAb with submillimolar affinity. Binding causes significant changes in the tertiary structure of mAb with no changes in its secondary structure. 2D 13 C- 1 H methyl NMR indicates that with increasing concentration of polysorbates, the Fab region showed a decrease in crosspeak volumes. In addition to volume changes, PS20 caused significant changes in the chemical shifts compared to no changes in the case of PS80. No such changes in crosspeak volumes or chemical shifts were observed in the case of Fc region, indicating that polysorbates predominantly affect the Fab region compared to the Fc region. This differential effect of polysorbates on the Fab and Fc regions was because of the lesser thermodynamic stability of the Fab compared to the Fc. These results further indicate that PS80 is the preferred polysorbate for this mAb formulation, because it offers higher protection against aggregation, causes lesser structural perturbation, and has weaker binding affinity with fewer binding sites compared to PS20. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

PubMed

Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

2004-12-21

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Peri-implant and systemic effects of high-/low-affinity bisphosphonate-hydroxyapatite composite coatings in a rabbit model with peri-implant high bone turnover

PubMed Central

2012-01-01

Background Hydroxyapatite (HA) coatings composed with bisphosphonates (BPs) which have high mineral-binding affinities have been confirmed to successfully enhance implant stability. However, few previous studies focused on HA coatings composed with low-affinity BPs or on systemic effects of locally released BPs. Methods In this long-term study, we developed two kinds of BP-HA composite coatings using either high-affinity BP (alendronate, ALN) or low-affinity BP (risedronate, RIS). Thirty-six rabbits were divided into three groups according to different coating applications (group I: HA, group II: ALN-HA, and group III: RIS-HA). Implants were inserted into the proximal region of the medullary cavity of the left tibiay. At insertion, 2 × 108 wear particles were injected around implants to induce a peri-implant high bone turnover environment. Both local (left tibias) and systemic (right tibias and lumbar vertebrae) inhibitory effect on bone resorption were compared, including bone-implant integration, bone architecture, bone mineral density (BMD), implant stability, and serum levels of bone turnover markers. Results The results indicated that ALN-HA composite coating, which could induce higher bone-implant contact (BIC) ratio, bone mass augmentation, BMD, and implant stability in the peri-implant region, was more potent on peri-implant bone, while RIS-HA composite coating, which had significant systemic effect, was more potent on non-peri-implant bone, especially lumbar vertebrae. Conclusions It is instructive and meaningful to further clinical studies that we could choose different BP-HA composite coatings according to the patient’s condition. PMID:22686414

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

PubMed Central

2011-01-01

Background Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity. Results To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin. Conclusions These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity. PMID:22185675

2'β-Fluoro-Tricyclo Nucleic Acids (2'F-tc-ANA): Thermal Duplex Stability, Structural Studies, and RNase H Activation.

PubMed

Istrate, Alena; Katolik, Adam; Istrate, Andrei; Leumann, Christian J

2017-08-01

We describe the synthesis, thermal stability, structural and RNase H activation properties of 2'β-fluoro-tricyclo nucleic acids (2'F-tc-ANA). Three 2'F-tc-ANA nucleosides (T, 5Me C and A) were synthesized starting from a previously described fluorinated tricyclo sugar intermediate. NMR analysis and quantum mechanical calculations indicate that 2'F-tc-ANA nucleosides prefer sugar conformations in the East and South regions of the pseudorotational cycle. UV-melting experiments revealed that non-consecutive insertions of 2'F-tc-ANA units in DNA reduce the affinity to DNA and RNA complements. However, an oligonucleotide with five contiguous 2'F-tc-ANA-T insertions exhibits increased affinity to complementary RNA. Moreover, a fully modified 10-mer 2'F-tc-ANA oligonucleotide paired to both DNA (+1.6 °C/mod) and RNA (+2.5 °C/mod) with significantly higher affinity compared to corresponding unmodified DNA, and similar affinity compared to corresponding tc-DNA. In addition, CD spectroscopy and molecular dynamics simulations indicate that the conformation of the 2'F-tc-ANA/RNA duplex is similar to that of a DNA/RNA duplex. Moreover, in some sequence contexts, 2'F-tc-ANA promotes RNase H-mediated cleavage of a complementary RNA strand. Taken together, 2'F-tc-ANA represents a nucleic acid analogue that offers the advantage of high RNA affinity while maintaining the ability to activate RNase H, and can be considered a prospective candidate for gene silencing applications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zolpidem displays heterogeneity in its binding to the nonhuman primate benzodiazepine receptor in vivo.

PubMed

Schmid, L; Bottlaender, M; Fuseau, C; Fournier, D; Brouillet, E; Mazière, M

1995-10-01

The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [11C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [11C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and approximately 100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for < 32% of the specific [11C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.

Genetic variation in South Indian castes: evidence from Y-chromosome, mitochondrial, and autosomal polymorphisms

PubMed Central

Watkins, WS; Thara, R; Mowry, BJ; Zhang, Y; Witherspoon, DJ; Tolpinrud, W; Bamshad, MJ; Tirupati, S; Padmavati, R; Smith, H; Nancarrow, D; Filippich, C; Jorde, LB

2008-01-01

Background Major population movements, social structure, and caste endogamy have influenced the genetic structure of Indian populations. An understanding of these influences is increasingly important as gene mapping and case-control studies are initiated in South Indian populations. Results We report new data on 155 individuals from four Tamil caste populations of South India and perform comparative analyses with caste populations from the neighboring state of Andhra Pradesh. Genetic differentiation among Tamil castes is low (RST = 0.96% for 45 autosomal short tandem repeat (STR) markers), reflecting a largely common origin. Nonetheless, caste- and continent-specific patterns are evident. For 32 lineage-defining Y-chromosome SNPs, Tamil castes show higher affinity to Europeans than to eastern Asians, and genetic distance estimates to the Europeans are ordered by caste rank. For 32 lineage-defining mitochondrial SNPs and hypervariable sequence (HVS) 1, Tamil castes have higher affinity to eastern Asians than to Europeans. For 45 autosomal STRs, upper and middle rank castes show higher affinity to Europeans than do lower rank castes from either Tamil Nadu or Andhra Pradesh. Local between-caste variation (Tamil Nadu RST = 0.96%, Andhra Pradesh RST = 0.77%) exceeds the estimate of variation between these geographically separated groups (RST = 0.12%). Low, but statistically significant, correlations between caste rank distance and genetic distance are demonstrated for Tamil castes using Y-chromosome, mtDNA, and autosomal data. Conclusion Genetic data from Y-chromosome, mtDNA, and autosomal STRs are in accord with historical accounts of northwest to southeast population movements in India. The influence of ancient and historical population movements and caste social structure can be detected and replicated in South Indian caste populations from two different geographic regions. PMID:19077280

Personality Types and Affinity for Computers

DTIC Science & Technology

1991-03-01

differences on personality dimensions between the respondents, and to explore the relationship between these differences and computer affinity. The results...between the respondents, and to explore the relationship between these differences and computer affinity. The results revealed no significant differences...type to this measure of computer affinity. 2 II. LITERATURZ REVIEW The interest of this study was the relationship between a person’s psychological

Molecular Interaction Between Salivary Proteins and Food Tannins.

PubMed

Silva, Mafalda Santos; García-Estévez, Ignacio; Brandão, Elsa; Mateus, Nuno; de Freitas, Victor; Soares, Susana

2017-08-09

Polyphenols interaction with salivary proteins (SP) has been related with organoleptic features such as astringency. The aim of this work was to study the interaction between some human SP and tannins through two spectroscopic techniques, fluorescence quenching, and saturation transfer difference-nuclear magnetic resonance (STD-NMR). Generally, the results showed a significant interaction between SP and both condensed tannins and ellagitannins. Herein, STD-NMR proved to be a useful tool to map tannins' epitopes of binding, while fluorescence quenching allowed one to discriminate binding affinities. Ellagitannins showed the greatest binding constants values (K SV from 20.1 to 94.1 mM -1 ; K A from 0.7 to 8.3 mM -1 ) in comparison with procyanidins (K SV from 5.4 to 40.0 mM -1 ; K A from 1.1 to 2.7 mM -1 ). In fact, punicalagin was the tannin that demonstrated the highest affinity for all three SP. Regarding SP, P-B peptide was the one with higher affinity for ellagitannins. On the other hand, cystatins showed in general the lower K SV and K A values. In the case of condensed tannins, statherin was the SP with the highest affinity, contrasting with the other two SP. Altogether, these results are evidence that the distinct SP present in the oral cavity have different abilities to interact with food tannins class.

Selective Targeting of High-Affinity LFA-1 Does Not Augment Costimulation Blockade in a Nonhuman Primate Renal Transplantation Model.

PubMed

Samy, K P; Anderson, D J; Lo, D J; Mulvihill, M S; Song, M; Farris, A B; Parker, B S; MacDonald, A L; Lu, C; Springer, T A; Kachlany, S C; Reimann, K A; How, T; Leopardi, F V; Franke, K S; Williams, K D; Collins, B H; Kirk, A D

2017-05-01

Costimulation blockade (CoB) via belatacept is a lower-morbidity alternative to calcineurin inhibitor (CNI)-based immunosuppression. However, it has higher rates of early acute rejection. These early rejections are mediated in part by memory T cells, which have reduced dependence on the pathway targeted by belatacept and increased adhesion molecule expression. One such molecule is leukocyte function antigen (LFA)-1. LFA-1 exists in two forms: a commonly expressed, low-affinity form and a transient, high-affinity form, expressed only during activation. We have shown that antibodies reactive with LFA-1 regardless of its configuration are effective in eliminating memory T cells but at the cost of impaired protective immunity. Here we test two novel agents, leukotoxin A and AL-579, each of which targets the high-affinity form of LFA-1, to determine whether this more precise targeting prevents belatacept-resistant rejection. Despite evidence of ex vivo and in vivo ligand-specific activity, neither agent when combined with belatacept proved superior to belatacept monotherapy. Leukotoxin A approached a ceiling of toxicity before efficacy, while AL-579 failed to significantly alter the peripheral immune response. These data, and prior studies, suggest that LFA-1 blockade may not be a suitable adjuvant agent for CoB-resistant rejection. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia

D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoinedmore » by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.« less

Preliminary evaluation of new polymer matrix for solid-phase extraction of nonylphenol from water samples.

PubMed

Guerreiro, António; Soares, Ana; Piletska, Elena; Mattiasson, Bo; Piletsky, Sergey

2008-03-31

Molecularly imprinted (MIP) and blank polymers with affinity for nonylphenol were designed using computational modelling. Chromatographic tests demonstrated higher affinity of imprinted polymers towards the template nonylphenol as compared with blank polymers. The performance of both polymers in solid-phase extraction was however very similar. Both blank and imprinted polymers appeared to be suitable for the removal and pre-concentration of nonylphenol from contaminated water samples with 99% efficiency of the recovery. The commercial resins PH(EC) (Biotage) and C18 (Varian) tested in the same conditions used for comparative purposes had recovery rate <84%. The polymer capacity for nonylphenol was 231 mg g(-1) for blank and 228 mg g(-1) for MIP. The synthesised materials can have significance for sample pre-concentration and environmental analysis of this class of compounds.

A Family of Finite-Dimensional Representations of Generalized Double Affine Hecke Algebras of Higher Rank

NASA Astrophysics Data System (ADS)

Fu, Yuchen; Shelley-Abrahamson, Seth

2016-06-01

We give explicit constructions of some finite-dimensional representations of generalized double affine Hecke algebras (GDAHA) of higher rank using R-matrices for U_q(sl_N). Our construction is motivated by an analogous construction of Silvia Montarani in the rational case. Using the Drinfeld-Kohno theorem for Knizhnik-Zamolodchikov differential equations, we prove that the explicit representations we produce correspond to Montarani's representations under a monodromy functor introduced by Etingof, Gan, and Oblomkov.

Sorption of Cu(2+) on humic acids sequentially extracted from a sediment.

PubMed

Yang, Kun; Miao, Gangfen; Wu, Wenhao; Lin, Daohui; Pan, Bo; Wu, Fengchang; Xing, Baoshan

2015-11-01

In addition to the diverse properties of humic acids (HAs) extracted from different soils or sediments, chemical compositions, functional groups and structures of HAs extracted from a single soil or sediment could also be diverse and thus significantly affect sorption of heavy metals, which is a key process controlling the transfer, transformation and fate of heavy metals in the environment. In this study, we sequentially extracted four HA fractions from a single sediment and conducted the sorption experiments of Cu(2+) on these HA fractions. Our results showed that aromaticity and acidic group content of HA fraction decreased with increasing extraction. Earlier extracted HA fraction had higher sorption capacity and affinity for Cu(2+). There were two fractions of adsorbed Cu(2+) on HAs, i.e., ion exchanged fraction and surface bonded fraction, which can be captured mechanically by the bi-Langmuir model with good isotherm fitting. The ion exchanged fraction had larger sorption capacity but lower sorption affinity, compared with the surface bonded fraction. The dissociated carboxyl groups of HAs were responsible for both fractions of Cu(2+) sorption, due to the more Cu(2+) sorption on the earlier extracted HA fraction with more carboxyl groups and at higher pH. The intensive competition between H(+) and the exchangeable Cu(2+) could result in the decrease of ion exchanged capacity and affinity for Cu(2+) on HAs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of mitochondrial energy production in cardiac cells of rainbow trout (Oncorhynchus mykiss).

PubMed

Birkedal, R; Gesser, H

2004-04-01

In skinned rat cardiac fibres, mitochondrial affinity for endogenous ADP generated by creatine kinase and Ca2+-activated ATPases is higher than for exogenous ADP added to the surrounding medium, suggesting that mitochondria are functionally coupled to creatine kinase and ATPases. Such a coupling may be weaker or absent in ectothermic vertebrate cardiac cells, because they typically have less elaborate intracellular membrane structures, higher glycolytic capacity and lower working temperature. Therefore, we examined skinned cardiac fibres from rainbow trout at 10 degrees C. The apparent mitochondrial affinity for endogenous ADP was obtained by stimulation with ATP and recording of the release of ADP into the surrounding medium. The apparent affinity for endogenous ADP was much higher than for exogenous ADP suggesting a functional coupling between mitochondria and ATPases. The apparent affinity for exogenous ADP and ATP was increased by creatine or an increase in Ca2+-activity, which should increase intrafibrillar turnover of ATP to ADP. In conclusion, ADP seems to be channelled from creatine kinase and ATPases to mitochondria without being released to the surrounding medium. Thus, despite difference in structure, temperature and metabolic capacity, trout myocardium resembles that of rat with regard to the regulation of mitochondrial respiration. Copyright 2004 Springer-Verlag

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

NASA Astrophysics Data System (ADS)

Hoang Thi Thu, Duyen; Razavi, Bahar S.

2016-04-01

Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil: Critical review of estimation criteria and approaches. Soil Biology & Biochemistry 67, 192-211.

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Molindone: higher doses needed to block pergolide-induced elevation of serum corticosterone than to elevate dopamine metabolites in brain.

PubMed

Fuller, R W; Snoddy, H D

1983-12-05

Molindone at a dose of 3 mg/kg i.p. in rats prevented pergolide-induced decreases in brain DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid), causing instead significant increases in these dopamine metabolites when given in combination with pergolide. Molindone alone at 3 mg/kg caused two-fold or greater increases in DOPAC and HVA and at doses as low as 0.3 mg/kg caused significant increases in these metabolites. However, molindone at 3 mg/kg and lower doses was without effect on pergolide-induced elevation of serum corticosterone, though a higher dose of molindone, 10 mg/kg, significantly antagonized this increase in corticosterone. These data support earlier findings with molindone, suggesting it has greater affinity for presynaptic dopamine autoreceptors than for postsynaptic dopamine receptors.

Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics.

PubMed

Chen, Chien-Lun; Lin, Tsung-Shih; Tsai, Cheng-Han; Wu, Chih-Ching; Chung, Ting; Chien, Kun-Yi; Wu, Maureen; Chang, Yu-Sun; Yu, Jau-Song; Chen, Yi-Ting

2013-06-24

In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Relative changes in the abundance of branchial Na(+)/K(+)-ATPase alpha-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities.

PubMed

Tang, Cheng-Hao; Chiu, Yu-Huei; Tsai, Shu-Chuan; Lee, Tsung-Han

2009-08-01

Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos), the euryhaline teleost, exhibited adaptive changes in branchial Na(+)/K(+)-ATPase (NKA) activity with different Na(+) and K(+) affinities. Since alteration of activity and ion-affinity may be influenced by changes in different isoforms of NKA alpha-subunit (i.e., the catalytic subunit), it is, thus, intriguing to compare the patterns of protein abundance of three major NKA alpha-isoform-like proteins (i.e., alpha1, alpha2, and alpha3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance of three NKA alpha-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), as well as hypersaline water (HSW, 60 per thousand) were analyzed by immunoblotting. In the acclimation experiments, the SW group revealed significantly higher levels of NKA alpha1- and alpha3-like proteins than the FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSW revealed the abundance of branchial NKA alpha1-like and alpha3-like proteins decreased significantly after 96 and 12 hr, respectively, and no significant difference was found in NKA alpha2-like protein. Furthermore, when fish were transferred from SW to FW, the amounts of NKA alpha1- and alpha3-like proteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in the abundance of branchial NKA alpha1- and alpha3-like proteins may fulfill the requirements of altering NKA activity with different Na(+) or K(+) affinity for euryhaline milkfish acclimated to environments of various salinities. 2009 Wiley-Liss, Inc.

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Hooking horseradish peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization

NASA Astrophysics Data System (ADS)

Peng, Ye; Ling-Ling, Hu; Yu-Zhi, Du; Yong-Juan, Xu; Hua-Gang, Ni; Cong, Chen; Xiao-Lin, Lu; Xiao-Jun, Huang

2017-05-01

A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.

Attenuated total reflection fourier transform infrared spectroscopy towards disclosing mechanism of bacterial adhesion on thermally stabilized titanium nano-interfaces.

PubMed

Gopal, Judy; Chun, Sechul; Doble, Mukesh

2016-08-01

Titanium is widely used as medical implant material and as condenser material in the nuclear industry where its integrity is questioned due to its susceptibility to bacterial adhesion. A systematic investigation on the influence of thermally (50-800 °C) stabilized titanium (TS-Ti) nano oxide towards bacterial adhesion was carried out. The results showed that below 350 °C significant bacterio-phobicity was observed, while above 500 °C significant affinity towards bacterial cells was recorded. Conventional characterization tools such as HR-TEM and XRD did not provide much insight on the changes occurring on the oxide film with heat treatment, however, attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) of the surface showed significant changes in the spectral pattern as a function of increasing heat treatment. It was observed that elevated OH, N-H and C=O groups and rutile titania on the TS-Ti oxide films led to higher affinity for bacterial adhesion. On the other hand low temperature TS-Ti nanooxide films (<350 °C) showed high C-H groups and decreased OH groups on their surface, which possibly contributed towards their bacterio-phobicity. The TS-Ti nanooxide film grown at 50 °C was observed to be the most efficient anti-bacterial adhesion interface, while the 800 °C interface was the one showing highest affinity towards bacterial adhesion. This study confirms the successful application of ATR-FTIR technique for nano-oxide film characterization and towards understanding the variations in bacterial interaction of such nano interfaces.

Student Engagement and Neoliberalism: Mapping an Elective Affinity

ERIC Educational Resources Information Center

Zepke, Nick

2015-01-01

The purpose of this article is to argue that student engagement, an important area for research about learning and teaching in formal higher education, has an elective affinity with neoliberalism, a hegemonic ideology in many countries of the developed world. The paper first surveys an extensive research literature examining student engagement and…

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu

Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5more » interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.« less

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

DOE PAGES

Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu; ...

2014-10-23

Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5more » interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.« less

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

PubMed

Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

2017-11-28

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. Copyright © 2017 Du et al.

Increased hemoglobin O2 affinity protects during acute hypoxia

PubMed Central

Yalcin, Ozlem

2012-01-01

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O2) transport and O2 utilization. Although decreasing hemoglobin (Hb) O2 affinity would favor the release of O2 to the tissues, increasing Hb O2 affinity would augment arterial O2 saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O2 affinity will augment O2 transport during severe hypoxia (10 and 5% inspired O2) compared with normal Hb O2 affinity. RBC Hb O2 affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O2 affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O2 (Po2). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po2 at which the Hb is 50% saturated with O2 by 12.6 mmHg. During 10 and 5% O2 hypoxia, 5HMF increased arterial blood O2 saturation by 35 and 48% from the vehicle group, respectively. During 5% O2 hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po2 was three times higher in the 5HMF group compared with the control group at 5% O2 hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O2 affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

A novel phagocytic receptor (CgNimC) from Pacific oyster Crassostrea gigas with lipopolysaccharide and gram-negative bacteria binding activity.

PubMed

Wang, Weilin; Liu, Rui; Zhang, Tao; Zhang, Ran; Song, Xuan; Wang, Lingling; Song, Linsheng

2015-03-01

Phagocytosis is an evolutionarily conserved process to ingest the invading microbes and apoptotic or necrotic corpses, playing vital roles in defensing invaders and maintenance of normal physiological conditions. In the present study, a new Nimrod family phagocytic receptor with three EGF-like domains was identified in Pacific oyster Crassostrea gigas (designated CgNimC). CgNimC shared homology with other identified multiple EGF-like domain containing proteins. The mRNA transcripts of CgNimC were mainly distributed in mantle and hemocytes. Its relative expression level in hemocytes was significantly (P < 0.01) up-regulated after the injection of bacteria Vibrio anguillarum. Different to the NimC in Drosophila and Anopheles gambiae, the recombinant protein of CgNimC (rCgNimC) could bind directly to two gram-negative bacteria V. anguillarum and Vibrio splendidus, but not to gram-positive bacteria Staphylococci aureus, Micrococcus luteus or fungi Yarrowia lipolytica and Pichia pastoris. The affinity of rCgNimC toward M. luteus and Y. lipolytica was enhanced when the microorganisms were pre-incubated with the cell free hemolymph. rCgNimC exhibited higher affinity to lipopolysaccharide (LPS) and relatively lower affinity to peptidoglycan (PGN), while no affinity to glucan (GLU). After the CgNimC receptor was blocked by anti-rCgNimC antibody in vitro, the phagocytic rate of hemocytes toward two gram-negative bacteria V. anguillarum and V. splendidus was reduced significantly (P < 0.05), but no significant change of phagocytic rate was observed toward M. luteus and Y. lipolytica. All these results implied that CgNimC, with significant binding capability to LPS and gram-negative bacteria, was a novel phagocytic receptor involved in immune response of Pacific oyster. Further, it was speculated that receptors of Nimrod family might function as a phagocytic receptor to recognize PAMPs on the invaders and its recognition could be promoted by opsonization of molecules in hemolymph. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Naik, Pradeep K.

2014-07-01

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3 kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8 kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2 kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1 kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

Biogeographical Interpretation of Elevational Patterns of Genus Diversity of Seed Plants in Nepal

PubMed Central

Li, Miao; Feng, Jianmeng

2015-01-01

This study tests if the biogeographical affinities of genera are relevant for explaining elevational plant diversity patterns in Nepal. We used simultaneous autoregressive (SAR) models to investigate the explanatory power of several predictors in explaining the diversity-elevation relationships shown in genera with different biogeographical affinities. Delta akaike information criterion (ΔAIC) was used for multi-model inferences and selections. Our results showed that both the total and tropical genus diversity peaked below the mid-point of the elevational gradient, whereas that of temperate genera had a nearly symmetrical, unimodal relationship with elevation. The proportion of temperate genera increased markedly with elevation, while that of tropical genera declined. Compared to tropical genera, temperate genera had wider elevational ranges and were observed at higher elevations. Water-related variables, rather than mid-domain effects (MDE), were the most significant predictors of elevational patterns of tropical genus diversity. The temperate genus diversity was influenced by energy availability, but only in quadratic terms of the models. Though climatic factors and mid-domain effects jointly explained most of the variation in the diversity of temperate genera with elevation, the former played stronger roles. Total genus diversity was most strongly influenced by climate and the floristic overlap of tropical and temperate floras, while the influences of mid-domain effects were relatively weak. The influences of water-related and energy-related variables may vary with biogeographical affinities. The elevational patterns may be most closely related to climatic factors, while MDE may somewhat modify the patterns. Caution is needed when investigating the causal factors underlying diversity patterns for large taxonomic groups composed of taxa of different biogeographical affinities. Right-skewed diversity-elevation patterns may be produced by the differential response of taxa with varying biogeographical affinities to climatic factors and MDE. PMID:26488164

Biogeographical Interpretation of Elevational Patterns of Genus Diversity of Seed Plants in Nepal.

PubMed

Li, Miao; Feng, Jianmeng

2015-01-01

This study tests if the biogeographical affinities of genera are relevant for explaining elevational plant diversity patterns in Nepal. We used simultaneous autoregressive (SAR) models to investigate the explanatory power of several predictors in explaining the diversity-elevation relationships shown in genera with different biogeographical affinities. Delta akaike information criterion (ΔAIC) was used for multi-model inferences and selections. Our results showed that both the total and tropical genus diversity peaked below the mid-point of the elevational gradient, whereas that of temperate genera had a nearly symmetrical, unimodal relationship with elevation. The proportion of temperate genera increased markedly with elevation, while that of tropical genera declined. Compared to tropical genera, temperate genera had wider elevational ranges and were observed at higher elevations. Water-related variables, rather than mid-domain effects (MDE), were the most significant predictors of elevational patterns of tropical genus diversity. The temperate genus diversity was influenced by energy availability, but only in quadratic terms of the models. Though climatic factors and mid-domain effects jointly explained most of the variation in the diversity of temperate genera with elevation, the former played stronger roles. Total genus diversity was most strongly influenced by climate and the floristic overlap of tropical and temperate floras, while the influences of mid-domain effects were relatively weak. The influences of water-related and energy-related variables may vary with biogeographical affinities. The elevational patterns may be most closely related to climatic factors, while MDE may somewhat modify the patterns. Caution is needed when investigating the causal factors underlying diversity patterns for large taxonomic groups composed of taxa of different biogeographical affinities. Right-skewed diversity-elevation patterns may be produced by the differential response of taxa with varying biogeographical affinities to climatic factors and MDE.

The SAMPL4 host-guest blind prediction challenge: an overview.

PubMed

Muddana, Hari S; Fenley, Andrew T; Mobley, David L; Gilson, Michael K

2014-04-01

Prospective validation of methods for computing binding affinities can help assess their predictive power and thus set reasonable expectations for their performance in drug design applications. Supramolecular host-guest systems are excellent model systems for testing such affinity prediction methods, because their small size and limited conformational flexibility, relative to proteins, allows higher throughput and better numerical convergence. The SAMPL4 prediction challenge therefore included a series of host-guest systems, based on two hosts, cucurbit[7]uril and octa-acid. Binding affinities in aqueous solution were measured experimentally for a total of 23 guest molecules. Participants submitted 35 sets of computational predictions for these host-guest systems, based on methods ranging from simple docking, to extensive free energy simulations, to quantum mechanical calculations. Over half of the predictions provided better correlations with experiment than two simple null models, but most methods underperformed the null models in terms of root mean squared error and linear regression slope. Interestingly, the overall performance across all SAMPL4 submissions was similar to that for the prior SAMPL3 host-guest challenge, although the experimentalists took steps to simplify the current challenge. While some methods performed fairly consistently across both hosts, no single approach emerged as consistent top performer, and the nonsystematic nature of the various submissions made it impossible to draw definitive conclusions regarding the best choices of energy models or sampling algorithms. Salt effects emerged as an issue in the calculation of absolute binding affinities of cucurbit[7]uril-guest systems, but were not expected to affect the relative affinities significantly. Useful directions for future rounds of the challenge might involve encouraging participants to carry out some calculations that replicate each others' studies, and to systematically explore parameter options.

Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease.

PubMed

Bonaventura, Celia; Godette, Gerald; Ferruzzi, Giulia; Tesh, Shirley; Stevens, Robert D; Henkens, Robert

2002-07-10

Factors which govern transnitrosation reactions between hemoglobin (Hb) and low molecular weight thiols may define the extent to which S-nitrosated Hb (SNO-Hb) plays a role in NO in the control of blood pressure and other NO-dependent reactions. We show that exposure to S-nitrosylated cysteine (CysNO) produces equivalent levels of SNO-Hb for Hb A(0) and sickle cell Hb (Hb S), although these proteins differ significantly in the electron affinity of their heme groups as measured by their anaerobic redox potentials. Dolphin Hb, a cooperative Hb with a redox potential like that of Hb S, produces less SNO-Hb, indicating that steric considerations outweigh effects of altered electron affinity at the active-site heme groups in control of SNO-Hb formation. Examination of oxygen binding at 5-20 mM heme concentrations revealed increases due to S-nitrosation in the apparent oxygen affinity of both Hb A(0) and Hb S, similar to increases seen at lower heme concentrations. As observed at lower heme levels, deoxygenation is not sufficient to trigger release of NO from SNO-Hb. A sharp increase in apparent oxygen affinity occurs for unmodified Hb S at concentrations above 12.5 mM, its minimum gelling concentration. This affinity increase still occurs in 30 and 60% S-nitrosated samples, but at higher heme concentration. This oxygen binding behavior is accompanied by decreased gel formation of the deoxygenated protein. S-nitrosation is thus shown to have an effect similar to that reported for other SH-group modifications of Hb S, in which R-state stabilization opposes Hb S aggregation.

Investigation of the Binding Profiles of AZD2184 and Thioflavin T with Amyloid-β(1-42) Fibril by Molecular Docking and Molecular Dynamics Methods.

PubMed

Kuang, Guanglin; Murugan, N Arul; Tu, Yaoquan; Nordberg, Agneta; Ågren, Hans

2015-09-03

Detecting deposits of amyloid β fibrils in the brain is of paramount importance for an early diagnosis of Alzheimer's disease. A number of PET tracers have been developed for amyloid imaging, but many suffer from poor specificity and large signal to background ratio. Design of tracers with specificity and improved binding affinity requires knowledge about various potential binding sites in the amyloid β fibril available for the tracers and the nature of the local microenvironment of these sites. In this study we investigate the local structure of fibrils using two important probes, namely, thioflavin T (a fluorescent probe) and AZD2184 (a PET tracer). The target structures for amyloid-β(1-42) fibril are based on reported NMR solution models. By explicitly considering the effect of fibril flexibility on the available binding sites for all these models, the binding affinity of these probes has been investigated. The binding profiles of AZD2184 and thioflavin T were studied by molecular docking and molecular dynamics simulation methods. The two compounds were found to bind at the same sites of the fibril: three of which are within the fibril, and one is on the two sides of the Met35 residue on the surface. The binding affinity of AZD2184 and thioflavin T is found to be higher at the core sites than on the surface due to more contact residues. The binding affinity of AZD2184 is much higher than that of thioflavin T at every site due to electrostatic interaction and spatial restriction, which is in good agreement with experimental observation. However, the structural change of thioflavin T is much more significant than that of AZD2184, which is the chemical basis for its usage as a fluorescent probe. The ramifications of these results for the design and optimization of PET radioligands and fluorescent probes are briefly discussed.

A radiogallium-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging.

PubMed

Sano, Kohei; Masuda, Ryo; Hisada, Hayato; Oishi, Shinya; Shimokawa, Kenta; Ono, Masahiro; Fujii, Nobutaka; Saji, Hideo; Mukai, Takahiro

2014-03-01

We have developed a novel radiogallium (Ga)-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga-DOTA core, Ga-DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga-DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga-DOTA-TZ1. (67)Ga-DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with (67)Ga-DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga-DOTA could be effective in detecting CXCR4-expressing tumors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Removal of selected pharmaceuticals from aqueous solution using magnetic chitosan: sorption behavior and mechanism.

PubMed

Zhang, Yalei; Shen, Zhe; Dai, Chaomeng; Zhou, Xuefei

2014-11-01

A novel-modified magnetic chitosan adsorbent was used to remove selected pharmaceuticals, i.e., diclofenac (DCF) and clofibric acid (CA) and carbamazepine (CBZ), from aqueous solutions. The characterization of magnetic chitosan was achieved by scanning electron and transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, vibrating sample magnetometer, and nitrogen sorption analysis. The magnetic chitosan had effective sorption affinity for DCF and CA but no sorption of CBZ was observed. The sorption capacities of CA and DCF in the individual solutions were 191.2 and 57.5 mg/g, respectively. While in mixed solution, DCF showed higher sorption affinity. Sorption kinetics indicated a quick equilibrium reached within 2 min. Lower solution pH values were found to be advantageous for the adsorption process. The sorption efficacy of CA declined significantly with increasing inorganic salt concentration. However, sorption performance of DCF was stable under different ionic strength conditions.

LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

PubMed

Marshall, J C; Shakespear, R A; Odell, W D

1976-11-01

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

On the vanishing couplings in ADE affine Toda field theories

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saitoh, Y.; Shimada, T.

In this paper, the authors show that certain vanishing couplins in the ADE affine Toda field theories remain vanishing even after higher-order corrections are included. This is a requisite property for the Lagrangian formulation of the theory. The authors develop a new perturbative formulation and treat affine Toda field theories as a massless theory with exponential interaction terms. The authors shown that the nonrenormalization comes from the Dynkin automorphism of the Lie algebra associated with these theories. A charge balance conditions plays an important role in our scheme. The all-order nonrenormalization of vanishing couplings in [bar A][sub n] affine Todamore » field theory is also proved in a standard massive scheme.« less

Mixed infection of Galleria mellonella with two entomopathogenic nematode (Nematoda: Rhabditida) species: Steinernema affine benefits from the presence of Steinernema kraussei.

PubMed

Půza, Vladimír; Mrácek, Zdenek

2009-09-01

The interactions of two sympatric entomopathogenic nematodes Steinernema affine and Steinernema. kraussei were studied in a series of laboratory experiments. Single species, simultaneous and sequential infections of Galleria mellonella were performed in Eppendorf tubes and the invasion rate, nematode progeny production and the number of hosts producing nematode progeny were observed. The invasion rate of S. affine was not affected by the mixed infection whereas the invasion of the latter species was strongly reduced. S. affine out-competed S. kraussei in all treatments and the progeny production of the latter species occurred only sporadically. In comparison to single species infections, per-host progeny production of S. affine was affected only in the treatments with a low dose of S. affine, where it was 30-50% lowered. Generally, in the presence of the latter species, S. affine was able to infect and multiply in a higher number of hosts in comparison to single species infection, especially at a low inoculation rate. S. affine invaded and multiplied also in hosts already infected and even killed by S. kraussei producing a normal amount of progeny. Generally the results suggest that the interactions between steinernematid species can be more complex, including a positive effect of one species on another.

Canine Comfort: Pet Affinity Buffers the Negative Impact of Ambivalence over Emotional Expression on Perceived Social Support.

PubMed

Bryan, Jennifer L; Quist, Michelle C; Young, Chelsie M; Steers, Mai-Ly N; Foster, Dawn W; Lu, Qian

2014-10-01

This study evaluated pet affinity as a buffer between ambivalence over emotional expression (AEE) and social support. AEE occurs when one desires to express emotions but is reluctant to do so and is related to negative psychological outcomes. Individuals high in AEE may have difficulty receiving social support and thus may not gain accompanying benefits. Social support has been associated with positive health outcomes, and pet support is positively associated with human social support. The present study explores the potential protective effect of pet affinity. One hundred ninety-eight undergraduate dog owners completed measures assessing perceived social support, pet affinity, and AEE. AEE was expected to be negatively associated with social support, and pet affinity was expected to buffer the negative effects of AEE on social support. We found that AEE was negatively associated with perceived social support. An interaction between pet affinity and AEE emerged such that the negative association between AEE and social support was weaker among those higher in pet affinity. Thus, at high levels of AEE, those who felt a close connection with their pets reported more perceived social support than those less connected with their pets. Overall, these findings emphasize the potential benefits of pet affinity.

Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola.

PubMed

Kazieva, Ekaterina; Yamamoto, Yoko; Tajima, Yoshinori; Yokoyama, Keiichi; Katashkina, Joanna; Nishio, Yousuke

2017-09-01

The inhibition of mevalonate kinase (MVK) by downstream metabolites is an important mechanism in the regulation of isoprenoid production in a broad range of organisms. The first feedback-resistant MVK was previously discovered in the methanogenic archaeon Methanosarcinamazei. Here, we report the cloning, expression, purification, kinetic characterization and inhibition analysis of MVKs from two other methanogens, Methanosaetaconcilii and Methanocellapaludicola. Similar to the M. mazei MVK, these enzymes were not inhibited by diphosphomevalonate (DPM), dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), geranylpyrophosphate (GPP) or farnesylpyrophosphate (FPP). However, they exhibited significantly higher affinity to mevalonate and higher catalytic efficiency than the previously characterized enzyme.

Enhanced Adsorption of p-Arsanilic Acid from Water by Amine-Modified UiO-67 as Examined Using Extended X-ray Absorption Fine Structure, X-ray Photoelectron Spectroscopy, and Density Functional Theory Calculations.

PubMed

Tian, Chen; Zhao, Jian; Ou, Xinwen; Wan, Jieting; Cai, Yuepeng; Lin, Zhang; Dang, Zhi; Xing, Baoshan

2018-03-20

p-Arsanilic acid ( p-ASA) is an emerging organoarsenic pollutant comprising both inorganic and organic moieties. For the efficient removal of p-ASA, adsorbents with high adsorption affinity are urgently needed. Herein, amine-modified UiO-67 (UiO-67-NH 2 ) metal-organic frameworks (MOFs) were synthesized, and their adsorption affinities toward p-ASA were 2 times higher than that of the pristine UiO-67. Extended X-ray absorption fine structure (EXAFS), X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculation results revealed adsorption through a combination of As-O-Zr coordination, hydrogen bonding, and π-π stacking, among which As-O-Zr coordination was the dominant force. Amine groups played a significant role in enhancing the adsorption affinity through strengthening the As-O-Zr coordination and π-π stacking, as well as forming new adsorption sites via hydrogen bonding. UiO-67-NH 2 s could remove p-ASA at low concentrations (<5 mg L -1 ) in simulated natural and wastewaters to an arsenic level lower than that of the drinking water standard of World Health Organization (WHO) and the surface water standard of China, respectively. This work provided an emerging and promising method to increase the adsorption affinity of MOFs toward pollutants containing both organic and inorganic moieties, via modifying functional groups based on the pollutant structure to achieve synergistic adsorption effect.

Student Engagement, Ideological Contest and Elective Affinity: The Zepke Thesis Reviewed

ERIC Educational Resources Information Center

Trowler, Paul

2015-01-01

This paper takes up issues raised in two articles by Nick Zepke and portrayed here as "the Zepke thesis". This thesis argues that the literature on, interest in and practices around student engagement in higher education have an elective affinity with neo-liberal ideology. At one level this paper counters many of the assertions that…

Supporting Collaboration between K-12 and Higher Education: Year One, Interim Report One

ERIC Educational Resources Information Center

College Board Advocacy & Policy Center, 2012

2012-01-01

In April 2012, more than 100 educators and administrators from across the nation came together in Reston, Virginia, for a full day of meetings and discussions to launch the College Board Advocacy & Policy Center Affinity Network. The Affinity Network's mission is to facilitate students' successful transition from high school to college by: (1)…

Free energy changes and components implicit in the MWC allosteric model for the cooperative oxygen binding of hemoglobin.#

PubMed Central

Bucci, Enrico

2013-01-01

Hill’s plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energies changes of the two affinities (ΔGL) is the free energy of binding cooperativity. Notably ΔGL is positive in favor of the T form, that moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyHb, with a positive ΔGW also in favor of the T form . Increasing protein concentration shifts the isotherms to the right indicating the formation of intermediate polymeric forms. Enthalpy of the intermediates show a strong absorption of heat at the third oxygenation step due to polymers formation with quinary, and above, structures. The disassembly of intermediate polymers releases energy with a negative ΔG that compensates and allow the positivity of ΔGL. High energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity . PMID:23710673

Different modes of interaction by TIAR and HuR with target RNA and DNA

PubMed Central

Kim, Henry S.; Wilce, Matthew C. J.; Yoga, Yano M. K.; Pendini, Nicole R.; Gunzburg, Menachem J.; Cowieson, Nathan P.; Wilson, Gerald M.; Williams, Bryan R. G.; Gorospe, Myriam; Wilce, Jacqueline A.

2011-01-01

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U–rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2′-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways. PMID:21233170

Different modes of interaction by TIAR and HuR with target RNA and DNA.

PubMed

Kim, Henry S; Wilce, Matthew C J; Yoga, Yano M K; Pendini, Nicole R; Gunzburg, Menachem J; Cowieson, Nathan P; Wilson, Gerald M; Williams, Bryan R G; Gorospe, Myriam; Wilce, Jacqueline A

2011-02-01

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

IgE low affinity receptor (CD23) expression, Plasmodium falciparum specific IgE and tumor necrosis factor-alpha production in Thai uncomplicated and severe falciparum malaria patients.

PubMed

Kumsiri, Ratchanok; Troye-Blomberg, Marita; Pattanapanyasat, Kovit; Krudsood, Srivicha; Maneerat, Yaowapa

2016-02-01

Previous studies have suggested that Plasmodium falciparum (P. falciparum) specific IgE in the form of immune complexes crosslinking the low-affinity receptor (CD23) on monocyte results in tumor necrosis factor (TNF)-α and nitric oxide (NO) production. However, the roles of these parameters in severity and immune protection are still unclear. This study aimed to determine the association between CD23 expression on monocytes, plasma soluble CD23 (sCD23), total IgE, malaria-specific IgE and IgG, and TNF-α levels in P. falciparum infected patients. We evaluated 64 uncomplicated (UC) and 25 severe patients (S), admitted at the Hospital for Tropical Diseases, Mahidol University, and 34 healthy controls (C) enrolled in 2001. Flow cytometry and enzyme linked immunosorbent assays (ELISA) demonstrated that trends of the CD23 expression, levels of sCD23 and specific IgE were higher in the S group as compared to those in the UC and C groups. Plasma levels of P. falciparum specific IgE in the UC (p=0.011) and S groups (p=0.025) were significantly higher than those in C group. In contrast the TNF-α levels tended to be higher in the UC than those in the S (p=0.343) and significantly higher than those in C (p=0.004) groups. The specific IgG levels in UC were significantly higher than those in S and C (p<0.001) groups. At admission, a strong significant negative correlation was found between specific IgG and sCD23 (r=-0.762, p=0.028), and TNF-α and IgE-IgG complexes (r=-0.715, p=0.002). Significant positive correlations between levels of specific IgE and TNF-α (r=0.575, p=0.010); and sCD23 (r=0.597, p=0.000) were also observed. In conclusion, our data suggest that CD23 expression and malaria-specific IgE levels may be involved in the severity of the disease while TNF-α and the malaria-specific IgG may correlate with protection against falciparum malaria. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Promotion of pro-osteogenic responses by a bioactive ceramic coating.

PubMed

Aniket; Young, Amy; Marriott, Ian; El-Ghannam, Ahmed

2012-12-01

The objective of this study was to analyze the responses of bone-forming osteoblasts to Ti-6Al-4V implant material coated with silica-calcium phosphate nanocomposite (SCPC50). Osteoblast differentiation at the interface with SCPC50-coated Ti-6Al-4V was correlated to the adsorption of high amount of serum proteins, high surface affinity to fibronectin, Ca uptake from and P and Si release into the medium. SCPC50-coated Ti-6Al-4V adsorbed significantly more serum protein (p < 0.05) than control uncoated substrates. Moreover, Western blot analysis showed that the SCPC50 coating had a high affinity for serum fibronectin. Protein conformation analyses by FTIR showed that the ratio of the area under the peak for amide I/amide II bands was significantly higher (p < 0.05) on the surface of SCPC50-coated substrates than that on the surface of the control uncoated substrates. Moreover, ICP - OES analyses indicated that SCPC50-coated substrates withdrew Ca ions from, and released P and Si ions into, the tissue culture medium, respectively. In conjunction with the favorable protein adsorption and modifications in medium composition, MC3T3-E1 osteoblast-like cells attached to SCPC50-coated substrates expressed 10-fold higher level of mRNA encoding osteocalcin and had significantly higher production of osteopontin and osteocalcin proteins than cells attached to the uncoated Ti-6A1-4V substrates. In addition, osteoblast-like cells attached to the SCPC50-coated substrates produced significantly lower levels of the inflammatory and osteoclastogenic cytokines, IL-6, IL-12p40, and RANKL than those attached to uncoated Ti-6Al-4V substrates. These results suggest that SCPC50 coating could enhance bone integration with orthopedic and maxillofacial implants while minimizing the induction of inflammatory bone cell responses. Copyright © 2012 Wiley Periodicals, Inc.

A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

PubMed Central

Ikeda, Tohru; Kasai, Michiyuki; Tatsukawa, Eri; Kamitakahara, Masanobu; Shibata, Yasuaki; Yokoi, Taishi; Nemoto, Takayuki K; Ioku, Koji

2014-01-01

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism. PMID:24286277

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

PubMed Central

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

Affinity States of Striatal Dopamine D2 Receptors in Antipsychotic-Free Patients with Schizophrenia

PubMed Central

Kubota, Manabu; Nagashima, Tomohisa; Takano, Harumasa; Kodaka, Fumitoshi; Fujiwara, Hironobu; Takahata, Keisuke; Moriguchi, Sho; Higuchi, Makoto; Okubo, Yoshiro; Takahashi, Hidehiko; Ito, Hiroshi

2017-01-01

Abstract Background Dopamine D2 receptors are reported to have high-affinity (D2High) and low-affinity (D2Low) states. Although an increased proportion of D2High has been demonstrated in animal models of schizophrenia, few clinical studies have investigated this alteration of D2High in schizophrenia in vivo. Methods Eleven patients with schizophrenia, including 10 antipsychotic-naive and 1 antipsychotic-free individuals, and 17 healthy controls were investigated. Psychopathology was assessed by Positive and Negative Syndrome Scale, and a 5-factor model was used. Two radioligands, [11C]raclopride and [11C]MNPA, were employed to quantify total dopamine D2 receptor and D2High, respectively, in the striatum by measuring their binding potentials. Binding potential values of [11C]raclopride and [11C]MNPA and the binding potential ratio of [11C]MNPA to [11C]raclopride in the striatal subregions were statistically compared between the 2 diagnostic groups using multivariate analysis of covariance controlling for age, gender, and smoking. Correlations between binding potential and Positive and Negative Syndrome Scale scores were also examined. Results Multivariate analysis of covariance demonstrated a significant effect of diagnosis (schizophrenia and control) on the binding potential ratio (P=.018), although the effects of diagnosis on binding potential values obtained with either [11C]raclopride or [11C]MNPA were nonsignificant. Posthoc test showed that the binding potential ratio was significantly higher in the putamen of patients (P=.017). The Positive and Negative Syndrome Scale “depressed” factor in patients was positively correlated with binding potential values of both ligands in the caudate. Conclusions The present study indicates the possibilities of: (1) a higher proportion of D2High in the putamen despite unaltered amounts of total dopamine D2 receptors; and (2) associations between depressive symptoms and amounts of caudate dopamine D2 receptors in patients with schizophrenia. PMID:29016872

Rabbit anti-rabies immunoglobulins production and evaluation.

PubMed

Liu, Xinjian; Liu, Qiongqiong; Feng, Xiaomin; Tang, Qi; Wang, Zhongcan; Li, Suqing; Feng, Zhenqing; Zhu, Jin; Guan, Xiaohong

2011-04-01

Due to the disadvantages of human and equine rabies immunoglobulin, it is necessary to develop a substitute for HRIG and ERIG, especially for those people living in the developing countries. Because of higher affinity and lower immunogenicity of rabbit's immunoglobulins, anti-rabies immunoglobulins specific to rabies virus were produced in rabbits as a bioreactor, and had been characterized by ELISA, affinity assay, immunofluorescence assay (IFA), immunocytochemistry, rapid fluorescent focus inhibition test (RFFIT). ELISA, affinity assay and IFA showed that rabbit RIG (RRIG) bound specifically to rabies virions. RFFIT result showed that RRIG has neutralization activity. This result was confirmed in vivo in a Kunming mouse challenge model and the protection rate of the treatment with RRIG was higher (25%) than that offered by HRIG when mice were challenged with a lethal RV dose. Our results demonstrate that RRIG is safe and efficacious as a candidate drug to replace rabies immunoglobulin in post-exposure prophylaxis.

Effect of plasticizer on moisture sorption isotherm of sugar palm (Arenga Pinnata) starch film

NASA Astrophysics Data System (ADS)

Jatmiko, Tri Hadi; Poeloengasih, Crescentiana D.; Prasetyo, Dwi Joko; Rosyida, Vita Taufika

2016-02-01

The effect of plasticizer type (glycerol, sorbitol) and plasticizer concentrations (30, 35, 40, 45% w/w polymer) on the moisture sorption isotherm characteristics of sugar palm (Arenga pinnata) starch films were investigated. Moisture affinity of sugar palm starch films was influenced by the plasticizer type and plasticizer concentration. The affinity of the glycerol plasticized film is stronger than that of sorbitol plasticized film. Sugar palm starch film with a higher concentration of glycerol absorbs more moisture with higher initial absorption rate than that of with sorbitol. Films with higher plasticizer concentration of glycerol and sorbitol show higher equilibrium moisture contents at the given relative humidity. The moisture sorption isotherm characteristic of sugar palm starch films can be described very well with the semi empirical 4 parameter Peleg's model.

Formation of (HCOO – )(H 2 SO 4 ) Anion Clusters: Violation of Gas-Phase Acidity Predictions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Gao-Lei; Wang, Xue-Bin; Valiev, Marat

2017-08-10

Sulfuric acid is commonly known to be a strong acid and, by all counts, should readily donate its proton to formate, which has much higher proton affinity. This conventional wisdom is challenged in this work, where temperature-dependent negative ion photoelectron spectroscopy (NIPES) and theoretical studies demonstrate the existence of (HCOO?)(H2SO4) pair at the energy slightly below the conventional (HCOOH)(HSO4?) structure. Analysis of quantum-mechanical calculations indicates that large proton affinity barrier (~36 kcal/mol), favoring proton transfer to formate, is offset by the gain in inter-molecular interaction energy between HCOO? and H2SO4 through the formation of two strong hydrogen bonds. However, thismore » stabilization comes with severe entropic penalty, requiring the two species in the precise align-ment. As a result, the population of (HCOO?)(H2SO4) drops significantly at higher temperatures, rendering (HCOOH)(HSO4?) to be the dominant species. This phe-nomenon is consistent with the NIPES data, which shows depletion in the spectra assigned to (HCOO?)(H2SO4), and has also been verified by ab initio molecular dynamics simulations.« less

Cu0-loaded SBA-15@ZnO with improved electrical properties and affinity towards hydrogen

NASA Astrophysics Data System (ADS)

Bouazizi, N.; Louhichi, S.; Ouargli, R.; Bargougui, R.; Vieillard, J.; Derf, F. Le; Azzouz, A.

2017-05-01

A core-shell material was prepared using SBA-15 crystallites as cores for the growth of a ZnO shell, followed by Cu0 dispersion. The resulting Cu/SBA-15@ZnO nanostructure displayed higher specific surface area (SSA) and higher number of smaller pores as compared to the starting materials. Dispersion of fine Cu0NPs induced a compaction of the host matrice and a marked decay of the hydrophilic character, explained in terms of the involvement of terminal hydroxyl groups in competitive sbnd HO:Cu interaction at the expense of H-bridges with water. Heating at 400-450 °C seems to trigger ZnO dehydroxylation with possible self-polycondensation and/or the formation of Si-O-Zn bridges. This is an additional explanation of the significant SSA increase and decrease in the average pore diameter. Both ZnO and Cu0NP incorporation induced shifts in the UV-vis absorption band towards higher wavelengths, indicating a decrease in the optical band gap energy and an improvement of the conductance properties. As compared to ZnO, Cu0NPs produced stronger improvement of the conductance, which was found to increase with higher frequencies. Cu/SBA-15@ZnO also displayed higher affinity towards hydrogen as compared to SBA-15@ZnO and SBA-15 at ambient conditions. These outstanding properties combined to an appreciable thermal stability are worth to be prone to deeper investigations, because they can open promising prospects for Cu/SBA-15@ZnO as sensor, electrode material, electrocatalyst and/or hydrogen capture matrice.

ROLE OF GENETIC SUSCEPTIBILITY TO LATENT ADENOVIRAL INFECTION AND DECREASED LUNG FUNCTION

PubMed Central

Kasuga, Ikuma; Hogg, James C.; Paré, Peter D.; Hayashi, Shizu; Sedgwick, Edward G.; Ruan, Jian; Wallace, Alison M.; He, Jian-Qing; Zhang, Xiaozhu; Sandford, Andrew J.

2009-01-01

Background Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin β5 (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function. Methods Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay. Results E1A positive individuals have a lower FEV1 compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV1 level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes. Conclusions Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD. PMID:19502044

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts.

PubMed

Carayannopoulos, M O; Potter, K N; Li, Y; Natvig, J B; Capra, J D

2000-04-01

The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.

Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

PubMed

Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

2014-01-01

Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

NASA Astrophysics Data System (ADS)

Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

2016-04-01

Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center

PubMed Central

Schwickert, Tanja A.; Victora, Gabriel D.; Fooksman, David R.; Kamphorst, Alice O.; Mugnier, Monica R.; Gitlin, Alexander D.; Dustin, Michael L.

2011-01-01

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide–major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T–B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells. PMID:21576382

Modern affinity reagents: Recombinant antibodies and aptamers.

PubMed

Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

2015-12-01

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement.

PubMed

Gorris, Hans H; Bade, Steffen; Röckendorf, Niels; Fránek, Milan; Frey, Andreas

2011-08-17

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

Affinity-seeking, social loneliness, and social avoidance among Facebook users.

PubMed

Lemieux, Robert; Lajoie, Sean; Trainor, Nathan E

2013-04-01

This study explored the relations between use of the social networking site Facebook and scores on affinity-seeking, social loneliness, and social avoidance by 313 college students. Social loneliness and social avoidance, but not affinity-seeking, were positively and statistically significantly related to time spent using Facebook. The number of close Facebook friends was negatively and statistically significantly related to social loneliness and social avoidance. Women perceived Facebook as a more integral part of daily interactions than did men. 38% of the 283 Facebook members indicated their accounts contained information and/or a picture that could embarrass them, with men having significantly more embarrassing content than women. The findings are discussed within the context of social compensation.

Disposition in the rat of buprenorphine administered parenterally and as a subcutaneous implant.

PubMed

Pontani, R B; Vadlamani, N L; Misra, A L

1985-04-01

Disposition of [15, 16(n)-3H]buprenorphine in the rat has been investigated after a single 0.2 mg/kg i.v. bolus dose and continuous administration via a s.c. implantable long-acting delivery system. After the i.v. injection, the tri-exponential decay of drug from brain occurred with t1/2 values of 0.6, 2.3 and 7.2 h, respectively (plasma t1/2 0.5, 1.4 h, third phase not estimated due to sustained concn.) Decay of drug from another high-affinity binding site in brain occurred with t1/2 values of 1.1 and 68.7 h, respectively. Fat and lung had higher concn. than other tissues and plasma. No metabolites of drug were detected in brain. Unmetabolized drug excreted in urine and faeces one week after i.v. injection were 1.9 and 22.4% of dose, respectively, and 92% of the dose was accounted for in one week. Urinary metabolites (%) were: conjugated buprenorphine 0.9; norbuprenorphine (free 9.4, conjugated 5.2); tentative 6-O-desmethylnorbuprenorphine (free 5.4, conjugated 15.9). Peak plasma concn. of buprenorphine occurred four weeks after s.c. implantation of a long-acting 10 mg 3H-buprenorphine pellet, and apparent dissociation half-lives of drug from low- and high-affinity binding sites in brain were 4.6 and 6.8 weeks, respectively. Fat, spleen and skeletal muscle had higher concn. than other tissues and plasma. No significant difference in brain morphine concn. was observed in placebo and nonlabelled buprenorphine-pelleted animals after a 2 mg/kg i.v. challenge dose of 3H-morphine. This study emphasizes the importance of high-affinity binding of buprenorphine in brain and subsequent slow dissociation as a prime factor in its prolonged agonist/antagonist effects and higher potency than other narcotic agonists.

Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α

PubMed Central

Real Hernandez, Luis M.; Fan, Junfeng; Johnson, Michelle H.; Gonzalez de Mejia, Elvira

2015-01-01

Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1α promotes. PMID:26413797

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

DOE PAGES

Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu; ...

2014-10-23

Chitin is a fungal microbe-associated molecular pattern (MAMP) that is recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor in plants and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in the plant chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, only AtLYK4/AtLYK5-2 doublemore » mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. Furthermore, the data suggest that AtLYK5 is the primary plant receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant innate immunity.« less

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

PubMed Central

Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu; Nguyen, Cuong T; Jedrzejczak, Robert P; Joachimiak, Andrzej; Stacey, Gary

2014-01-01

Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity. DOI: http://dx.doi.org/10.7554/eLife.03766.001 PMID:25340959

The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Yangrong; Liang, Yan; Tanaka, Kiwamu

Chitin is a fungal microbe-associated molecular pattern (MAMP) that is recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor in plants and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in the plant chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, only AtLYK4/AtLYK5-2 doublemore » mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. Furthermore, the data suggest that AtLYK5 is the primary plant receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant innate immunity.« less

Development and In Vitro Characterization of a Gemcitabine-loaded MUC4-targeted Immunoliposome Against Pancreatic Ductal Adenocarcinoma.

PubMed

Urey, Carlos; Hilmersson, Katarzyma Said; Andersson, Bodil; Ansari, Daniel; Andersson, Roland

2017-11-01

Pancreatic Ductal adeno-carcinoma (PDAC) is a devastating disease. Gemcitabine is the standard chemotherapeutic agent against PDAC but has only limited effectiveness. The aim of the study was to develop and study the targeting affinity and in vitro antiproliferative effect of a MUC4-targeted gemcitabine-loaded immuno-liposome for treatment of PDAC. Gemcitabine-loaded immunoliposomes were developed by grafting anti-MUC4 antibodies to the liposomal surface. Targeting affinity was compared in vitro between immunoliposomes and non-targeted liposomes and anti-proliferative effect was compared in vitro between free drug, non-targeted liposomal gemcitabine and MUC4-targeted immunoliposomal gemcitabine on a MUC4-positive pancreatic cancer cell line, Capan-1. Development of a MUC4-targeted immunoliposome was confirmed and characterized by immunoblots and size characterization. The MUC4-targeted immunoliposome showed a significantly higher targeting affinity compared to the non-targeted liposomes and also showed an improved antiproliferative effect compared to free and non-targeted liposomal drug. Successful development and characterization of a MUC4-targeted immunoliposome shows promising results for a targeted treatment and improved retention of gemcitabine for treatment of PDAC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces.

PubMed

Sankaran, Shrikrishnan; Cavatorta, Emanuela; Huskens, Jurriaan; Jonkheijm, Pascal

2017-09-05

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV 2+ ). The binding affinity of the knottins with CB[8] and MV 2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Antianxiety effect of cannabis: involvement of central benzodiazepine receptors.

PubMed

Sethi, B B; Trivedi, J K; Kumar, P; Gulati, A; Agarwal, A K; Sethi, N

1986-01-01

The present work, involving clinical, behavioral, and biochemical studies, was undertaken to elucidate the probable mechanism of the observed antianxiety effects of cannabis. The population for the clinical study consisted of 50 male chronic cannabis users who were otherwise healthy and 50 matched controls. When evaluated on Taylor's Manifest Anxiety Scale (TMA), these subjects had low anxiety scores as compared with the controls. To explore the possible interaction of cannabis with the benzodiazepine receptors, behavioral and biochemical studies in mice were devised, involving acute and chronic cannabis administration. Behavioral study revealed that mice under chronic cannabis treatment scored significantly higher on foot shock-induced aggression, but this was significantly blocked by benzodiazepine receptor antagonist. Furthermore, chronic cannabis treatment significantly (p less than 0.001) increased the frequency of licking response periodically punished by shocks. This confirms the antianxiety effect of cannabis, which also appears to be mediated through a benzodiazepine receptor, as it was reduced significantly (p less than 0.001) by a benzodiazepine receptor blocker. Specific 3H-diazepam binding was carried out in frontal cortex to assess both the population and affinity of benzodiazepine receptors. Our results indicate that acute cannabis treatment has no significant effect, whereas chronic cannabis treatment significantly increased 3H-diazepam binding as compared with controls. Scatchard analysis further reveals that increased affinity is responsible for increased binding to these receptors. It is therefore our contention that the antianxiety effect of cannabis is mediated through central benzodiazepine receptors.

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

PubMed Central

Monarez, Roberto R.; Macdonald, Clinton C.; Dass, Brinda

2006-01-01

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified Kd values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than τCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements. PMID:17029590

Organically functionalized mesoporous SBA-15 as sorbents for removal of selected pharmaceuticals from water.

PubMed

Bui, Tung Xuan; Kang, Seo-Young; Lee, Sang-Hyup; Choi, Heechul

2011-10-15

Mesoporous silica SBA-15 and its postfunctionalized counterparts with hydroxymethyl (HM-SBA-15), aminopropyl (AP-SBA-15), and trimethylsilyl (TMS-SBA-15) were prepared and characterized by powder X-ray diffraction, N(2) adsorption-desorption measurement, Fourier-transform infrared spectroscopy, and elemental analysis. The removal of a mixture of 12 selected pharmaceuticals was investigated by batch adsorption experiments onto SBA-15 and the grafted materials. SBA-15 showed to have moderate adsorption affinity with amino-containing (atenolol, trimethoprim) and hydrophobic pharmaceuticals, but it displayed minimal adsorption affinity toward hydrophilic compounds. HM-SBA-15 was analogous with SBA-15 in terms of the adsorption efficiency toward all pharmaceuticals. AP-SBA-15 exhibited an increase in the adsorption of two acidic compounds (clofibric acid, diclofenac) but a decrease in the adsorption of estrone and the two amino-containing compounds. Among the grafted materials, TMS-SBA-15 had the highest adsorption affinity toward most pharmaceuticals. Moreover, the adsorption of nine pharmaceuticals to TMS-SBA-15 was significantly higher than that to SBA-15; seven of which showed the removal percentages from 70.6% to 98.9% onto TMS-SBA-15. The number of pharmaceuticals showing high adsorption efficiency onto TMS-SBA-15 did not alter significantly as the pH changed in the range of 5.5-7.6. The results suggest that TMS-SBA-15 is a promising material for the removal of pharmaceuticals from aqueous phase, especially for the treatment of wastewater from drug manufacturers. Copyright © 2011 Elsevier B.V. All rights reserved.

T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate.

PubMed

Ise, Wataru; Fujii, Kentaro; Shiroguchi, Katsuyuki; Ito, Ayako; Kometani, Kohei; Takeda, Kiyoshi; Kawakami, Eiryo; Yamashita, Kazuo; Suzuki, Kazuhiro; Okada, Takaharu; Kurosaki, Tomohiro

2018-04-17

Higher- or lower-affinity germinal center (GC) B cells are directed either to plasma cell or GC recycling, respectively; however, how commitment to the plasma cell fate takes place is unclear. We found that a population of light zone (LZ) GC cells, Bcl6 lo CD69 hi expressing a transcription factor IRF4 and higher-affinity B cell receptors (BCRs) or Bcl6 hi CD69 hi with lower-affinity BCRs, favored the plasma cell or recycling GC cell fate, respectively. Mechanistically, CD40 acted as a dose-dependent regulator for Bcl6 lo CD69 hi cell formation. Furthermore, we found that expression of intercellular adhesion molecule 1 (ICAM-1) and signaling lymphocytic activation molecule (SLAM) in Bcl6 lo CD69 hi cells was higher than in Bcl6 hi CD69 hi cells, thereby affording more stable T follicular helper (Tfh)-GC B cell contacts. These data support a model whereby commitment to the plasma cell begins in the GC and suggest that stability of Tfh-GC B cell contacts is key for plasma cell-prone GC cell formation. Copyright © 2018. Published by Elsevier Inc.

Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities

NASA Astrophysics Data System (ADS)

Yao, Yuyu; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

2016-03-01

The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)- d-GlcNAc, a single chain variable fragment and α- d-Gal-(1→2)-[α- d-Abe-(1→3)]-α- d-Man-OCH3, cholera toxin B subunit homopentamer with β- d-Gal-(1→3)-β- d-GalNAc-(1→4)[α- d-Neu5Ac-(2→3)]-β- d-Gal-(1→4)-β- d-Glc, and a fragment of galectin 3 and α- l-Fuc-(1→2)-β- d-Gal-(1→3)-β- d-GlcNAc-(1→3)-β- d-Gal-(1→4)-β- d-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

Inter- and intra-variability of seed germination traits of Carpobrotus edulis N.E.Br. and its hybrid C. affine acinaciformis.

PubMed

Podda, Lina; Santo, Andrea; Mattana, Efisio; Mayoral, Olga; Bacchetta, Gianluigi

2018-06-22

Invasions by alien Carpobrotus spp. have been recognized as one of the most severe threats to Mediterranean-climate coastal ecosystems and Carpobrotus is considered one of the most widespread invasive alien genera in the Mediterranean Basin. The aims of this study were to characterize seed germination of both C. edulis and its hybrid C. affine acinaciformis, in terms of photoperiod, temperature and salinity. Inter- and intra-specific variability in the responses to photoperiod (12/12 h light and total darkness), constant temperatures (5, 10, 15, 20, 25°C) and an alternating temperature regime (25/10°C), salt stress (0, 125, 250, 500 mM NaCl) and the recovery of seed germination were evaluated for two seed lots of C. edulis and two of its hybrid C. affine acinaciformis. All the tested seed lots achieved higher germination percentages in the light, respect to total darkness. In relation to temperature, the two C. edulis seed lots did not show a preference, while the two C. affine acinaciformis seed lots differed in their germination response, one germinating more at the lowest temperatures (5 and 10°C) and one at the highest (20 and 25°C). For all the seed lots, highest germination occurred without NaCl (0 mM) and germination decreased with increasing salinity. Different germination requirements in saline substrate were not detected for C. edulis, while were observed for C. affine acinaciformis. Marked differences were detected in recovery responses between the two taxa. C. edulis demonstrated to have the ability to germinate in a wide time window during the year. This study identified significant differences in seed production, seed mass, germination requirements (temperature) and salinity tolerance for both C. edulis and C. affine acinaciformis. Our results indicated the extreme versatility of the hybrid forms to germinate in a wide range of natural conditions and habitats. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

PubMed Central

O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

1997-01-01

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses. PMID:9334373

Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities.

PubMed

Yao, Yuyu; Richards, Michele R; Kitova, Elena N; Klassen, John S

2016-03-01

The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-D-GlcNAc, a single chain variable fragment and α-D-Gal-(1→2)-[α-D-Abe-(1→3)]-α-D-Man-OCH3, cholera toxin B subunit homopentamer with β-D-Gal-(1→3)-β-D-GalNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal-(1→4)-β-D-Glc, and a fragment of galectin 3 and α-L-Fuc-(1→2)-β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-β-D-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

Measurements of the talus in the assessment of population affinity.

PubMed

Bidmos, Mubarak A; Dayal, Manisha R; Adegboye, Oyelola A

2018-06-01

As part of their routine work, forensic anthropologists are expected to report population affinity as part of the biological profile of an individual. The skull is the most widely used bone for the estimation of population affinity but it is not always present in a forensic case. Thus, other bones that preserve well have been shown to give a good indication of either the sex or population affinity of an individual. In this study, the potential of measurements of the talus was investigated for the purpose of estimating population affinity in South Africans. Nine measurements from two hundred and twenty tali of South African Africans (SAA) and South African Whites (SAW) from the Raymond A. Dart Collection of Human Skeletons were used. Direct and step-wise discriminant function and logistic regression analyses were carried out using SPSS and SAS. Talar length was the best single variable for discriminating between these two groups for males while in females the head height was the best single predictor. Average accuracies for correct population affinity classification using logistic regression analysis were higher than those obtained from discriminant function analysis. This study was the first of its type to employ discriminant function analyses and logistic regression analyses to estimate the population affinity of an individual from the talus. Thus these equations can now be used by South African anthropologists when estimating the population affinity of dismembered or damaged or incomplete skeletal remains of SAA and SAW. Copyright © 2018 Elsevier B.V. All rights reserved.

Impaired locomotor activity and exploratory behavior in mice lacking histamine H1 receptors

PubMed Central

Inoue, Isao; Yanai, Kazuhiko; Kitamura, Daisuke; Taniuchi, Ichiro; Kobayashi, Takashi; Niimura, Kaku; Watanabe, Takehiko; Watanabe, Takeshi

1996-01-01

From pharmacological studies using histamine antagonists and agonists, it has been demonstrated that histamine modulates many physiological functions of the hypothalamus, such as arousal state, locomotor activity, feeding, and drinking. Three kinds of receptors (H1, H2, and H3) mediate these actions. To define the contribution of the histamine H1 receptors (H1R) to behavior, mutant mice lacking the H1R were generated by homologous recombination. In brains of homozygous mutant mice, no specific binding of [3H]pyrilamine was seen. [3H]Doxepin has two saturable binding sites with higher and lower affinities in brains of wild-type mice, but H1R-deficient mice showed only the weak labeling of [3H]doxepin that corresponds to lower-affinity binding sites. Mutant mice develop normally, but absence of H1R significantly increased the ratio of ambulation during the light period to the total ambulation for 24 hr in an accustomed environment. In addition, mutant mice significantly reduced exploratory behavior of ambulation and rearings in a new environment. These results indicate that through H1R, histamine is involved in circadian rhythm of locomotor activity and exploratory behavior as a neurotransmitter. PMID:8917588

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

Advancing Peptide-Based Biorecognition Elements for Biosensors Using in-Silico Evolution.

PubMed

Xiao, Xingqing; Kuang, Zhifeng; Slocik, Joseph M; Tadepalli, Sirimuvva; Brothers, Michael; Kim, Steve; Mirau, Peter A; Butkus, Claire; Farmer, Barry L; Singamaneni, Srikanth; Hall, Carol K; Naik, Rajesh R

2018-05-25

Sensors for human health and performance monitoring require biological recognition elements (BREs) at device interfaces for the detection of key molecular biomarkers that are measurable biological state indicators. BREs, including peptides, antibodies, and nucleic acids, bind to biomarkers in the vicinity of the sensor surface to create a signal proportional to the biomarker concentration. The discovery of BREs with the required sensitivity and selectivity to bind biomarkers at low concentrations remains a fundamental challenge. In this study, we describe an in-silico approach to evolve higher sensitivity peptide-based BREs for the detection of cardiac event marker protein troponin I (cTnI) from a previously identified BRE as the parental affinity peptide. The P2 affinity peptide, evolved using our in-silico method, was found to have ∼16-fold higher affinity compared to the parent BRE and ∼10 fM (0.23 pg/mL) limit of detection. The approach described here can be applied towards designing BREs for other biomarkers for human health monitoring.

Hemoglobin Function in Stored Blood.

DTIC Science & Technology

1974-08-01

States during 1973. Several advantages over ACA) are important. Blood stored in CPD maintains higher ./ levels of 2,3-DPG (2,3- diphosphoglycerate ) and a...survival and ATP levels in stored blood is explained by the several functions of ATP which are necessary for cell viability. However, ATP levels do...not correlate with oxygen affinity during storage. Levels of 2,3-DPG determine oxygen affinity and thus hemoglobin function. (12,13) When normal levels

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

Microaerobic DO-induced microbial mechanisms responsible for enormous energy saving in upflow microaerobic sludge blanket reactor.

PubMed

Zheng, Shaokui; Cui, Cancan; Quan, Ying; Sun, Jian

2013-07-01

This study experimentally examined the microaerobic dissolved oxygen (DO)-induced microbial mechanisms that are responsible for enormous energy savings in the upflow microaerobic sludge blanket reactor (UMSB) for domestic wastewater treatment. Phylogenetic and kinetic analyses (as determined by clone library analyses and sludge oxygen affinity analyses) showed that the microaerobic conditions in the UMSB led to the proliferation and dominance of microaerophilic bacteria that have higher oxygen affinities (i.e., lower sludge oxygen half-saturation constant values), which assured efficient COD and NH3-N removals and sludge granulation in the UMSB similar as those achieved in the aerobic control. However, the microaerobic DO level in the UMSB achieved significant short-cut nitrification, a 50-90% reduction in air supply, and an 18-28% reduction in alkali consumption. Furthermore, the disappearance of sludge bulking in the UMSB when it was dominated by "bulking-induced" filamentous bacteria should be attributed to its upflow column-type configuration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics.

PubMed

Gul, Ahmet; Erman, Burak

2018-01-16

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

Removal of furan and phenolic compounds from simulated biomass hydrolysates by batch adsorption and continuous fixed-bed column adsorption methods.

PubMed

Lee, Sang Cheol; Park, Sunkyu

2016-09-01

It has been proposed to remove all potential inhibitors and sulfuric acid in biomass hydrolysates generated from dilute-acid pretreatment of biomass, based on three steps of sugar purification process. This study focused on its first step in which furan and phenolic compounds were selectively removed from the simulated hydrolysates using activated charcoal. Batch adsorption experiments demonstrated that the affinity of activated charcoal for each component was highest in the order of vanillic acid, 4-hydroxybenzoic acid, furfural, acetic acid, sulfuric acid, and xylose. The affinity of activated charcoal for furan and phenolic compounds proved to be significantly higher than that of the other three components. Four separation strategies were conducted with a combination of batch adsorption and continuous fixed-bed column adsorption methods. It was observed that xylose loss was negligible with near complete removal of furan and phenolic compounds, when at least one fixed-bed column adsorption was implemented in the strategy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

NASA Astrophysics Data System (ADS)

Gul, Ahmet; Erman, Burak

2018-03-01

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP.

PubMed

Belov, Sergey; Buneva, Valentina N; Nevinsky, Georgy A

2017-10-01

Myelin basic protein (MBP) is a major protein of myelin-proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12-mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti-MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K d  = 0.51-0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10 -1 to 2.3 × 10 -4  M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K d , M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192-fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins. Copyright © 2017 John Wiley & Sons, Ltd.

Interrelationship among Fe-His Bond Strengths, Oxygen Affinities, and Intersubunit Hydrogen Bonding Changes upon Ligand Binding in the β Subunit of Human Hemoglobin: The Alkaline Bohr Effect.

PubMed

Nagatomo, Shigenori; Okumura, Miki; Saito, Kazuya; Ogura, Takashi; Kitagawa, Teizo; Nagai, Masako

2017-03-07

Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O 2 affinity changes were investigated via 1 H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O 2 to the α subunits is forbidden in the mutant Hbs, the O 2 affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe-His stretching frequency of the β subunits was shifted to higher values. Thus, it was confirmed for the β subunits that the stronger the Fe-His bond, the higher the O 2 affinity. It was found in this study that the quaternary structure of α(Fe 3+ )β(Fe 2+ -CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspβ94-Hisβ146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisβ146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94-Trpβ37 hydrogen bond in the hinge region and the Tyrα42-Aspβ99 hydrogen bond in the switch region of the α 1 -β 2 interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe-His bond of the β subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the β subunit.

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics.

PubMed

Miyanoiri, Youhei; Kobayashi, Hisanori; Imai, Takao; Watanabe, Michinao; Nagata, Takashi; Uesugi, Seiichi; Okano, Hideyuki; Katahira, Masato

2003-10-17

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT1A receptors.

PubMed

Tordera, Rosa M; Monge, Antonio; Del Río, Joaquín; Lasheras, Berta

2002-05-03

It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT1A receptors and devoid of high affinity at other receptors studied, with the only exception of alpha1-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [3H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01-0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT1A receptor function, 0.01-0.1 microM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT1A receptors; much lower doses (0.01-0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1-5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT1A receptor partial agonist, and with a remarkable activity in an animal model of depression with high predictive validity.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Design and synthesis of novel pyrimidine analogs as highly selective, non-covalent BTK inhibitors.

PubMed

Kawahata, Wataru; Asami, Tokiko; Irie, Takayuki; Sawa, Masaaki

2018-01-15

BTK is a promising target for the treatment of multiple diseases such as B cell malignances, asthma, and rheumatoid arthritis. Here, we report the discovery of a series of novel pyrimidine analogs as potent, highly selective, non-covalent inhibitors of BTK. Compound 25d demonstrated higher affinity to an unactivated conformation of BTK that resulted in an excellent kinase selectivity. Compound 25d showed a good oral bioavailability in mice, and significantly inhibits the PCA reaction in mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins

NASA Astrophysics Data System (ADS)

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A.; Jiménez, M. Consuelo

2018-06-01

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α1-acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222.

Selectivity of arsenite interaction with zinc finger proteins.

PubMed

Zhao, Linhong; Chen, Siming; Jia, Liangyuan; Shu, Shi; Zhu, Pingping; Liu, Yangzhong

2012-08-01

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ≫ C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.

Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

PubMed

Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

2014-09-01

Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Muscarinic receptor occupation and receptor activation in the guinea-pig ileum by some acetamides related to oxotremorine.

PubMed Central

Ringdahl, B.

1984-01-01

The dissociation constants (KD values) and relative efficacies of seven acetamide analogues of oxotremorine, including two enantiomeric pairs, at muscarinic receptors in the guinea-pig isolated ileum were determined. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. All of the compounds studied had lower affinities than oxotremorine, but some had substantially higher relative efficacies. Replacement of the pyrrolidine ring of N-methyl-N-(4- pyrrolidino -2- butynyl )acetamide(I), the parent compound in the series, by a dimethylamino or a trimethylammonium group decreased the affinity 32 and 4.5 fold, respectively, whereas the relative efficacy increased 5.7-8.3 times. There was no correlation between relative efficacies and affinities of the compounds. The structural requirements for high affinity and high efficacy appeared to be quite different. PMID:6733356

Screening for Natural Inhibitors of Topoisomerases I from Rhamnus davurica by Affinity Ultrafiltration and High-Performance Liquid Chromatography–Mass Spectrometry

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Topoisomerase I (Topo I) catalyzes topological interconversion of duplex DNA during DNA replication and transcription, and has been deemed as important antineoplastic targets. In this study, the fraction R.d-60 from ethyl acetate extracts of Rhamnus davurica showed higher inhibitory rates against SGC-7901 and HT-29 compared with the R.d-30 fraction in vitro. However, the specific active components of R.d-60 fraction remain elusive. To this end, a method based on bio-affinity ultrafiltration and high performance liquid chromatography/electrospray mass spectrometry (HPLC- ESI-MS/MS) was developed to rapidly screen and identify the Topo I inhibitors in this fraction. The enrichment factors (EFs) were calculated to evaluate the binding affinities between the bioactive constituents and Topo I. As a result, eight ligands were identified and six of which with higher EFs showed more potential antitumor activity. Furthermore, antiproliferative assays in vitro (IC50 values) with two representative candidates (apigenin, quercetin) against SGC-7901, HT-29 and Hep G2 cells were conducted and further validated. Finally, the structure-activity relationships revealed that flavones contain a C2-C3 double bond of C ring exhibited higher bio-affinities to Topo I than those without it. This integrated method combining Topo I ultrafiltration with HPLC-MS/MS proved to be very efficient in rapid screening and identification of potential Topo I inhibitors from the complex extracts of medicinal plants, and could be further explored as a valuable high-throughput screening platform in the early drug discovery stage. PMID:28919906

A General Strategy for Targeting Drugs to Bone.

PubMed

Jahnke, Wolfgang; Bold, Guido; Marzinzik, Andreas L; Ofner, Silvio; Pellé, Xavier; Cotesta, Simona; Bourgier, Emmanuelle; Lehmann, Sylvie; Henry, Chrystelle; Hemmig, René; Stauffer, Frédéric; Hartwieg, J Constanze D; Green, Jonathan R; Rondeau, Jean-Michel

2015-11-23

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sulfide binding properties of truncated hemoglobins.

PubMed

Nicoletti, Francesco P; Comandini, Alessandra; Bonamore, Alessandra; Boechi, Leonardo; Boubeta, Fernando Martin; Feis, Alessandro; Smulevich, Giulietta; Boffi, Alberto

2010-03-16

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

A comprehensive study of layer-specific morphological changes in the microstructure of carotid arteries under uniaxial load.

PubMed

Krasny, Witold; Morin, Claire; Magoariec, Hélène; Avril, Stéphane

2017-07-15

The load bearing properties of large blood vessels are principally conferred by collagen and elastin networks and their microstructural organization plays an important role in the outcomes of various arterial pathologies. In particular, these fibrous networks are able to rearrange and reorient spatially during mechanical deformations. In this study, we investigate for the first time whether these well-known morphological rearrangements are the same across the whole thickness of blood vessels, and subsequently if the underlying mechanisms that govern these rearrangements can be predicted using affine kinematics. To this aim, we submitted rabbit carotid samples to uniaxial load in three distinct deformation directions, while recording live images of the 3D microstructure using multiphoton microscopy. Our results show that the observed realignment of collagen and elastin in the media layer, along with elastin of the adventitia layer, remained limited to small angles that can be predicted by affine kinematics. We show also that collagen bundles of fibers in the adventitia layer behaved in significantly different fashion. They showed a remarkable capacity to realign in the direction of the load, whatever the loading direction. Measured reorientation angles of the fibers were significantly higher than affine predictions. This remarkable property of collagen bundles in the adventitia was never observed before, it shows that the medium surrounding collagen in the adventitia undergoes complex deformations challenging traditional hyperelastic models based on mixture theories. The biomechanical properties of arteries are conferred by the rearrangement under load of the collagen and elastin fibers making up the arterial microstructure. Their kinematics under deformation is not yet characterized for all fiber networks. In this respect we have submitted samples of arterial tissue to uniaxial tension, simultaneously to confocal imaging of their microstructure. Our method allowed identifying for the first time the remarkable ability of adventitial collagen fibers to reorient in the direction of the load, achieving reorientation rotations that exceeded those predicted by affine kinematics, while all other networks followed the affine kinematics. Our results highlight new properties of the microstructure, which might play a role in the outcomes of vascular pathologies like aneurysms. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Opposing intermolecular tuning of Ca2+ affinity for Calmodulin by its target peptides

NASA Astrophysics Data System (ADS)

Cheung, Margaret

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca2+) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on binding between CaM and two specific targets, Ca2+/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca2+ signaling pathways in neurons. It was shown experimentally that Ca2+/CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide, which binds to Ca2+-free CaM or Ca2+/CaM with similar binding affinity. Unlike CaM-CaMKII peptide that allowed structure determination by crystallography, the structural description of CaM-Ng peptide is unknown due to low binding affinity, therefore, we computationally generated an ensemble of CaM-Ng peptide structures by matching the changes in the chemical shifts of CaM upon Ng peptide binding from nuclear magnetic resonance experiments. We computed the changes in Ca2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca2+ for CaM in the presence of Ng by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca2+ binding loops particularly at C-domain of CaM, enabling Ca2+release. In contrast, CaMKII increases Ca2+ affinity for the C-domain of CaM by stabilizing the two Ca2+ binding loops.

Fast-match on particle swarm optimization with variant system mechanism

NASA Astrophysics Data System (ADS)

Wang, Yuehuang; Fang, Xin; Chen, Jie

2018-03-01

Fast-Match is a fast and effective algorithm for approximate template matching under 2D affine transformations, which can match the target with maximum similarity without knowing the target gesture. It depends on the minimum Sum-of-Absolute-Differences (SAD) error to obtain the best affine transformation. The algorithm is widely used in the field of matching images because of its fastness and robustness. In this paper, our approach is to search an approximate affine transformation over Particle Swarm Optimization (PSO) algorithm. We treat each potential transformation as a particle that possesses memory function. Each particle is given a random speed and flows throughout the 2D affine transformation space. To accelerate the algorithm and improve the abilities of seeking the global excellent result, we have introduced the variant system mechanism on this basis. The benefit is that we can avoid matching with huge amount of potential transformations and falling into local optimal condition, so that we can use a few transformations to approximate the optimal solution. The experimental results prove that our method has a faster speed and a higher accuracy performance with smaller affine transformation space.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study.

PubMed

Steck, Andrea K; Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J; Yu, Liping

2016-07-01

Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects.

Impact of antipsychotic medication on transcranial direct current stimulation (tDCS) effects in schizophrenia patients.

PubMed

Agarwal, Sri Mahavir; Bose, Anushree; Shivakumar, Venkataram; Narayanaswamy, Janardhanan C; Chhabra, Harleen; Kalmady, Sunil V; Varambally, Shivarama; Nitsche, Michael A; Venkatasubramanian, Ganesan; Gangadhar, Bangalore N

2016-01-30

Transcranial direct current stimulation (tDCS) has generated interest as a treatment modality for schizophrenia. Dopamine, a critical pathogenetic link in schizophrenia, is also known to influence tDCS effects. We evaluated the influence of antipsychotic drug type (as defined by dopamine D2 receptor affinity) on the impact of tDCS in schizophrenia. DSM-IV-TR-diagnosed schizophrenia patients [N=36] with persistent auditory hallucinations despite adequate antipsychotic treatment were administered add-on tDCS. Patients were divided into three groups based on the antipsychotic's affinity to D2 receptors. An auditory hallucinations score (AHS) was measured using the auditory hallucinations subscale of the Psychotic Symptom Rating Scales (PSYRATS). Add-on tDCS resulted in a significant reduction inAHS. Antipsychotic drug type had a significant effect on AHS reduction. Patients treated with high affinity antipsychotics showed significantly lesser improvement compared to patients on low affinity antipsychotics or a mixture of the two. Furthermore, a significant sex-by-group interaction occurred; type of medication had an impact on tDCS effects only in women. Improvement differences could be due to the larger availability of the dopamine receptor system in patients taking antipsychotics with low D2 affinity. Sex-specific differences suggest potential estrogen-mediated effects. This study reports a first-time observation on the clinical utility of antipsychotic drug type in predicting tDCS effects in schizophrenia. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

PubMed

Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

2016-03-01

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

Exploring Strategies for Labeling Viruses with Gold Nanoclusters through Non-equilibrium Molecular Dynamics Simulations.

PubMed

Pohjolainen, Emmi; Malola, Sami; Groenhof, Gerrit; Häkkinen, Hannu

2017-09-20

Biocompatible gold nanoclusters can be utilized as contrast agents in virus imaging. The labeling of viruses can be achieved noncovalently but site-specifically by linking the cluster to the hydrophobic pocket of a virus via a lipid-like pocket factor. We have estimated the binding affinities of three different pocket factors of echovirus 1 (EV1) in molecular dynamics simulations combined with non-equilibrium free-energy calculations. We have also studied the effects on binding affinities with a pocket factor linked to the Au 102 pMBA 44 nanocluster in different protonation states. Although the absolute binding affinities are over-estimated for all the systems, the trend is in agreement with recent experiments.3 Our results suggest that the natural pocket factor (palmitic acid) can be replaced by molecules pleconaril (drug) and its derivative Kirtan1 that have higher estimated binding affinities. Our results also suggest that including the gold nanocluster does not decrease the affinity of the pocket factor to the virus, but the affinity is sensitive to the protonation state of the nanocluster, i.e., to pH conditions. The methodology introduced in this work helps in the design of optimal strategies for gold-virus bioconjugation for virus detection and manipulation.

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

PubMed Central

Tharakaraman, Kannan; Robinson, Luke N.; Hatas, Andrew; Chen, Yi-Ling; Siyue, Liu; Raguram, S.; Sasisekharan, V.; Wogan, Gerald N.; Sasisekharan, Ram

2013-01-01

Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen–antibody interfaces to predict protein–protein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1–3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein–protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies. PMID:23569282

Anti-neuropeptide Y plasma immunoglobulins in relation to mood and appetite in depressive disorder.

PubMed

Garcia, Frederico D; Coquerel, Quentin; do Rego, Jean-Claude; Cravezic, Aurore; Bole-Feysot, Christine; Kiive, Evelyn; Déchelotte, Pierre; Harro, Jaanus; Fetissov, Sergueï O

2012-09-01

Depression and eating disorders are frequently associated, but the molecular pathways responsible for co-occurrence of altered mood, appetite and body weight are not yet fully understood. Neuropeptide Y (NPY) has potent antidepressant and orexigenic properties and low central NPY levels have been reported in major depression. In the present study, we hypothesized that in patients with major depression alteration of mood, appetite and body weight may be related to NPY-reactive autoantibodies (autoAbs). To test this hypothesis, we compared plasma levels and affinities of NPY-reactive autoAbs between patients with major depression and healthy controls. Then, to evaluate if changes of NPY autoAb properties can be causally related to altered mood and appetite, we developed central and peripheral passive transfer models of human autoAbs in mice and studied depressive-like behavior in forced-swim test and food intake. We found that plasma levels of NPY IgG autoAbs were lower in patients with moderate but not with mild depression correlating negatively with the Montgomery-Åsberg Depression Rating Scale scores and with immobility time of the forced-swim test in mice after peripheral injection of autoAbs. No significant differences in NPY IgG autoAb affinities between patients with depression and controls were found, but higher affinity of IgG autoAbs for NPY was associated with lower body mass index and prevented NPY-induced orexigenic response in mice after their central injection. These data suggest that changes of plasma levels of anti-NPY autoAbs are relevant to altered mood, while changes of their affinity may participate in altered appetite and body weight in patients with depressive disorder. Copyright © 2012 Elsevier Ltd. All rights reserved.

Binding affinity and decontamination of dermal decontamination gel to model chemical warfare agent simulants.

PubMed

Cao, Yachao; Elmahdy, Akram; Zhu, Hanjiang; Hui, Xiaoying; Maibach, Howard

2018-05-01

Six chemical warfare agent simulants (trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate) were studied in in vitro human skin to explore relationship between dermal penetration/absorption and the mechanisms of simulant partitioning between stratum corneum (SC) and water as well as between dermal decontamination gel (DDGel) and water. Both binding affinity to and decontamination of simulants using DDGel were studied. Partition coefficients of six simulants between SC and water (Log P SC/w ) and between DDGel and water (Log P DDGel/w ) were determined. Results showed that DDGel has a similar or higher binding affinity to each simulant compared to SC. The relationship between Log P octanol/water and Log P SC/w as well as between Log P octanol/water and Log P DDGel/w demonstrated that partition coefficient of simulants correlated to their lipophilicity or hydrophilicity. Decontamination efficiency results with DDGel for these simulants were consistent with binding affinity results. Amounts of percentage dose of chemicals in DDGel of trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate were determined to be 61.15, 85.67, 75.91, 53.53, 89.89 and 76.58, with corresponding amounts absorbed in skin of 0.96, 0.65, 1.68, 0.72, 0.57 and 1.38, respectively. In vitro skin decontamination experiments coupled with a dermal absorption study demonstrated that DDGel can efficiently remove chemicals from skin surface, back-extract from the SC, and significantly reduced chemical penetration into skin or systemic absorption for all six simulants tested. Therefore, DDGel offers a great potential as a NextGen skin Decon platform technology for both military and civilian use. Copyright © 2018 John Wiley & Sons, Ltd.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko

2014-04-18

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystalmore » structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.« less

Prediction of the binding affinities of peptides to class II MHC using a regularized thermodynamic model

PubMed Central

2010-01-01

Background The binding of peptide fragments of extracellular peptides to class II MHC is a crucial event in the adaptive immune response. Each MHC allotype generally binds a distinct subset of peptides and the enormous number of possible peptide epitopes prevents their complete experimental characterization. Computational methods can utilize the limited experimental data to predict the binding affinities of peptides to class II MHC. Results We have developed the Regularized Thermodynamic Average, or RTA, method for predicting the affinities of peptides binding to class II MHC. RTA accounts for all possible peptide binding conformations using a thermodynamic average and includes a parameter constraint for regularization to improve accuracy on novel data. RTA was shown to achieve higher accuracy, as measured by AUC, than SMM-align on the same data for all 17 MHC allotypes examined. RTA also gave the highest accuracy on all but three allotypes when compared with results from 9 different prediction methods applied to the same data. In addition, the method correctly predicted the peptide binding register of 17 out of 18 peptide-MHC complexes. Finally, we found that suboptimal peptide binding registers, which are often ignored in other prediction methods, made significant contributions of at least 50% of the total binding energy for approximately 20% of the peptides. Conclusions The RTA method accurately predicts peptide binding affinities to class II MHC and accounts for multiple peptide binding registers while reducing overfitting through regularization. The method has potential applications in vaccine design and in understanding autoimmune disorders. A web server implementing the RTA prediction method is available at http://bordnerlab.org/RTA/. PMID:20089173

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

PubMed Central

Xi, Hongjuan; Kumar, Sunil; Dosen-Micovic, Ljiljana; Arya, Dev P.

2013-01-01

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA12-x-dT12-x-dT12-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA).2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the Tm for triplex decreases with increasing pH value in the presence of neomycin, while the Tm for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA12-x-dT12-x-dT12-3′, respectively. (4) The specific heat capacity change (ΔCp) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔCp ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔCp is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔCp is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC50 (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove. PMID:20167243

H 2-saturation of high affinity H 2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

DOE PAGES

Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G.; ...

2016-03-10

Soil microbial communities are continuously exposed to H 2 diffusing into the soil from the atmosphere. N 2-fixing nodules represent a peculiar microniche in soil where H 2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H 2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H 2 exposure from the atmosphere and N 2-fixing nodules. Biphasic kinetic parameters governing H 2 oxidation activity in soil changed drastically upon elevated H 2 exposure, corresponding to a slight but significant decay ofmore » high affinity H 2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H 2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H 2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H 2 exposure, suggesting that H 2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H 2-rich environments exert a direct influence on soil H 2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.« less

Mechanism of increased clearance of glycated albumin by proximal tubule cells

PubMed Central

Wagner, Mark C.; Myslinski, Jered; Pratap, Shiv; Flores, Brittany; Rhodes, George; Campos-Bilderback, Silvia B.; Sandoval, Ruben M.; Kumar, Sudhanshu; Patel, Monika; Ashish

2016-01-01

Serum albumin is the most abundant plasma protein and has a long half-life due to neonatal Fc receptor (FcRn)-mediated transcytosis by many cell types, including proximal tubule cells of the kidney. Albumin also interacts with, and is modified by, many small and large molecules. Therefore, the focus of the present study was to address the impact of specific known biological albumin modifications on albumin-FcRn binding and cellular handling. Binding at pH 6.0 and 7.4 was performed since FcRn binds albumin strongly at acidic pH and releases it after transcytosis at physiological pH. Equilibrium dissociation constants were measured using microscale thermophoresis. Since studies have shown that glycated albumin is excreted in the urine at a higher rate than unmodified albumin, we studied glucose and methylgloxal modified albumins (21 days). All had reduced affinity to FcRn at pH 6.0, suggesting these albumins would not be returned to the circulation via the transcytotic pathway. To address why modified albumin has reduced affinity, we analyzed the structure of the modified albumins using small-angle X-ray scattering. This analysis showed significant structural changes occurring to albumin with glycation, particularly in the FcRn-binding region, which could explain the reduced affinity to FcRn. These results offer an explanation for enhanced proximal tubule-mediated sorting and clearance of abnormal albumins. PMID:26887834

Interaction of KRAS G-quadruplex with natural polyphenols: A spectroscopic analysis with molecular modeling.

PubMed

Pattanayak, Rudradip; Basak, Pijush; Sen, Srikanta; Bhattacharyya, Maitree

2016-08-01

Researchers are endeavoring to find out new therapeutics for curing cancer and G-quadruplex DNA has already been identified as a prospective one in this venture. Stabilizing G-quadruplex structures of telomere has emerged to be an important strategy in this context. Mutation in KRAS is mostly responsible for pancreatic, lung and colon cancer. In this present study we explored binding and conformational behaviour of G-quadruplex with different ligands by utilizing several biophysical techniques. Natural polyphenols like Curcumin and Ellagic acid were observed to bind with the G-quadruplex and enhance the melting temperature significantly indicating higher stability. UV-vis spectroscopy confirms formation of G quadruplex-ligand complex for both the compounds with specific binding affinity. Fluorimetric studies revealed that Ellagic acid had stronger binding affinity, 1.10×10(5)M(-1) compared to Curcumin, 1.6×10(4)M(-1) towards G-quadruplex. Interestingly, Curcumin provides greater stability by stacking on the top of the quadruplex structure with the help of the loops compared to Ellagic acid as is evident by docking studies. The keto form of curcumin showed stronger affinity than the enol form. We have developed a general model to estimate the influence of the ligands towards stabilizing the G-quadruplex subsequently characterizing the binding profile to enlighten prospective therapeutics. Copyright © 2016. Published by Elsevier B.V.

Covalent Organic Frameworks as a Decorating Platform for Utilization and Affinity Enhancement of Chelating Sites for Radionuclide Sequestration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Qi; Aguila, Briana; Earl, Lyndsey D.

The potential consequences of nuclear events and the complexity of nuclear waste management motivate the development of selective solid-phase sorbents to provide enhanced protection. In this paper, it is shown that 2D covalent organic frameworks (COFs) with unique structures possess all the traits to be well suited as a platform for the deployment of highly efficient sorbents such that they exhibit remarkable performance, as demonstrated by uranium capture. The chelating groups laced on the open 1D channels exhibit exceptional accessibility, allowing significantly higher utilization efficiency. In addition, the 2D extended polygons packed closely in an eclipsed fashion bring chelating groupsmore » in adjacent layers parallel to each other, which may facilitate their cooperation, thereby leading to high affinity toward specific ions. As a result, the amidoxime-functionalized COFs far outperform their corresponding amorphous analogs in terms of adsorption capacities, kinetics, and affinities. Specifically, COF-TpAb-AO is able to reduce various uranium contaminated water samples from 1 ppm to less than 0.1 ppb within several minutes, well below the drinking water limit (30 ppb), as well as mine uranium from spiked seawater with an exceptionally high uptake capacity of 127 mg g -1. Finally, these results delineate important synthetic advances toward the implementation of COFs in environmental remediation.« less

Experimental determination and modeling of arsenic complexation with humic and fulvic acids.

PubMed

Fakour, Hoda; Lin, Tsair-Fuh

2014-08-30

The complexation of humic acid (HA) and fulvic acid (FA) with arsenic (As) in water was studied. Experimental results indicate that arsenic may form complexes with HA and FA with a higher affinity for arsenate than for arsenite. With the presence of iron oxide based adsorbents, binding of arsenic to HA/FA in water was significantly suppressed, probably due to adsorption of As and HA/FA. A two-site ligand binding model, considering only strong and weak site types of binding affinity, was successfully developed to describe the complexation of arsenic on the two natural organic fractions. The model showed that the numbers of weak sites were more than 10 times those of strong sites on both HA and FA for both arsenic species studied. The numbers of both types of binding sites were found to be proportional to the HA concentrations, while the apparent stability constants, defined for describing binding affinity between arsenic and the sites, are independent of the HA concentrations. To the best of our knowledge, this is the first study to characterize the impact of HA concentrations on the applicability of the ligand binding model, and to extrapolate the model to FA. The obtained results may give insights on the complexation of arsenic in HA/FA laden groundwater and on the selection of more effective adsorption-based treatment methods for natural waters. Copyright © 2014 Elsevier B.V. All rights reserved.

H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

PubMed Central

Piché-Choquette, Sarah; Tremblay, Julien; Tringe, Susannah G.

2016-01-01

Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities. PMID:26989620

Melanin-Based Coatings as Lead-Binding Agents

PubMed Central

Sono, Karin; Lye, Diane; Moore, Christine A.; Boyd, W. Christopher; Gorlin, Thomas A.; Belitsky, Jason M.

2012-01-01

Interactions between metal ions and different forms of melanin play significant roles in melanin biochemistry. The binding properties of natural melanin and related synthetic materials can be exploited for nonbiological applications, potentially including water purification. A method for investigating metal ion-melanin interactions on solid support is described, with lead as the initial target. 2.5 cm discs of the hydrophobic polymer PVDF were coated with synthetic eumelanin from the tyrosinase-catalyzed polymerization of L-dopa, and with melanin extracted from human hair. Lead (Pb2+) binding was quantified by atomic absorption spectroscopy (flame mode), and the data was well fit by the Langmuir model. Langmuir affinities ranged from 3.4 · 103 to 2.2 · 104 M−1. At the maximum capacity observed, the synthetic eumelanin coating bound ~9% of its mass in lead. Binding of copper (Cu2+), zinc (Zn2+), and cadmium (Cd2+) to the synthetic-eumelanin-coated discs was also investigated. Under the conditions tested, the Langmuir affinities for Zn2+, Cd2+, and Cu2+ were 35%, 53%, and 77%, respectively, of the Langmuir affinity for Pb2+. The synthetic-eumelanin-coated discs have a slightly higher capacity for Cu2+ on a per mole basis than for Pb2+, and lower capacities for Cd2+ and Zn2+. The system described can be used to address biological questions and potentially be applied toward melanin-based water purification. PMID:22611345

Covalent Organic Frameworks as a Decorating Platform for Utilization and Affinity Enhancement of Chelating Sites for Radionuclide Sequestration

DOE PAGES

Sun, Qi; Aguila, Briana; Earl, Lyndsey D.; ...

2018-03-27

The potential consequences of nuclear events and the complexity of nuclear waste management motivate the development of selective solid-phase sorbents to provide enhanced protection. In this paper, it is shown that 2D covalent organic frameworks (COFs) with unique structures possess all the traits to be well suited as a platform for the deployment of highly efficient sorbents such that they exhibit remarkable performance, as demonstrated by uranium capture. The chelating groups laced on the open 1D channels exhibit exceptional accessibility, allowing significantly higher utilization efficiency. In addition, the 2D extended polygons packed closely in an eclipsed fashion bring chelating groupsmore » in adjacent layers parallel to each other, which may facilitate their cooperation, thereby leading to high affinity toward specific ions. As a result, the amidoxime-functionalized COFs far outperform their corresponding amorphous analogs in terms of adsorption capacities, kinetics, and affinities. Specifically, COF-TpAb-AO is able to reduce various uranium contaminated water samples from 1 ppm to less than 0.1 ppb within several minutes, well below the drinking water limit (30 ppb), as well as mine uranium from spiked seawater with an exceptionally high uptake capacity of 127 mg g -1. Finally, these results delineate important synthetic advances toward the implementation of COFs in environmental remediation.« less

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma

PubMed Central

Sánchez-Prieto, Ricardo; Saada, Sofiane; Naves, Thomas; Guillaudeau, Angélique; Perraud, Aurélie; Sindou, Philippe; Lacroix, Aurélie; Descazeaud, Aurélien; Lalloué, Fabrice; Jauberteau, Marie-Odile

2016-01-01

p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC. PMID:27120782

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

PubMed

Piekielna, Justyna; De Marco, Rossella; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tömböly, Csaba; Artali, Roberto; Janecka, Anna

2016-05-01

The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016. © 2016 Wiley Periodicals, Inc.

Effect of the dilution rate on microbial competition: r-strategist can win over k-strategist at low substrate concentration.

PubMed

Winkler, Mari-K H; Boets, Pieter; Hahne, Birk; Goethals, Peter; Volcke, Eveline I P

2017-01-01

The conditions present in both in vitro and in vivo ecosystems determine the microbial population harbouring it. One commonly accepted theory is that a species with a high substrate affinity and low growth rate (k-strategist) will win the competition against a second species with a lower substrate affinity and higher growth rate (r-strategist) if both species are subjected to low substrate concentrations. In this study two nitrite oxidizing bacteria (NOB), Nitrospira defluvii (k-strategist) and Nitrobacter vulgaris (r-strategist), were cultivated in a continuous reactor systems. The minimal hydraulic retention time (HRT) required for maintaining the slower growing Nitrospira was first determined. A reactor containing Nitrobacter was set to the same HRT and Nitrospira was injected to evaluate the effect of the dilution rate on the competition between both species. By following the microbial population dynamics with qPCR analysis, it was shown that not only the substrate affinity drives the competition between k- and r-strategists but also the dilution rate. Experimental data and numerical simulations both revealed that the washout of Nitrobacter was significantly delayed at dilution rates close to the μmax of Nitrospira. The competition could be even reverted towards Nitrobacter (r-strategist) despite of low nitrite concentrations and dilution rates lower than the μmax of Nitrospira.

Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

PubMed

Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

2016-03-01

Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

PubMed

Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

2017-11-05

14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

Three Decades of Implementation Research in Higher Education: Limitations and Prospects of Theory Development

ERIC Educational Resources Information Center

Kohoutek, Jan

2013-01-01

The article adopts a comparative approach to review three periods of theory development in research into higher education policy implementation. Given the conceptual affinity between Cerych and Sabatier's 1986 seminal study into higher education policy implementation and public policy implementation theory, the field of public policy is chosen for…

Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

PubMed

Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

2016-09-01

Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

PubMed Central

Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

2010-01-01

The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

PubMed

Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

2011-05-25

Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

PubMed Central

Cerezo, Miguel; Camañes, Gemma; Flors, Víctor; Primo-Millo, Eduardo

2007-01-01

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO3−. The Vmax for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks. Additionally, we studied the regulation of root NO3− uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO3− and down-regulated by the N status and by NO3− itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status. The use of various metabolic uncouplers or inhibitors indicated that NO3− net uptake mediated by iHATS and LATS was an active transport system in both rootstocks. PMID:19516998

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

PubMed

Rogez-Florent, Tiphaine; Goossens, Laurence; Drucbert, Anne-Sophie; Duban-Deweer, Sophie; Six, Perrine; Depreux, Patrick; Danzé, Pierre-Marie; Goossens, Jean-François; Foulon, Catherine

2016-10-15

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic evidence on the origins of Indian caste populations.

PubMed

Bamshad, M; Kivisild, T; Watkins, W S; Dixon, M E; Ricker, C E; Rao, B B; Naidu, J M; Prasad, B V; Reddy, P G; Rasanayagam, A; Papiha, S S; Villems, R; Redd, A J; Hammer, M F; Nguyen, S V; Carroll, M L; Batzer, M A; Jorde, L B

2001-06-01

The origins and affinities of the approximately 1 billion people living on the subcontinent of India have long been contested. This is owing, in part, to the many different waves of immigrants that have influenced the genetic structure of India. In the most recent of these waves, Indo-European-speaking people from West Eurasia entered India from the Northwest and diffused throughout the subcontinent. They purportedly admixed with or displaced indigenous Dravidic-speaking populations. Subsequently they may have established the Hindu caste system and placed themselves primarily in castes of higher rank. To explore the impact of West Eurasians on contemporary Indian caste populations, we compared mtDNA (400 bp of hypervariable region 1 and 14 restriction site polymorphisms) and Y-chromosome (20 biallelic polymorphisms and 5 short tandem repeats) variation in approximately 265 males from eight castes of different rank to approximately 750 Africans, Asians, Europeans, and other Indians. For maternally inherited mtDNA, each caste is most similar to Asians. However, 20%-30% of Indian mtDNA haplotypes belong to West Eurasian haplogroups, and the frequency of these haplotypes is proportional to caste rank, the highest frequency of West Eurasian haplotypes being found in the upper castes. In contrast, for paternally inherited Y-chromosome variation each caste is more similar to Europeans than to Asians. Moreover, the affinity to Europeans is proportionate to caste rank, the upper castes being most similar to Europeans, particularly East Europeans. These findings are consistent with greater West Eurasian male admixture with castes of higher rank. Nevertheless, the mitochondrial genome and the Y chromosome each represents only a single haploid locus and is more susceptible to large stochastic variation, bottlenecks, and selective sweeps. Thus, to increase the power of our analysis, we assayed 40 independent, biparentally inherited autosomal loci (1 LINE-1 and 39 Alu elements) in all of the caste and continental populations (approximately 600 individuals). Analysis of these data demonstrated that the upper castes have a higher affinity to Europeans than to Asians, and the upper castes are significantly more similar to Europeans than are the lower castes. Collectively, all five datasets show a trend toward upper castes being more similar to Europeans, whereas lower castes are more similar to Asians. We conclude that Indian castes are most likely to be of proto-Asian origin with West Eurasian admixture resulting in rank-related and sex-specific differences in the genetic affinities of castes to Asians and Europeans.

Genetic Evidence on the Origins of Indian Caste Populations

PubMed Central

Bamshad, Michael; Kivisild, Toomas; Watkins, W. Scott; Dixon, Mary E.; Ricker, Chris E.; Rao, Baskara B.; Naidu, J. Mastan; Prasad, B.V. Ravi; Reddy, P. Govinda; Rasanayagam, Arani; Papiha, Surinder S.; Villems, Richard; Redd, Alan J.; Hammer, Michael F.; Nguyen, Son V.; Carroll, Marion L.; Batzer, Mark A.; Jorde, Lynn B.

2001-01-01

The origins and affinities of the ∼1 billion people living on the subcontinent of India have long been contested. This is owing, in part, to the many different waves of immigrants that have influenced the genetic structure of India. In the most recent of these waves, Indo-European-speaking people from West Eurasia entered India from the Northwest and diffused throughout the subcontinent. They purportedly admixed with or displaced indigenous Dravidic-speaking populations. Subsequently they may have established the Hindu caste system and placed themselves primarily in castes of higher rank. To explore the impact of West Eurasians on contemporary Indian caste populations, we compared mtDNA (400 bp of hypervariable region 1 and 14 restriction site polymorphisms) and Y-chromosome (20 biallelic polymorphisms and 5 short tandem repeats) variation in ∼265 males from eight castes of different rank to ∼750 Africans, Asians, Europeans, and other Indians. For maternally inherited mtDNA, each caste is most similar to Asians. However, 20%–30% of Indian mtDNA haplotypes belong to West Eurasian haplogroups, and the frequency of these haplotypes is proportional to caste rank, the highest frequency of West Eurasian haplotypes being found in the upper castes. In contrast, for paternally inherited Y-chromosome variation each caste is more similar to Europeans than to Asians. Moreover, the affinity to Europeans is proportionate to caste rank, the upper castes being most similar to Europeans, particularly East Europeans. These findings are consistent with greater West Eurasian male admixture with castes of higher rank. Nevertheless, the mitochondrial genome and the Y chromosome each represents only a single haploid locus and is more susceptible to large stochastic variation, bottlenecks, and selective sweeps. Thus, to increase the power of our analysis, we assayed 40 independent, biparentally inherited autosomal loci (1 LINE-1 and 39 Alu elements) in all of the caste and continental populations (∼600 individuals). Analysis of these data demonstrated that the upper castes have a higher affinity to Europeans than to Asians, and the upper castes are significantly more similar to Europeans than are the lower castes. Collectively, all five datasets show a trend toward upper castes being more similar to Europeans, whereas lower castes are more similar to Asians. We conclude that Indian castes are most likely to be of proto-Asian origin with West Eurasian admixture resulting in rank-related and sex-specific differences in the genetic affinities of castes to Asians and Europeans. PMID:11381027

Modeling the binding affinity of structurally diverse industrial chemicals to carbon using the artificial intelligence approaches.

PubMed

Gupta, Shikha; Basant, Nikita; Rai, Premanjali; Singh, Kunwar P

2015-11-01

Binding affinity of chemical to carbon is an important characteristic as it finds vast industrial applications. Experimental determination of the adsorption capacity of diverse chemicals onto carbon is both time and resource intensive, and development of computational approaches has widely been advocated. In this study, artificial intelligence (AI)-based ten different qualitative and quantitative structure-property relationship (QSPR) models (MLPN, RBFN, PNN/GRNN, CCN, SVM, GEP, GMDH, SDT, DTF, DTB) were established for the prediction of the adsorption capacity of structurally diverse chemicals to activated carbon following the OECD guidelines. Structural diversity of the chemicals and nonlinear dependence in the data were evaluated using the Tanimoto similarity index and Brock-Dechert-Scheinkman statistics. The generalization and prediction abilities of the constructed models were established through rigorous internal and external validation procedures performed employing a wide series of statistical checks. In complete dataset, the qualitative models rendered classification accuracies between 97.04 and 99.93%, while the quantitative models yielded correlation (R(2)) values of 0.877-0.977 between the measured and the predicted endpoint values. The quantitative prediction accuracies for the higher molecular weight (MW) compounds (class 4) were relatively better than those for the low MW compounds. Both in the qualitative and quantitative models, the Polarizability was the most influential descriptor. Structural alerts responsible for the extreme adsorption behavior of the compounds were identified. Higher number of carbon and presence of higher halogens in a molecule rendered higher binding affinity. Proposed QSPR models performed well and outperformed the previous reports. A relatively better performance of the ensemble learning models (DTF, DTB) may be attributed to the strengths of the bagging and boosting algorithms which enhance the predictive accuracies. The proposed AI models can be useful tools in screening the chemicals for their binding affinities toward carbon for their safe management.

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

PubMed

Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

1997-10-15

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

PubMed Central

Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

2015-01-01

AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

Chelating effect in short polymers for the design of bidentate binders of increased affinity and selectivity

PubMed Central

Fortuna, Sara; Fogolari, Federico; Scoles, Giacinto

2015-01-01

The design of new strong and selective binders is a key step towards the development of new sensing devices and effective drugs. Both affinity and selectivity can be increased through chelation and here we theoretically explore the possibility of coupling two binders through a flexible linker. We prove the enhanced ability of double binders of keeping their target with a simple model where a polymer composed by hard spheres interacts with a spherical macromolecule, such as a protein, through two sticky spots. By Monte Carlo simulations and thermodynamic integration we show the chelating effect to hold for coupling polymers whose radius of gyration is comparable to size of the chelated particle. We show the binding free energy of flexible double binders to be higher than that of two single binders and to be maximized when the binding sites are at distances comparable to the mean free polymer end-to-end distance. The affinity of two coupled binders is therefore predicted to increase non linearly and in turn, by targeting two non-equivalent binding sites, this will lead to higher selectivity. PMID:26496975

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-07-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed Central

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-01-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin. PMID:2544892

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome.

PubMed

Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping; Tumer, Nilgun E

2016-12-01

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Hypoxia delays hematopoiesis: retention of embryonic hemoglobin and erythrocytes in larval rainbow trout, Oncorhynchus mykiss, during chronic hypoxia exposure.

PubMed

Bianchini, Kristin; Wright, Patricia A

2013-12-01

In rainbow trout development, a switch occurs from high-affinity embryonic hemoglobin (Hb) and round, embryonic erythrocytes to lower-affinity adult Hb and oval, adult erythrocytes. Our study investigated the early ontogeny of rainbow trout blood properties and the hypoxia response. We hypothesized that hypoxia exposure would delay the ontogenetic turnover of Hb and erythrocytes because retention of high-affinity embryonic Hb would facilitate oxygen loading. To test this hypothesis we developed a method of efficiently extracting blood from individual embryos and larvae and optimized several techniques for measuring hematological parameters on microliter (0.5-2.0 μl) blood samples. In chronic hypoxia (30% of oxygen saturation), stage-matched embryos and larvae possessed half the Hb concentration, erythrocyte counts and hematocrit observed in normoxia. Hypoxia-reared larvae also had threefold to sixfold higher mRNA expression of the embryonic Hb α-1, β-1 and β-2 subunits relative to stage-matched normoxia-reared larvae. Furthermore, in hypoxia, the round embryonic erythrocytic shape persisted into later developmental stages. Despite these differences, Hb-oxygen affinity (P50), cooperativity and the Root effect were unaltered in hypoxia-reared O. mykiss. The data support our hypothesis that chronic hypoxia delays the ontogenetic turnover of Hb and erythrocytes, but without the predicted functional consequences (i.e. higher than expected P50). These results also suggest that the Hb-oxygen affinity is protected during development in chronic hypoxia to favor oxygen unloading at the tissues. We conclude that in early trout development, the blood-oxygen transport system responds very differently to chronic hypoxia relative to adults, possibly because respiration depends relatively more on oxygen diffusion than convection.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Two-phase positive inotropic effects of ouabain and the presence of multiple classes of ouabain binding sites in the ferret heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Y.C.; Akera, T.

1986-03-05

Characteristics of more than one class of ouabain receptors which appear to exist in ferret heart were examined. In isolated papillary muscle, 1 to 30 nM ouabain produced a positive inotropic effect in the presence of 5 ..mu..M propranolol and 2 ..mu..M phentolamine. Higher concentrations of ouabain (0.1 to 10 ..mu..M) produced an additional and prominent inotropic effect. In partially purified Na, K-ATPase, ouabain caused a monophasic inhibition; however, the concentration-inhibition curve spanned over 5 log units, indicating that ouabain is interacting with more than a single class of the enzyme. Scatchard analysis of specific /sup 3/H-ouabain binding revealed approximatelymore » equal abundance of high and low affinity binding sites. The K/sub D/ value for high affinity sites was approximately 20 nM whereas that for low affinity sites was about 45 times higher. When phosphoenzyme was formed in the presence of (..gamma..-/sup 32/P)-ATP, Mg/sup 2 +/ and Na/sup +/ and subjected to SDS gel electrophoresis, two distinct K/sup +/-sensitive bands with about 100,000 dalton molecular weight were detected. Molecular weight difference between these two bands was approximately 2500 dalton. Phosphorylation of either band was abolished by 1 ..mu..M ouabain suggesting that both bands may correspond to the high-affinity binding sites. These results indicate that high and low affinity ouabain binding sites exists in approximately equal abundance in the ferret heart, and that binding of ouabain to these sites cases Na,K-ATPase inhibition and the positive inotropic effect.« less

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

Evaluation of respiratory and cardiac motion correction schemes in dual gated PET/CT cardiac imaging.

PubMed

Lamare, F; Le Maitre, A; Dawood, M; Schäfers, K P; Fernandez, P; Rimoldi, O E; Visvikis, D

2014-07-01

Cardiac imaging suffers from both respiratory and cardiac motion. One of the proposed solutions involves double gated acquisitions. Although such an approach may lead to both respiratory and cardiac motion compensation there are issues associated with (a) the combination of data from cardiac and respiratory motion bins, and (b) poor statistical quality images as a result of using only part of the acquired data. The main objective of this work was to evaluate different schemes of combining binned data in order to identify the best strategy to reconstruct motion free cardiac images from dual gated positron emission tomography (PET) acquisitions. A digital phantom study as well as seven human studies were used in this evaluation. PET data were acquired in list mode (LM). A real-time position management system and an electrocardiogram device were used to provide the respiratory and cardiac motion triggers registered within the LM file. Acquired data were subsequently binned considering four and six cardiac gates, or the diastole only in combination with eight respiratory amplitude gates. PET images were corrected for attenuation, but no randoms nor scatter corrections were included. Reconstructed images from each of the bins considered above were subsequently used in combination with an affine or an elastic registration algorithm to derive transformation parameters allowing the combination of all acquired data in a particular position in the cardiac and respiratory cycles. Images were assessed in terms of signal-to-noise ratio (SNR), contrast, image profile, coefficient-of-variation (COV), and relative difference of the recovered activity concentration. Regardless of the considered motion compensation strategy, the nonrigid motion model performed better than the affine model, leading to higher SNR and contrast combined with a lower COV. Nevertheless, when compensating for respiration only, no statistically significant differences were observed in the performance of the two motion models considered. Superior image SNR and contrast were seen using the affine respiratory motion model in combination with the diastole cardiac bin in comparison to the use of the whole cardiac cycle. In contrast, when simultaneously correcting for cardiac beating and respiration, the elastic respiratory motion model outperformed the affine model. In this context, four cardiac bins associated with eight respiratory amplitude bins seemed to be adequate. Considering the compensation of respiratory motion effects only, both affine and elastic based approaches led to an accurate resizing and positioning of the myocardium. The use of the diastolic phase combined with an affine model based respiratory motion correction may therefore be a simple approach leading to significant quality improvements in cardiac PET imaging. However, the best performance was obtained with the combined correction for both cardiac and respiratory movements considering all the dual-gated bins independently through the use of an elastic model based motion compensation.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C; Munson, P J; Rodbard, D

1979-12-01

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

NASA Astrophysics Data System (ADS)

Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

2016-06-01

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

ECL-IAA and ECL-GADA Can Identify High-Risk Single Autoantibody-Positive Relatives in the TrialNet Pathway to Prevention Study

PubMed Central

Fouts, Alexandra; Miao, Dongmei; Zhao, Zhiyuan; Dong, Fran; Sosenko, Jay; Gottlieb, Peter; Rewers, Marian J.

2016-01-01

Abstract Background: Relatives with single positive islet autoantibodies have a much lower risk of progression to diabetes than those with multiple autoantibodies. Materials and Methods: TrialNet subjects positive for single autoantibody to insulin (mIAA) (n = 50) or single autoantibody to glutamic acid decarboxylase (GADA) (n = 50) were analyzed using new electrochemiluminescence (ECL) assays (ECL-IAA and ECL-GADA, respectively) at their initial visit and longitudinally over time. Affinity assays were performed on a subset of single autoantibody-positive subjects at initial and most recent visits. Results: After a mean follow-up of 5.3 years, 20 subjects developed type 1 diabetes. Among either single GADA or single mIAA subjects, those who were positive in the ECL assay showed higher affinity at the initial visit, and affinity results stayed consistent over time. No converting events from low to high or high to low affinity were seen over time. Conclusions: Confirmed positivity for ECL is associated with high affinity and can help staging of risk for type 1 diabetes in single autoantibody-positive subjects. PMID:26991969

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Higher spin black holes with soft hair

NASA Astrophysics Data System (ADS)

Grumiller, Daniel; Pérez, Alfredo; Prohazka, Stefan; Tempo, David; Troncoso, Ricardo

2016-10-01

We construct a new set of boundary conditions for higher spin gravity, inspired by a recent "soft Heisenberg hair"-proposal for General Relativity on three-dimensional Anti-de Sitter space. The asymptotic symmetry algebra consists of a set of affine û(1) current algebras. Its associated canonical charges generate higher spin soft hair. We focus first on the spin-3 case and then extend some of our main results to spin- N , many of which resemble the spin-2 results: the generators of the asymptotic W 3 algebra naturally emerge from composite operators of the û(1) charges through a twisted Sugawara construction; our boundary conditions ensure regularity of the Euclidean solutions space independently of the values of the charges; solutions, which we call "higher spin black flowers", are stationary but not necessarily spherically symmetric. Finally, we derive the entropy of higher spin black flowers, and find that for the branch that is continuously connected to the BTZ black hole, it depends only on the affine purely gravitational zero modes. Using our map to W -algebra currents we recover well-known expressions for higher spin entropy. We also address higher spin black flowers in the metric formalism and achieve full consistency with previous results.

Influences of Histidine-1 and Azaphenylalanine-4 on the Affinity, Anti-inflammatory, and Antiangiogenic Activities of Azapeptide Cluster of Differentiation 36 Receptor Modulators.

PubMed

Chignen Possi, Kelvine; Mulumba, Mukandila; Omri, Samy; Garcia-Ramos, Yesica; Tahiri, Houda; Chemtob, Sylvain; Ong, Huy; Lubell, William D

2017-11-22

Azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) exhibit promising affinity, selectivity, and modulator activity on the cluster of differentiation 36 receptor (CD36). For example, [A 1 , azaF 4 ]- and [azaY 4 ]-GHRP-6 (1a and 2b) were previously shown to bind selectively to CD36 and exhibited respectively significant antiangiogenic and slight angiogenic activities in a microvascular sprouting assay using choroid explants. The influences of the 1- and 4-position residues on the affinity, anti-inflammatory, and antiangiogenic activity of these azapeptides have now been studied in detail by the synthesis and analysis of a set of 25 analogues featuring Ala 1 or His 1 and a variety of aromatic side chains at the aza-amino acid residue in the 4-position. Although their binding affinities differed only by a factor of 17, the analogues exhibited significant differences in ability to modulate production of nitric oxide (NO) in macrophages and choroidal neovascularization.

Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity.

PubMed

Gladwin, M T; Schechter, A N; Shelhamer, J H; Pannell, L K; Conway, D A; Hrinczenko, B W; Nichols, J S; Pease-Fye, M E; Noguchi, C T; Rodgers, G P; Ognibene, F P

1999-10-01

Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion.

Biochemical Regulatory Features of Activation-Induced Cytidine Deaminase Remain Conserved from Lampreys to Humans

PubMed Central

King, Justin J.; Amemiya, Chris T.; Hsu, Ellen

2017-01-01

ABSTRACT Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties. PMID:28716949

Interaction of phenolic acids and their derivatives with human serum albumin: Structure-affinity relationships and effects on antioxidant activity.

PubMed

Zhang, Yunyue; Wu, Simin; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Ren, Fazheng; Zhang, Hao

2018-02-01

In this study, 111 phenolic acids and their derivatives were chosen to investigate their structure-affinity relationships when binding to human serum albumin (HSA), and effects on their antioxidant activity. A comprehensive mathematical model was employed to calculate the binding constants, using a fluorescence quenching method, and this was corrected for the inner-filter effect to improve accuracy. We found that a hydroxy group at the 2-position of the benzene ring exerted a positive effect on the affinities, while a 4-hydroxy substituent had a negative influence. Both methylation of the hydroxy groups and replacing the hydroxy groups with methyl groups at the 3- and 4-positions of the benzene ring enhanced the binding affinities. Hydrophobic force and hydrogen bonding were binding forces for the phenolic acids, and their methyl esters, respectively. The antioxidant activity of the HSA-phenolic acid interaction compounds was higher than that of the phenolic acids alone. Copyright © 2017. Published by Elsevier Ltd.

Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.

PubMed

Hultsch, Christina; Pawelke, Beate; Bergmann, Ralf; Wuest, Frank

2006-09-01

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

Peptide-nucleic acids (PNAs) with pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT) as a universal base: their synthesis and binding affinity for oligodeoxyribonucleotides.

PubMed

Hirano, Taisuke; Kuroda, Kenji; Kataoka, Masanori; Hayakawa, Yoshihiro

2009-07-21

Peptide-nucleic acids (PNAs) including pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT) as a nucleobase were synthesized, and their binding affinity for the complementary oligodeoxyribonucleotides was investigated. We found that PNAs with one or two PPT(s) and natural nucleobases (i.e., adenine, cytosine, guanine, or thymine) have excellent binding affinity for oligodeoxyribonucleotides with complementary bases at the positions facing the natural nucleobases, and with adenine, cytosine, guanine, and thymine at the positions opposite PPT in PNAs. The binding affinity of the PPT-containing PNA is higher than or comparable to that of a PNA consisting of all complementary natural nucleobases, viz. a PNA with a suitable natural nucleobase in place of PPT in the PPT-containing PNA. Consequently, it was concluded that PPT serves as a useful universal base that can recognize all natural nucleobases.

One-pot synthesis and sigma receptor binding studies of novel spirocyclic-2,6-diketopiperazine derivatives.

PubMed

Ghandi, Mehdi; Sherafat, Fatemeh; Sadeghzadeh, Masoud; Alirezapour, Behrouz

2016-06-01

New spirocyclic-2,6-diketopiperazine derivatives containing benzylpiperidine and cycloalkane moieties were synthesized by a one-pot two-step sequential Ugi/intramolecular N-amidation process in moderate to good yields. The in vitro ligand-binding profile studies performed on the sigma-1 and sigma-2 receptors revealed that the σ1 affinities and subtype selectivities of three spirocyclic piperidine derivatives are generally comparable to those of spirocycloalkane analogues. Compared to the low σ1 affinities obtained for cycloalkyl-substituted spirocyclic-2,6-diketopiperazines with n=2, those with n=1 proved to have optimal fitting with σ2 subtype by exhibiting higher affinities. Moreover, the best binding affinity and subtype selectivity was identified for compound 3c with Kiσ1=5.9±0.5nM and Kiσ2=563±21nM as well as 95-fold σ1/σ2 selectivity ratio, respectively. Copyright © 2016. Published by Elsevier Ltd.

Synthesis, modelling, and mu-opioid receptor affinity of N-3(9)-arylpropenyl-N-9(3)-propionyl-3,9-diazabicycl.

PubMed

Pinna, G A; Murineddu, G; Curzu, M M; Villa, S; Vianello, P; Borea, P A; Gessi, S; Toma, L; Colombo, D; Cignarella, G

2000-08-01

A series of N-3-arylpropenyl-N-9-propionyl-3,9-diazabicyclo[3.3.1]nonanes (1a-g) and of reverted N-3-propionyl-N-9-arylpropenyl isomers (2a-g), as homologues of the previously reported analgesic 3,8-diazabicyclo[3.2.1]octanes (I-II), were synthesized and evaluated for the binding affinity towards opioid receptor subtypes mu, delta and kappa. Compounds 1a-g and 2a-g exhibited a strong selective mu-affinity with Ki values in the nanomolar range, which favourably compared with those of I and II. In addition, contrary to the trend observed for DBO-I, II, the mu-affinity of series 2 is markedly higher than that of the isomeric series 1. This aspect was discussed on the basis of the conformational studies performed on DBN which allowed hypotheses on the mode of interaction of these compounds with the mu receptor.

Biogeographic affinity helps explain productivity-richness relationships at regional and local scales

USGS Publications Warehouse

Harrison, S.; Grace, J.B.

2007-01-01

The unresolved question of what causes the observed positive relationship between large-scale productivity and species richness has long interested ecologists and evolutionists. Here we examine a potential explanation that we call the biogeographic affinity hypothesis, which proposes that the productivity-richness relationship is a function of species' climatic tolerances that in turn are shaped by the earth's climatic history combined with evolutionary niche conservatism. Using botanical data from regions and sites across California, we find support for a key prediction of this hypothesis, namely, that the productivity-species richness relationship differs strongly and predictably among groups of higher taxa on the basis of their biogeographic affinities (i.e., between families or genera primarily associated with north-temperate, semiarid, or desert zones). We also show that a consideration of biogeographic affinity can yield new insights on how productivity-richness patterns at large geographic scales filter down to affect patterns of species richness and composition within local communities. ?? 2007 by The University of Chicago. All rights reserved.

Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

PubMed Central

Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

1992-01-01

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939

Distribution of Hydroxychloroquine in Lymphoid Tissue in a Rabbit Model for HIV Infection

PubMed Central

González-Hernández, Iliana; Aguirre-Cruz, Lucinda; Sotelo, Julio; López-Arellano, Raquel; Morales-Hipólito, Adriana

2014-01-01

Hydroxychloroquine has been proposed for HIV treatment; however, little is known about its disposition in the lymphatic system, where replication takes place. Therefore, its distribution in lymphoid tissues (Peyer's patches and popliteal, submandibular, femoral, splenic, and prescapular lymph nodes) was evaluated and compared with that in blood. Results showed a high affinity of hydroxychloroquine for all of these tissues, with higher affinity for the splenic and submandibular lymph nodes, suggesting its potential use as a coadjuvant in HIV therapy. PMID:24145523

Detection Method of TOXOPLASMA GONDII Tachyzoites

NASA Astrophysics Data System (ADS)

Eassa, Souzan; Bose, Chhanda; Alusta, Pierre; Tarasenko, Olga

2011-06-01

Tachyzoites are considered to be the most important stage of Toxoplasma gondii which causes toxoplasmosis. T. gondii is, an obligate intracellular parasite which infects a wide range of cells. The present study was designed to develop a method for an early detection of T. gondii tachyzoites. The method comprised of a binding assay which was analyzed using principal component and cluster analysis. Our data showed that glycoconjugates GC1, GC2, GC3 and GC10 exhibit a significantly higher binding affinity for T. gondii tachyzoites as compared to controls (T. gondii only, PAA only, GC 1, 2, 3, and 10 only).

Gas Phase Sensing of Alcohols by Metal Organic Framework–Polymer Composite Materials

PubMed Central

2017-01-01

Affinity layers play a crucial role in chemical sensors for the selective and sensitive detection of analytes. Here, we report the use of composite affinity layers containing Metal Organic Frameworks (MOFs) in a polymeric matrix for sensing purposes. Nanoparticles of NH2-MIL-53(Al) were dispersed in a Matrimid polymer matrix with different weight ratios (0–100 wt %) and drop-casted on planar capacitive transducer devices. These coated devices were electrically analyzed using impedance spectroscopy and investigated for their sensing properties toward the detection of a series of alcohols and water in the gas phase. The measurements indicated a reversible and reproducible response in all devices. Sensor devices containing 40 wt % NH2-MIL-53(Al) in Matrimid showed a maximum response for methanol and water. The sensor response time slowed down with increasing MOF concentration until 40 wt %. The half time of saturation response (τ0.5) increased by ∼1.75 times for the 40 wt % composition compared to devices coated with Matrimid only. This is attributed to polymer rigidification near the MOF/polymer interface. Higher MOF loadings (≥50 wt %) resulted in brittle coatings with a response similar to the 100 wt % MOF coating. Cross-sensitivity studies showed the ability to kinetically distinguish between the different alcohols with a faster response for methanol and water compared to ethanol and 2-propanol. The observed higher affinity of the pure Matrimid polymer toward methanol compared to water allows also for a higher uptake of methanol in the composite matrices. Also, as indicated by the sensing studies with a mixture of water and methanol, the methanol uptake is independent of the presence of water up to 6000 ppm of water. The NH2-MIL-53(Al) MOFs dispersed in the Matrimid matrix show a sensitive and reversible capacitive response, even in the presence of water. By tuning the precise compositions, the affinity kinetics and overall affinity can be tuned, showing the promise of this type of chemical sensors. PMID:28440621

The whereabouts of flower visitors: contrasting land-use preferences revealed by a country-wide survey based on citizen science.

PubMed

Deguines, Nicolas; Julliard, Romain; de Flores, Mathieu; Fontaine, Colin

2012-01-01

In the past decade, accumulating evidence of pollinator decline has raised concerns regarding the functioning of terrestrial ecosystems and the sustainability of crop production. Although land-use changes have been advanced as the major causes, the affinities of most wild pollinators with the main land-use types remain unknown. Filling this gap in our knowledge is a prerequisite to improving conservation and management programmes. We estimated the affinity of flower visitors with urban, agricultural and natural land-uses using data from a country-wide scale monitoring scheme based on citizen science (Spipoll). We tested whether the affinities differed among insect orders and according to insect frequency (frequent or infrequent). Our results indicate that the affinities with the three land-use types differed among insect orders. Apart from Hymenopterans, which appeared tolerant to the different land-uses, all flower visitors presented a negative affinity with urban areas and a positive affinity with agricultural and natural areas. Additionally, infrequent taxa displayed a lower affinity with urban areas and a higher affinity with natural areas than did frequent taxa. Within frequent taxa, Hymenoptera and Coleoptera included specialists of the three land-use types whereas Diptera and Lepidoptera contained specialists of all but urban areas. Our approach allowed the first standardised evaluation of the affinity of flower visitors with the main land-use types across a broad taxonomical range and a wide geographic scope. Our results suggest that the most detrimental land-use change for flower visitor communities is urbanisation. Moreover, our findings highlight the fact that agricultural areas have the potential to host highly diverse pollinator communities. We suggest that policy makers should, therefore, focus on the implementation of pollinator-friendly practices in agricultural lands. This may be a win-win strategy, as both biodiversity and crop production may benefit from healthier communities of flower visitors in these areas.

Ethnic Heterogeneity, Group Affinity, and State Higher Education Spending

ERIC Educational Resources Information Center

Foster, John M.; Fowles, Jacob

2018-01-01

A rich interdisciplinary literature exists exploring the determinants of state higher education funding policies. However, that work has collectively ignored an important finding from political economy literature: namely, that citizens' preferences regarding public spending are strongly influenced by the state's ethnic and racial context. Drawing…

A combined photophysical and computational study on the binding of mycophenolate mofetil and its major metabolite to transport proteins.

PubMed

Vendrell-Criado, Victoria; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2018-06-15

Binding of the immunosuppressive agent mycophenolate mofetil (MMP) and its pharmacologically active metabolite mycophenolic acid (MPA) to human serum albumin (HSA) and α 1 -acid glycoprotein (HAAG) has been investigated by means of an integrated approach involving selective excitation of the drug fluorophore, following their UV-A triggered fluorescence and docking studies. The formation of the protein/ligand complexes was evidenced by a dramatic enhancement of the fluorescence intensity and a hypsochromic shift of the emission band. In HSA, competitive studies using oleic acid as site I probe revealed site I as the main binding site of the ligands. Binding constants revealed that the affinity of the active metabolite by HSA is four-fold higher than its proactive form. Moreover, the affinity of MMP by HSA is three-fold higher than by HAAG. Docking studies revealed significant molecular binding differences in the binding of MMP and MPA to sub-domain IIA of HSA (site 1). For MPA, the aromatic moiety would be in close contact to Trp214 with the flexible chain pointing to the other end of the sub-domain; on the contrary, for MMP, the carboxylate group of the chain would be fixed nearby Trp214 through electrostatic interactions with residues Arg218 and Arg222. Copyright © 2018 Elsevier B.V. All rights reserved.

Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

PubMed Central

Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

2017-01-01

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

Adenosine transport systems on dissociated brain cells from mouse, guinea-pig, and rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnston, M.E.; Geiger, J.D.

1990-09-01

The kinetics and sodium dependence of adenosine transport were determined using an inhibitor-stop method on dissociated cell body preparations obtained from mouse, guinea-pig and rat brain. Transport affinity (KT) values for the high affinity adenosine transport systems KT(H) were significantly different between these three species; mean +/- SEM values were 0.34 +/- 0.1 in mouse, 0.9 +/- 0.2 in rat, and 1.5 +/- 0.5 microM in guinea-pig. The KT values for the low affinity transport system KT(L) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate (3H)adenosine (Vmax) than didmore » mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the KT(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between KT(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse, guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.« less

Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

PubMed Central

Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

2018-01-01

Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qibin; Tang, Ning; Brock, Jonathan W.

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resultedmore » in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.« less

Tailoring the affinity of organosilica membranes by introducing polarizable ethenylene bridges and aqueous ozone modification.

PubMed

Xu, Rong; Kanezashi, Masakoto; Yoshioka, Tomohisa; Okuda, Tetsuji; Ohshita, Joji; Tsuru, Toshinori

2013-07-10

Bis(triethoxysilyl)ethylene (BTESEthy) was used as a novel precursor to develop a microporous organosilica membrane via the sol-gel technique. Water sorption measurements confirmed that ethenylene-bridged BTESEthy networks had a higher affinity for water than that of ethane-bridged organosilica materials. High permeance of CO2 with high CO2/N2 selectivity was explained relative to the strong CO2 adsorption on the network with π-bond electrons. The introduction of polarizable and rigid ethenylene bridges in the network structure led to improved water permeability and high NaCl rejection (>98.5%) in reverse osmosis (RO). Moreover, the aqueous ozone modification promoted significant improvement in the water permeability of the membrane. After 60 min of ozone exposure, the water permeability reached 1.1 × 10(-12) m(3)/(m(2) s Pa), which is close to that of a commercial seawater RO membrane. Meanwhile, molecular weight cutoff measurements indicated a gradual increase in the effective pore size with ozone modification, which may present new options for fine-tuning of membrane pore sizes.

Kinetic characteristics of native γ-glutamylcysteine ligase in the aging housefly, Musca domestica L.☆

PubMed Central

Toroser, Dikran; Sohal, Rajindar S.

2010-01-01

The catalytic activity of γ-glutamylcysteine ligase (γ-GCL; EC 6.3.2.2) was compared between relatively young (4-day-old) and old (19-day-old) houseflies (Musca domestica) in order to understand the mechanism of putative deterioration of glutathione homeostasis during the aging process. Hanes–Woolf analyses ([S]/v vs [S]) indicated that γ-GCL had significantly higher affinities for its substrates in the young than in the old flies. The Km values in the young and old flies were, respectively, for glutamate 0.6 and 5.5 mM; for cysteine 0.3 and 4.6 mM; and for ATP 1.2 and 2.9 mM. Furthermore, young but not old flies exhibited substrate-dependent inhibition of γ-GCL activity at >5 mM cysteine indicating a loss of metabolic regulation during aging. The age-associated differences in the affinity of native γ-GCL towards its substrates suggest that de novo synthesis of glutathione would be relatively less efficient in the old houseflies. PMID:15596139

Cadmium mobility in sediments and soils from a coal mining area on Tibagi River watershed: environmental risk assessment.

PubMed

Galunin, Evgeny; Ferreti, Jeferson; Zapelini, Iago; Vieira, Isadora; Ricardo Teixeira Tarley, César; Abrão, Taufik; Santos, Maria Josefa

2014-01-30

The risk of cadmium contamination in the Tibagi River watershed (Parana State, Brazil) affected by past coal mining activities was assessed through sorption-desorption modeling for sediment and soil samples. The acidic character of the samples resulted in more competition between the cadmium ions and protons, thereby influencing the cadmium sorption-desorption. The sorption isotherms were fitted to the Langmuir and Freundlich single models and to the dual-site Langmuir-Freundlich (or Sips) model. The single-site models indicated a low-energy character of sorption sites on the sample sorption sites, whereas the dual-site model explained the availability of higher-affinity and lower-affinity non-specific sites. The correlation of the sorption and desorption constants with the physicochemical and mineralogical characteristics of the samples showed that the cadmium sorption behavior was significantly affected by the pH, point of zero charge, and also by the magnesium, aluminum, calcium and manganese amounts. Besides, the desorption rate and hysteresis index suggested a high risk of cadmium mobilization along the Tibagi River basin. Copyright © 2013 Elsevier B.V. All rights reserved.

CoVaccine HT™ adjuvant is superior to Freund's adjuvants in eliciting antibodies against the endogenous alarmin HMGB1.

PubMed

Lakhan, Nerissa; Stevens, Natalie E; Diener, Kerrilyn R; Hayball, John D

2016-12-01

Adjuvants are used to enhance the immune response against specific antigens for the production of antibodies, with the choice of adjuvant most critical for poorly immunogenic and self-antigens. This study quantitatively and qualitatively evaluated CoVaccine HT™ and Freund's adjuvants for eliciting therapeutic ovine polyclonal antibodies targeting the endogenous alarmin, high mobility group box-1 (HMGB1). Sheep were immunised with HMGB1 protein in CoVaccine HT™ or Freund's adjuvants, with injection site reactions and antibody titres periodically assessed. The binding affinity of antibodies for HMGB1 and their neutralisation activity was determined in-vitro, with in vivo activity confirmed using a murine model of endotoxemia. Results indicated that CoVaccine HT™ elicited significantly higher antibody tires with stronger affinity and more functional potency than antibodies induced with Freund's adjuvants. These studies provide evidence that CoVaccine HT™ is superior to Freund's adjuvants for the production of antibodies to antigens with low immunogenicity and supports the use of this alternative adjuvant for clinical and experimental use antibodies. Copyright © 2016 Elsevier B.V. All rights reserved.

Bioavailability of methyl parathion adsorbed on clay minerals and iron oxide.

PubMed

Cai, Peng; He, Xiaomin; Xue, Aifang; Chen, Hao; Huang, Qiaoyun; Yu, Jun; Rong, Xinming; Liang, Wei

2011-01-30

Adsorption, desorption and degradation by Pseudomonas putida of methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) on montmorillonite, kaolinite and goethite were studied. Metabolic activities of methyl parathion-degrading bacteria P. putida in the presence of minerals were also monitored by microcalorimetry to determine the degradation mechanism of methyl parathion. Montmorillonite presented higher adsorption capacity and affinity for methyl parathion than kaolinite and goethite. The percentage of degradation of methyl parathion adsorbed on minerals by P. putida was in the order of montmorillonite>kaolinite>goethite. The presence of minerals inhibited the exponential growth and the metabolic activity of P. putida. Among the examined minerals, goethite exhibited the greatest inhibitory effect on bacterial activity, while montmorillonite was the least depressing. The biodegradation of adsorbed methyl parathion by P. putida is apparently not controlled by the adsorption affinity of methyl parathion on minerals and may be mainly governed by the activity of the methyl parathion-degrading bacteria. The information obtained in this study is of fundamental significance for the understanding of the behavior of methyl parathion in soil environments. Copyright © 2010 Elsevier B.V. All rights reserved.

All-boron fullerene exhibits a strong affinity to inorganic anions

NASA Astrophysics Data System (ADS)

Colherinhas, Guilherme; Fileti, Eudes Eterno; Chaban, Vitaly V.

2017-03-01

Experimentally observed all-boron fullerene, B-80, inspires systematic investigation of its physical chemical properties and search for possible applications. We hereby report density functional theory calculations to characterize interactions of B-80 with the selected imidazolium room-temperature ionic liquids (RTILs), dimethylimidazolium nitrate and dimethylimidazolium hexafluorophosphate. Whereas the imidazolium cation exhibits a rather poor affinity to B-80, the inorganic anions form polar covalent bonds with the boron atom occupying a central position within a B-6 hexagon. Attachment of the RTIL ion pairs leads to a significant alteration of the electronic spectra, charge density distribution, valence and conduction molecular orbitals. The total binding energies keeping the RTIL@B80 complexes together range 200-250 kcal mol-1, being higher than the energies of many interactions in chemistry. The observed phenomenon predicts an excellent solubility of B-80 in the considered RTILs, but may also reveal a poor stability of B-80 in the polar media. Our results motivate further efforts in studying the behavior of the all-boron fullerene in polar environments.

Synthesis of β-galactosylamides as ligands of the peanut lectin. Insights into the recognition process.

PubMed

Cano, María Emilia; Varela, Oscar; García-Moreno, María Isabel; García Fernández, José Manuel; Kovensky, José; Uhrig, María Laura

2017-04-18

The synthesis of mono and divalent β-galactosylamides linked to a hydroxylated chain having a C2 symmetry axis derived from l-tartaric anhydride is reported. Reference compounds devoid of hydroxyl groups in the linker were also prepared from β-galactosylamine and succinic anhydride. After functionalization with an alkynyl residue, the resulting building blocks were grafted onto different azide-equipped scaffolds through the copper catalyzed azide-alkyne cycloaddition. Thus, a family of structurally related mono and divalent β-N-galactopyranosylamides was obtained and fully characterized. The binding affinities of the ligands towards the model lectin PNA were measured by the enzyme-linked lectin assay (ELLA). The IC 50 values were significantly higher than that of galactose but the presence of hydroxyl groups in the aglycone chain improved lectin recognition. Docking and molecular dynamics experiments were in accordance with the hypothesis that a hydroxyl group properly disposed in the linker could mimic the Glc O3 in the recognition process. On the other hand, divalent presentation of the ligands led to lectin affinity enhancements. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigation on the Enhanced Oxidation of Ferritic/Martensitic Steel P92 in Pure Steam

PubMed Central

Yuan, Juntao; Wu, Ximao; Wang, Wen; Zhu, Shenglong; Wang, Fuhui

2014-01-01

Oxidation of ferritic/martensitic steel P92 was investigated in pure oxygen and in pure steam at 600–800 °C by thermogravimetric analysis (TGA), optical microscopy (OM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The results showed that the oxidation of P92 was significantly enhanced and multilayer scale with an outer iron oxides layer formed in pure steam. At 700 °C, the gas switch markedly influenced the scaling kinetics and scale microstructure. It was supposed that the higher affinity of iron to steam would be attributed to the enhanced oxidation of P92 in pure steam, and the much easier transport of hydroxyl would account for the significant difference induced by gas switch. PMID:28788592

Influence of smectite hydration and swelling on atrazine sorption behavior.

PubMed

Chappell, Mark A; Laird, David A; Thompson, Michael L; Li, Hui; Teppen, Brian J; Aggarwal, Vaneet; Johnston, Cliff T; Boyd, Stephen A

2005-05-01

Smectites, clay minerals commonly found in soils and sediments, vary widely in their ability to adsorb organic chemicals. Recent research has demonstrated the importance of surface charge density and properties of exchangeable cations in controlling the affinity of smectites for organic molecules. In this study, we induced hysteresis in the crystalline swelling of smectites to test the hypothesis that the extent of crystalline swelling (or interlayer hydration status) has a large influence on the ability of smectites to adsorb atrazine from aqueous systems. Air-dried K-saturated Panther Creek (PC) smectite swelled less (d(001) = 1.38 nm) than never-dried K-PC (d(001) = 1.7 nm) when rehydrated in 20 mM KCl. Correspondingly, the air-dried-rehydrated K-PC had an order of magnitude greater affinity for atrazine relative to the never-dried K-PC. Both air-dried-rehydrated and never-dried Ca-PC expanded to approximately 2.0 nm in 10 mM CaCl2 and both samples had similar affinities for atrazine that were slightly lower than that of never-dried K-PC. The importance of interlayer hydration status in controlling sorption affinity was confirmed by molecular modeling, which revealed much greater interaction between interlayer water molecules and atrazine in a three-layer hydrate relative to a one-layer hydrate. The entropy change on moving atrazine from a fully hydrated state in the bulk solution to a partially hydrated state in the smectite interlayers is believed to be a major factor influencing sorption affinity. In an application test, choice of background solution (20 mM KCl versus 10 mM CaCl2) and air-drying treatments significantly affected atrazine sorption affinities for three-smectitic soils; however, the trends were not consistent with those observed for the reference smectite. Further, extending the initial rehydration time from 24 to 240 h (prior to adding atrazine) significantly decreased the soil's sorption affinity for atrazine. We conclude that interlayer hydration status has a large influence on the affinity of smectites for atrazine and that air-drying treatments have the potential to modify the sorption affinity of smectitic soils for organic molecules such as atrazine.

Size matters: influence of the size of nanoparticles on their interactions with ligands immobilized on the solid surface.

PubMed

Piletska, Elena V; Piletsky, Sergey A

2010-03-16

The correlation between the size of biotinylated nanoparticles and their affinity in relation to interactions with the solid surface was investigated. The silica particles with a diameter of 50-200 nm containing amino groups on the surface were labeled with different quantities of biotin. The affinity properties of biotinylated nanoparticles were studied using a Biacore 3000 instrument equipped with a streptavidin-coated sensor chip (SA chip). It was shown that the increase in the particle size from 50 to 200 nm reduced the affinity (K(D)) of biotin-streptavidin interactions from 1.2 x 10(-12) to 1.2 x 10(-10) M. It was found that the particles with higher concentrations of immobilized biotin on particle surfaces demonstrated stronger binding with streptavidin.

Committed to Learn: Student Engagement and Care in Higher Education

ERIC Educational Resources Information Center

Barnacle, Robyn; Dall'Alba, Gloria

2017-01-01

Efforts to evaluate and improve student engagement have been pervasive in higher education over recent years. Critics argue, however, that troubling affinities are evident between student engagement efforts and a neoliberal agenda which emphasises accountability through performativity. Neoliberalism manifests in policies that focus on the economic…

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

PubMed

Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

1990-04-15

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

The potentially beneficial central nervous system activity profile of ivacaftor and its metabolites.

PubMed

Schneider, Elena K; McQuade, Rachel M; Carbone, Vincenzo C; Reyes-Ortega, Felisa; Wilson, John W; Button, Brenda; Saito, Ayame; Poole, Daniel P; Hoyer, Daniel; Li, Jian; Velkov, Tony

2018-01-01

Ivacaftor-lumacaftor and ivacaftor are two new breakthrough cystic fibrosis transmembrane conductance modulators. The interactions of ivacaftor and its two metabolites hydroxymethylivacaftor (iva-M1) and ivacaftorcarboxylate (iva-M6) with neurotransmitter receptors were investigated in radioligand binding assays. Ivacaftor displayed significant affinity to the 5-hydroxytryptamine (5-HT; serotonin) 5-HT 2C receptor (p K i =6.06±0.03), β 3 -adrenergic receptor (p K i =5.71±0.07), δ-opioid receptor (p K i =5.59±0.06) and the dopamine transporter (p K i =5.50±0.20); iva-M1 displayed significant affinity to the 5-HT 2C receptor (p K i =5.81±0.04) and the muscarinic M3 receptor (p K i =5.70±0.10); iva-M6 displayed significant affinity to the 5-HT 2A receptor (p K i =7.33±0.05). The in vivo central nervous system activity of ivacaftor (40 mg·kg -1 intraperitoneally for 21 days) was assessed in a chronic mouse model of depression. In the forced swim test, the ivacaftor-treated group displayed decreased immobility (52.8±7.6 s), similarly to fluoxetine (33.8±11.0 s), and increased climbing/swimming activity (181.5±9.2 s). In the open field test, ivacaftor produced higher locomotor activity than the fluoxetine group, measured both as mean number of paw touches (ivacaftor 81.1±9.6 versus fluoxetine 57.9±9.5) and total distance travelled (ivacaftor 120.6±16.8 cm versus fluoxetine 84.5±16.0 cm) in 600 s. Treatment of 23 cystic fibrosis patients with ivacaftor-lumacaftor resulted in significant improvements in quality of life (including anxiety) in all five domains of the AweScoreCF questionnaire (p=0.092-0.096). Our findings suggest ivacaftor displays potential clinical anxiolytic and stimulating properties, and may have beneficial effects on mood.

Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

PubMed

Reichstetter, S; Ettinger, R A; Liu, A W; Gebe, J A; Nepom, G T; Kwok, W W

2000-12-15

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

Superbinder SH2 domains act as antagonists of cell signaling.

PubMed

Kaneko, Tomonori; Huang, Haiming; Cao, Xuan; Li, Xing; Li, Chengjun; Voss, Courtney; Sidhu, Sachdev S; Li, Shawn S C

2012-09-25

Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

Effective binding of perhalogenated closo-borates to serum albumins revealed by spectroscopic and ITC studies

NASA Astrophysics Data System (ADS)

Kuperman, Marina V.; Losytskyy, Mykhaylo Yu.; Bykov, Alexander Yu.; Yarmoluk, Sergiy M.; Zhizhin, Konstantin Yu.; Kuznetsov, Nikolay T.; Varzatskii, Oleg A.; Gumienna-Kontecka, Elzbieta; Kovalska, Vladyslava B.

2017-08-01

The interactions of boron cluster compounds closo-borates with biomolecules are widely studied due to their efficiency as agents for boron neutron capture therapy of cancer. In present work the binding abilities of anionic halogen closo-borates [B10Hal10]2- (Hal = Cl, Br, I) and [B12Hal12]2- (Hal = Cl, I) towards bovine and human serum albumins were investigated by spectroscopic and isothermal titration calorimetry (ITC) methods. The protein fluorescence quenching method and ITC studies confirmed the complex formation. The degree of protein fluorescence quenching increased from chlorine to iodine boron derivatives that is attributed to external heavy atom effect. The ITC data point on the existence in the protein structure of two types of binding sites: with higher and lower affinity to closo-borates. Albumin-closo-borate complex binding ratio, n (4-5 anions per protein molecule) is higher than for the parent hydrogen closo-borates (2 anions per protein molecule). Binding constants estimated by fluorescent and ITC methods indicate higher affinity of halogen closo-borates to albumins (K in the range of 104-106 M-1) comparing to that of the hydrogen closo-borate (K about 103 M-1). Due to their high affinity and high binding ratio to albumins halogen closo-borates are proposed for further studies as agents for boron neutron capture therapy.

Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

PubMed

O'Brien, Jeffrey; Shea, Kenneth J

2016-06-21

Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is in its early stages, these recent successes using only small libraries of functional monomers are most encouraging. It is likely that by expanding the chemical diversity of functional hydrogels and other polymers, a much broader range of NP-biomacromolecule affinity pairs will result. Since these robust, nontoxic polymers are readily synthesized in the chemistry laboratory, we believe the results presented in this Account offer a promising future for the development of low cost alternatives to more traditional protein affinity reagents such as antibodies.

Functional properties of myoglobins from five whale species with different diving capacities.

PubMed

Helbo, Signe; Fago, Angela

2012-10-01

Whales show an exceptionally wide range of diving capabilities and many express high amounts of the O(2) carrier protein myoglobin (Mb) in their muscle tissues, which increases their aerobic diving capacity. Although previous studies have mainly focused on the muscle Mb concentration and O(2) carrying capacity as markers of diving behavior in whales, it still remains unexplored whether whale Mbs differ in their O(2) affinities and nitrite reductase and peroxidase enzymatic activities, all functions that could contribute to differences in diving capacities. In this study, we have measured the functional properties of purified Mbs from five toothed whales and two baleen whales and have examined their correlation with average dive duration. Results showed that some variation in functional properties exists among whale Mbs, with toothed whale Mbs having higher O(2) affinities and nitrite reductase activities (similar to those of horse Mb) compared with baleen whale Mbs. However, these differences did not correlate with average dive duration. Instead, a significant correlation was found between whale Mb concentration and average duration and depth of dives, and between O(2) affinity and nitrite reductase activity when including horse Mb. Despite the fact that the functional properties showed little species-specific differences in vitro, they may still contribute to enhancing diving capacity as a result of the increased muscle Mb concentration found in extreme divers. In conclusion, Mb concentration rather than specific functional reactivities may support whale diving performance.

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity.

PubMed

Lee, Su Jin; Kim, So Yeon; Lee, Young Moo

2007-08-01

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.

Plasma binding of an alpha-blocking agent, nicergoline--affinity for serum albumin and native and modified alpha 1-acid glycoprotein.

PubMed

Robert, L; Migne, J; Santonja, R; Zini, R; Schmid, K; Tillement, J P

1983-06-01

The binding of nicergoline, an alpha-blocking drug, by human plasma proteins was studied using gel filtration, polyacrylamide gel electrophoresis, and equilibrium dialysis techniques. 3H-labeled nicergoline added to plasma was eluted together with two major protein fractions, one containing mainly serum albumin, the other glycoproteins such as alpha 1-acid glycoprotein (alpha 1-AG). Equilibrium dialysis experiments with pure human serum albumin and alpha 1-AG as well as with its chemically modified forms, desialylated, carboxymethylated, and both desialylated and carboxymethylated alpha 1-AG gave the following results: nicergoline has about a 4-fold higher affinity for alpha 1-AG than for serum albumin. There are two binding sites per molecule on serum albumin and one on alpha 1-AG. The binding parameters of alpha 1-AG were not significantly modified by desialylation or carboxymethylation. Only desialylated and carboxymethylated alpha 1-AG showed a decreased binding for nicergoline, suggesting conformational modifications induced by these combined treatments. The fact that desialylated alpha 1-AG keeps its affinity for nicergoline suggests the possibility of a selective introduction of this drug in cells possessing the Ashwell-type specific receptor for desialylated alpha 1-AG, for instance hepatocytes. Increased serum alpha 1-AG concentration induced by inflammatory reactions will also modify the distribution of bound nicergoline between serum albumin and alpha 1-AG and as a consequence its half-life and cell distribution.

Comparison of the pharmacokinetics of different analogs of 11C-labeled TZTP for imaging muscarinic M2 receptors with PET.

PubMed

Reid, Alicia E; Ding, Yu-Shin; Eckelman, William C; Logan, Jean; Alexoff, David; Shea, Colleen; Xu, Youwen; Fowler, Joanna S

2008-04-01

The only radiotracer available for the selective imaging of muscarinic M2 receptors in vivo is 3-(3-(3-[18F]fluoropropyl)thio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine) ([18F]FP-TZTP). We have prepared and labeled 3-(3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridne (FP-TZTP, 3) and two other TZTP derivatives with 11C at the methylpyridine moiety to explore the potential of using 11C-labeled FP-TZTP for positron emission tomography imaging of M2 receptors and to compare the effect of small structural changes on tracer pharmacokinetics (PK) in brain and peripheral organs. 11C-radiolabeled FP-TZTP, 3-(3-propylthio)-TZTP (6) and 3,3,3-(3-(3-trifluoropropyl)-TZTP (10) were prepared, and log D, plasma protein binding (PPB), affinity constants, time-activity curves (TACs), area under the curve (AUC) for arterial plasma, distribution volumes (DV) and pharmacological blockade in baboons were compared. Values for log D, PPB and affinity constants were similar for 3, 6 and 10. The fraction of parent radiotracer in the plasma was higher and the AUC lower for 10 than for 3 and 6. TACs for brain regions were similar for 3 and 6, which showed PK similar to the 18F tracer, while 10 showed slower uptake and little clearance over 90 min. DVs for 3 and 6 were similar to the 18F tracer but higher for 10. Uptake of the three tracers was significantly reduced by coinjection of unlabeled 3 and 6. Small structural variations on the TZTP structure greatly altered the PK in brain and behavior in blood with little change in the log D, PPB or affinity. The study suggests that 11C-radiolabeled 3 will be a suitable alternative to [18F]FP-TZTP for translational studies in humans.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

[Genetic-statistical analysis of environmental and genetic influences on the outcome of the juvenile and breeding performance tests for behaviour traits in Hovawart dogs].

PubMed

Boenigk, Katharina; Hamann, Henning; Distl, Ottmar

2006-01-01

The objective of the present study was to evaluate the importance of genetic and environmental sources of variation for results of behaviour tests recorded at juvenile and breeding performance tests in the Hovawart dog. For these analyses behaviour test results of 1882 (juvenile evaluation), respectively 929 dogs (breeding performance test) born in 1995 to 2000 had been used. Variance component estimation was performed for the traits appearance, play instinct, hunting affinity, group of people, shoot, acoustical and optical influences and temperament using multivariate linear animal models and Residual Maximum Likelihood (REML). The models included test-year-season, sex, litter size, age and inbreeding coefficient of the animal as fixed effects. Additive genetic effects of the animal, permanent environmental effect of the litter and the effect of the kennel were considered as random factors. The sex of the dog was significant for appearance, play instinct, hunting affinity, acoustical and optical influences of juvenile evaluation and for the traits temperament, play instinct, hunting affinity, acoustical and one of the optical influences of breeding performance test. The age of the dog at test significantly influenced the traits play instinct, hunting affinity and acoustical influences of juvenile evaluation and optical influences and hunting affinity of breeding performance test. All traits with exception of hunting affinity and group of people were significantly affected by the test-year-season. The inbreeding coefficient was significant for appearance of juvenile evaluation and play affinity of breeding performance test. The effect litter size did not influence any of the traits significantly. The estimated heritabilities for the behaviour traits of juvenile and breeding performance test ranged from h2 = 0.01 to h2 = 0.13, respectively h2 = 0.01 to h2 = 0.14, with standard errors of up to 0.03. The additive genetic correlations between most of the traits were moderately to highly positive (r(g) = 0.20 to r(g) = 1.0, respectively r(g) = 0.29 to r(g) = 1.0). Negative additive genetic correlations were only found for a few traits of juvenile (r(g) = -0.02 to r(g) = -0.58) and breeding performance test (r(g) = -0.28 to r(g) = -0.83). Progress in breeding for the behaviour traits investigated here may only be meaningful when information from all relatives is used in an animal model instead of selection based on the phenotype of the single animal.

Non-covalent interaction between dietary stilbenoids and human serum albumin: Structure-affinity relationship, and its influence on the stability, free radical scavenging activity and cell uptake of stilbenoids.

PubMed

Cao, Hui; Jia, Xueping; Shi, Jian; Xiao, Jianbo; Chen, Xiaoqing

2016-07-01

Dietary stilbenoids are associated with many benefits for human health, which depend on their bioavailability and bioaccessibility. The stilbenoid-human serum albumin (HSA) interactions are investigated to explore the structure-affinity relationship and influence on the stability, free radical scavenging activity and cell uptake of stilbenoids. The structure-affinity relationship of the stilbenoids-HSA interaction was found as: (1) the methoxylation enhanced the affinity, (2) an additional hydroxyl group increases the affinity and (3) the glycosylation significantly weakened the affinity. HSA obviously masked the free radical scavenging potential of stilbenoids. The stabilities of stilbenoids in different medium were determined as: HSA solution>human plasma>Dulbecco's modified Eagle's medium. It appears that the milk enhanced the cell uptake of stilbenoids with multi-hydroxyl groups and weakened the cell uptake of stilbenoids with methoxyl group on EA.hy 926 endothelial cells. The stilbenoids are hardly absorbed by human umbilical vein endothelial cells in the presence of milk. Copyright © 2016 Elsevier Ltd. All rights reserved.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Concentration-Dependent Multiple Binding Sites on Saliva-Treated Hydroxyapatite for Streptococcus sanguis

PubMed Central

Gibbons, R. J.; Moreno, E. C.; Etherden, I.

1983-01-01

The influence of bacterial cell concentration on estimates of the number of binding sites and the affinity for the adsorption of a strain of Streptococcus sanguis to saliva-treated hydroxyapatite was determined, and the possible presence of multiple binding sites for this organism was tested. The range of concentrations of available bacteria varied from 4.7 × 106 to 5,960 × 106 cells per ml. The numbers of adsorbed bacteria increased over the entire range tested, but a suggestion of a break in an otherwise smooth adsorption isotherm was evident. Values for the number of binding sites and the affinity varied considerably depending upon the range of available bacterial concentrations used to estimate them; high correlation coefficients were obtained in all cases. The use of low bacterial cell concentrations yielded lower values for the number of sites and much higher values for the affinity constant than did the use of high bacterial cell concentrations. When data covering the entire range of bacterial concentrations were employed, values for the number of sites and the affinity were similar to those obtained by using only high bacterial cell concentrations. The simplest explanation for these results is that there are multiple binding sites for S. sanguis on saliva-treated hydroxyapatite surfaces. When present in low concentration, the streptococci evidently attach to more specific high-affinity sites which become saturated when higher bacterial concentrations are employed. The possibility of multiple binding sites was substantiated by comparing estimates of the adsorption parameters from a computer-simulated isotherm with those derived from the experimentally generated isotherm. A mathematical model describing bacterial adsorption to binary binding sites was further evidence for the existence of at least two classes of binding sites for S. sanguis. Far fewer streptococci adsorbed to experimental pellicles prepared from saliva depleted of bacterial aggregating activity when low numbers of streptococci were used, but the magnitude of this difference was considerably less when high streptococcal concentrations were employed. This suggests an association between salivary components which possess bacterial-aggregating activity and bacterial adsorption to high-affinity specific binding sites on saliva-treated hydroxyapatite surfaces. PMID:6822416

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

PubMed

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

2015-09-01

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

PubMed Central

2014-01-01

Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. Results Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNβ in the RAW264.7 challenged with LPS. Conclusion Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action. PMID:24602493

Naftopidil for the treatment of urinary symptoms in patients with benign prostatic hyperplasia

PubMed Central

Masumori, Naoya

2011-01-01

Naftopidil, approved only in Japan, is an α1-adrenergic receptor antagonist (α1-blocker) used to treat lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH). Different from tamsulosin hydrochloride and silodosin, in that it has higher and extremely higher affinity respectively, for the α1A-adrenergic receptor subtype than for the α1D type, naftopidil has distinct characteristics because it has a three times greater affinity for the α1D-adrenergic receptor subtype than for the α1A subtype. Although well-designed large-scale randomized controlled studies are lacking and the optimal dosage of naftopidil is not always completely determined, previous reports from Japan have shown that naftopidil has superior efficacy to a placebo and comparable efficacy to other α1-blockers such as tamsulosin. On the other hand, the incidences of ejaculatory disorders and intraoperative floppy iris syndrome induced by naftopidil may be lower than for tamsulosin and silodosin having high affinity for the α1A-adrenergic receptor subtype. However, it remains unknown if the efficacy and safety of naftopidil in Japanese is applicable to white, black and Hispanic men having LUTS/BPH in western countries. PMID:21753885

Poliovirus antibody titres, relative affinity, and neutralising capacity in maternal milk.

PubMed

Zaman, S; Carlsson, B; Morikawa, A; Jeansson, S; Narayanan, I; Thiringer, K; Jalil, F; Hanson, L A

1993-02-01

Varying titres of secretory IgA antibodies to poliovirus type 1 were found previously in the milk of unvaccinated, lactating Pakistani mothers during two different years, reflecting the antigenic exposure on mucosal membranes. To study further the changes in the extent and the form of antigenic exposure reflected in the human milk, human milk samples from Pakistani, Indian, Japanese, and Swedish mothers were collected. The quality and the neutralising capacity of the antibodies was also studied. Secretory IgA, IgG, and IgM antibodies to poliovirus type 1 were determined using enzyme linked immunosorbent assay (ELISA) and relative affinity was measured in ELISA by elution with potassium thiocyanide. Microneutralisation tests were also performed. The higher secretory IgA antibody titres to poliovirus type 1 in the unvaccinated, naturally exposed Pakistani and Indian mothers' milk, compared with the Swedish and Japanese mothers, presumably reflect the epidemiological situation in these countries. Neutralising capacity and the relative antibody affinity seemed to be higher both in the Pakistani mothers and the group without natural exposure but only given inactivated poliovirus vaccine, that is the Swedish mothers, than the group meeting only live vaccine strains, that is the Japanese mothers.

Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

PubMed Central

Emala, C. W.; Aryana, A.; Hirshman, C. A.

1996-01-01

1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry.

PubMed

Sweadner, K J

1985-09-25

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.

Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

PubMed

Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

2016-03-14

The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Influence of N-ethylmaleimide on cholinoceptors and responses in longitudinal muscles from guinea-pig ileum.

PubMed Central

Aronstam, R. S.; Carrier, G. O.

1982-01-01

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea-pig ileum was determined from its inhibition of the binding of [3H]-3-quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 +/- 0.02 and 11 +/- 1 microM; 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N-ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mM N-ethylmaleimide for 20 min at 35 degrees C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 +/- 0.01 and 9 +/- 2 microM, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mM N-ethylmaleimide for 30s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose-response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N-ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane. PMID:7126999

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

PubMed

Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte; Bräuner-Osborne, Hans

2005-01-01

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

PubMed Central

2018-01-01

Background Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1) is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs) and the role of CHT1 in visceral hypersensitivity. Methods Repetitive water avoidance stress (WAS) was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD) was determined, and the abdominal withdrawal reflex (AWR) and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3), the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity. PMID:29849603

Serotonergic ergoline derivatives.

PubMed

Mantegani, S; Brambilla, E; Caccia, C; Damiani, G; Fornaretto, M G; McArthur, R A; Varasi, M

1998-05-05

Novel classes of 13- and 14-tertbutyl-ergoline derivatives were prepared, and characterised in vitro for their affinity for adrenergic, dopaminergic and serotonergic binding sites. This study particularly examines the importance of the presence and the position of the tert-butyl group in conferring either significant 5-HT1A or 5-HT2 affinity and selectivity respectively.

The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening

PubMed Central

Fu, Junjie; Lee, Timothy; Qi, Xin

2014-01-01

G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity. PMID:24941874

PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA

PubMed Central

Zengeya, Thomas; Li, Ming; Rozners, Eriks

2011-01-01

Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target. PMID:21333533

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

The influence of antibody fragment format on phage display based affinity maturation of IgG

PubMed Central

Steinwand, Miriam; Droste, Patrick; Frenzel, Andrè; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

2014-01-01

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab. PMID:24262918

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

PubMed

Thom, George; Burrell, Matthew; Haqqani, Arsalan S; Yogi, Alvaro; Lessard, Etienne; Brunette, Eric; Delaney, Christie; Baumann, Ewa; Callaghan, Deborah; Rodrigo, Natalia; Webster, Carl I; Stanimirovic, Danica B

2018-04-02

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having K D s of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Co-delivery of siRNA and hypericin into cancer cells by hyaluronic acid modified PLGA-PEI nanoparticles.

PubMed

Li, Yanan; Zhang, Junling; Wang, Buhai; Shen, Yan; Ouahab, Ammar

2016-01-01

Malignant tumors cause more death because of the resistance of the hypoxic cancer cell toward radiotherapy. Targeting for hypoxic cancer area and gene silencing to overcome the hypoxia are two kinds of important therapeutic strategies for treating tumors. In order to explore the combined effects of gene therapy and hypericin (Hy) on tumor cells, hypoxia-inducible factor 1 alpha (HIF-1α) small interfering ribonucleic acid (siRNA) was transfected into the hypoxic human nasopharyngeal carcinoma (CNE2) cells using Hy-encapsulated nanocomplexes (Hy-HPP NPs) as a carrier which would achieve dual targeting to the tumor necrosis area. NPs were prepared by emulsion-diffusion-evaporation method. Formulations were evaluated by conducting in vitro physicochemical studies, electrophoresis, in vivo study, and biochemical studies. Hy-loaded nanoparticles with a mean size of around 160 nm was able to enhance the accumulation in the tumors by enhanced permeability and retention effect. The electrophoresis confirmed the good stability of siRNA/Hy-HPP NPs in the presence of phosphate-buffered saline (pH 7.4), competitive heparin, and RNase. The results of transfection showed that the uptake of siRNA was significantly increased up to 50% in CNE2 cells. The level of the HIF-1α with Hy-encapsulated nanocomplexes was significantly reduced to 30% in the transfected CNE2 cells. In vivo studies, the carrier exhibited higher intensity at the tumor tissue cells and higher affinity toward the necrotic tumor tissue. Results demonstrated that Hy-HPP NPs could significantly enhance the tranfection efficiency of siRNA, suggesting Hy-encapsulated nanoparticle as an efficient gene carrier. The co-delivery of HIF-1α siRNA (siHIF-1α) and Hy could efficiently decrease the level of HIF-1α and increase the affinity toward necrotic tissues. Hence, this is a promising strategy for further application in radiotherapy.

What Future for Student Engagement in Neo-Liberal Times?

ERIC Educational Resources Information Center

Zepke, Nick

2015-01-01

The paper first examines the context that has given student engagement a very strong profile in higher education. It identifies neo-liberalism as the driving force in the present higher education context and argues that student engagement enjoys an elective affinity with it. While neo-liberalism is dominant, student engagement will be strong. But…

Investigating the Relationships between Approaches to Learning, Learner Identities and Academic Achievement in Higher Education

ERIC Educational Resources Information Center

Herrmann, K. J.; Bager-Elsborg, A.; McCune, V.

2017-01-01

This paper considers relationships between approaches to learning, learner identities, self-efficacy beliefs and academic achievement in higher education. In addition to already established survey instruments, a new scale, "subject area affinity," was developed. The scale explores the extent to which students identify with their area of…

[Serological affinity of some species of nonpathogenic corynebacteria].

PubMed

Furtat, I M; Nohina, T M; Mikhal's'kyĭ, L O; Vedenieieva, O A

2002-01-01

Serological peculiarities of the species strains Corynebacterium glutamicum, C. ammoniagenes, C. vitaeruminis, C. variabilis and strain of Corynebacterium sp. (Brevibacterium stationis) UCM Ac-719 have been investigated with the help of immunoenzyme analysis ELISA with the use of mice immune serum, specific to C. ammoniagenes UCM Ac-732T, C. vitaeruminis UCM Ac-718T, C. variabilis UCM Ac-717T, C. glutamicum UCM Ac-733 and Corynebacterium sp. UCM Ac-719. It has been established that the species of nonpathogenic corynebacteria differ between themselves as to the degree of serological affinity. C. variabilis, C. ammoniagenes and C. glutamicum are the least similar as to this indication. Weak antigenic relations have been revealed in C. vitaeruminis and C. ammoniagenes. The latter displayed the higher, as compared with other strains, affinity for Corynebacterium sp. UCM Ac-719. The highest degree of serological affinity within the species was registered in strains C. glutamicum and C. variabilis. Data obtained evidence that the ELISA method permits conducting the high-reliability species diagnosis of nonpathogenic corynebacteria on the basis of their antigenic characteristics.

Structure-affinity relationship of the interaction between phenolic acids and their derivatives and β-lactoglobulin and effect on antioxidant activity.

PubMed

Wu, Simin; Zhang, Yunyue; Ren, Fazheng; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Zhang, Hao

2018-04-15

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of β-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for β-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-β-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between β-lactoglobulin and phenolic acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

State-dependent block of CNG channels by dequalinium.

PubMed

Rosenbaum, Tamara; Gordon-Shaag, Ariela; Islas, León D; Cooper, Jeremy; Munari, Mika; Gordon, Sharona E

2004-03-01

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.

Biasing hydrogen bond donating host systems towards chemical warfare agent recognition.

PubMed

Hiscock, Jennifer R; Wells, Neil J; Ede, Jayne A; Gale, Philip A; Sambrook, Mark R

2016-10-12

A series of neutral ditopic and negatively charged, monotopic host molecules have been evaluated for their ability to bind chloride and dihydrogen phosphate anions, and neutral organophosphorus species dimethyl methylphosphonate (DMMP), pinacolyl methylphosphonate (PMP) and the chemical warfare agent (CWA) pinacolyl methylphosphonofluoridate (GD, soman) in organic solvent via hydrogen bonding. Urea, thiourea and boronic acid groups are shown to bind anions and neutral guests through the formation of hydrogen bonds, with the urea and thiourea groups typically exhibiting higher affinity interactions. The introduction of a negative charge on the host structure is shown to decrease anion affinity, whilst still allowing for high stability host-GD complex formation. Importantly, the affinity of the host for the neutral CWA GD is greater than for anionic guests, thus demonstrating the potential for selectivity reversal based on charge repulsion.

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

NASA Astrophysics Data System (ADS)

Li, Ke Sherry; Chu, Phillip Y.; Fourie-O'Donohue, Aimee; Srikumar, Neha; Kozak, Katherine R.; Liu, Yichin; Tran, John C.

2018-05-01

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.

Enhancing Community Detection By Affinity-based Edge Weighting Scheme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Andy; Sanders, Geoffrey; Henson, Van

Community detection refers to an important graph analytics problem of finding a set of densely-connected subgraphs in a graph and has gained a great deal of interest recently. The performance of current community detection algorithms is limited by an inherent constraint of unweighted graphs that offer very little information on their internal community structures. In this paper, we propose a new scheme to address this issue that weights the edges in a given graph based on recently proposed vertex affinity. The vertex affinity quantifies the proximity between two vertices in terms of their clustering strength, and therefore, it is idealmore » for graph analytics applications such as community detection. We also demonstrate that the affinity-based edge weighting scheme can improve the performance of community detection algorithms significantly.« less

A comparison of the properties of polyurethane immobilised Sphagnum moss, seaweed, sunflower waste and maize for the biosorption of Cu, Pb, Zn and Ni in continuous flow packed columns.

PubMed

Zhang, Yue; Banks, Charles

2006-02-01

The biosorption of Cu, Pb, Zn and Ni from a mixed solution of the metals was investigated in continuous flow packed columns containing polyurethane immobilised biomass. The characteristics and biosorption properties of Sphagnum moss, the brown seaweed Ascophyllum nodosum, waste biomass from the preparation of sunflower oil, and whole plant maize were compared. All the biomass types showed a preference for the sequestration of Pb followed by Cu, with Ni and Zn having roughly equal affinity. With continuous metal loading to the column there was an initial binding of all metals and then a displacement of the lower affinity metals by those with a high affinity. This led to a chromatographic effect in the column with breakthrough concentrations for low-affinity metals higher than the concentration in the feed. A similar phenomenon was found on desorption using acidic solutions where low-affinity metals were desorbed preferentially. The results also indicated that despite competitive displacement of one metal species by another the biomass appeared to succeed in retaining some low-affinity metal species indicating that there may be selective sites present with different affinity characteristics. When using a multi-metal solution with Cu, Pb, Zn and Ni at equal 10 mgl(-1) concentrations as column influent, the total quantities of metal sequestered were: seaweed, 117.3 mg g(-1); sunflower waste, 33.2 mg g(-1); Sphagnum moss, 32.5 mg g(-1); and maize, 2.3 mg g(-1). The use of an acid base potentiometric titration showed a relationship between the number of acid functional groups and biosorption capacity, although this was not proportional for the biomass types studied. It can, however, be used in conjunction with a simple classification of metals into high and low-affinity bands to make a preliminary assessment of a biosorption system.

Evaluation of respiratory and cardiac motion correction schemes in dual gated PET/CT cardiac imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lamare, F., E-mail: frederic.lamare@chu-bordeaux.fr; Fernandez, P.; CNRS, INCIA, UMR 5287, F-33400 Talence

Purpose: Cardiac imaging suffers from both respiratory and cardiac motion. One of the proposed solutions involves double gated acquisitions. Although such an approach may lead to both respiratory and cardiac motion compensation there are issues associated with (a) the combination of data from cardiac and respiratory motion bins, and (b) poor statistical quality images as a result of using only part of the acquired data. The main objective of this work was to evaluate different schemes of combining binned data in order to identify the best strategy to reconstruct motion free cardiac images from dual gated positron emission tomography (PET)more » acquisitions. Methods: A digital phantom study as well as seven human studies were used in this evaluation. PET data were acquired in list mode (LM). A real-time position management system and an electrocardiogram device were used to provide the respiratory and cardiac motion triggers registered within the LM file. Acquired data were subsequently binned considering four and six cardiac gates, or the diastole only in combination with eight respiratory amplitude gates. PET images were corrected for attenuation, but no randoms nor scatter corrections were included. Reconstructed images from each of the bins considered above were subsequently used in combination with an affine or an elastic registration algorithm to derive transformation parameters allowing the combination of all acquired data in a particular position in the cardiac and respiratory cycles. Images were assessed in terms of signal-to-noise ratio (SNR), contrast, image profile, coefficient-of-variation (COV), and relative difference of the recovered activity concentration. Results: Regardless of the considered motion compensation strategy, the nonrigid motion model performed better than the affine model, leading to higher SNR and contrast combined with a lower COV. Nevertheless, when compensating for respiration only, no statistically significant differences were observed in the performance of the two motion models considered. Superior image SNR and contrast were seen using the affine respiratory motion model in combination with the diastole cardiac bin in comparison to the use of the whole cardiac cycle. In contrast, when simultaneously correcting for cardiac beating and respiration, the elastic respiratory motion model outperformed the affine model. In this context, four cardiac bins associated with eight respiratory amplitude bins seemed to be adequate. Conclusions: Considering the compensation of respiratory motion effects only, both affine and elastic based approaches led to an accurate resizing and positioning of the myocardium. The use of the diastolic phase combined with an affine model based respiratory motion correction may therefore be a simple approach leading to significant quality improvements in cardiac PET imaging. However, the best performance was obtained with the combined correction for both cardiac and respiratory movements considering all the dual-gated bins independently through the use of an elastic model based motion compensation.« less

Antibody Affinity Against 2009 A/H1N1 Influenza and Pandemrix Vaccine Nucleoproteins Differs Between Childhood Narcolepsy Patients and Controls.

PubMed

Lind, Alexander; Freyhult, Eva; Ramelius, Anita; Olsson, Tomas; Arnheim-Dahlström, Lisen; Lamb, Favelle; Khademi, Mohsen; Ambati, Aditya; Maeurer, Markus; Lima Bomfim, Izaura; Fink, Katharina; Fex, Malin; Törn, Carina; Elding Larsson, Helena; Lernmark, Åke

2017-10-01

Increased narcolepsy incidence was observed in Sweden following the 2009 influenza vaccination with Pandemrix ® . A substitution of the 2009 nucleoprotein for the 1934 variant has been implicated in narcolepsy development. The aims were to determine (a) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (NP-CA2009) and Pandemrix-[A/Puerto Rico/8/1934(H1N1)] (NP-PR1934) nucleoproteins in 43 patients and 64 age-matched controls; (b) antibody affinity in reciprocal competitive assays in 11 childhood narcolepsy patients compared with 21 age-matched controls; and (c) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (H1N1 NS1), not a component of the Pandemrix vaccine. In vitro transcribed and translated 35 S-methionine-labeled H1N1 influenza A virus proteins were used in radiobinding reciprocal competition assays to estimate antibody levels and affinity (Kd). Childhood patients had higher NP-CA2009 (p = 0.0339) and NP-PR1934 (p = 0.0246) antibody levels compared with age-matched controls. These childhood controls had lower NP-CA2009 (p = 0.0221) and NP-PR1934 (p = 0.00619) antibodies compared with controls 13 years or older. In contrast, in patients 13 years or older, the levels of NP-PR1934 (p = 0.279) and NP-CA2009 (p = 0.0644) antibodies did not differ from the older controls. Childhood antibody affinity (Kd) against NP-CA2009 was comparable between controls (68 ng/mL) and patients (74 ng/mL; p = 0.21) with NP-CA2009 and NP-PR1934 displacement (controls: 165 ng/mL; patients: 199 ng/mL; p = 0.48). In contrast, antibody affinity against NP-PR1934 was higher in controls with either NP-PR1934 (controls: 9 ng/mL; patients: 20 ng/mL; p = 0.0031) or NP-CA2009 (controls: 14 ng/mL; patients: 23 ng/mL; p = 0.0048). A/H1N1-NS1 antibodies were detected in 0/43 of the narcolepsy patients compared with 3/64 (4.7%) controls (p = 0.272). Similarly, none (0/11) of the childhood patients and 1/21 (4.8%) of the childhood controls had A/H1N1-NS1 antibodies. The higher antibody affinities against NP-PR1934 in controls suggest better protection against wild-type virus. In contrast, the reduced NP-PR1934 antibody affinities among childhood narcolepsy patients suggest poor protection from the wild-type A/H1N1 virus and possibly increased risk for viral damage.

Affinity-aware checkpoint restart

DOE PAGES

Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

2014-12-08

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Affinity-aware checkpoint restart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saini, Ajay; Rezaei, Arash; Mueller, Frank

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

PubMed

Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Seppä, Titta; Akter, Sultana; Khan, Liton Md Ferdhos; Lamminmäki, Urpo

2014-09-19

Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Use of Whatman-41 filters in air quality sampling networks (with applications to elemental analysis)

NASA Technical Reports Server (NTRS)

Neustadter, H. E.; Sidik, S. M.; King, R. B.; Fordyce, J. S.; Burr, J. C.

1974-01-01

The operation of a 16-site parallel high volume air sampling network with glass fiber filters on one unit and Whatman-41 filters on the other is reported. The network data and data from several other experiments indicate that (1) Sampler-to-sampler and filter-to-filter variabilities are small; (2) hygroscopic affinity of Whatman-41 filters need not introduce errors; and (3) suspended particulate samples from glass fiber filters averaged slightly, but not statistically significantly, higher than from Whatman-41-filters. The results obtained demonstrate the practicability of Whatman-41 filters for air quality monitoring and elemental analysis.

α-Chymotrypsin in water-ethanol mixtures: Effect of preferential interactions

NASA Astrophysics Data System (ADS)

Sirotkin, Vladimir A.; Kuchierskaya, Alexandra A.

2017-12-01

We investigated preferential interactions of α-chymotrypsin with water-ethanol mixtures at 25 °C. Our approach is based on the analysis of residual enzyme activity and water/alcohol sorption. There are three concentration regimes. α-Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity is close to 100%. α-Chymotrypsin has a higher affinity for alcohol than for water at intermediate water content. Residual enzyme activity is close to zero in this concentration range. At low water content, ethanol is preferentially excluded from the protein surface. This results in preferential hydration of α-chymotrypsin and significant residual catalytic activity (∼50%) in water-poor ethanol.

Robust feature matching via support-line voting and affine-invariant ratios

NASA Astrophysics Data System (ADS)

Li, Jiayuan; Hu, Qingwu; Ai, Mingyao; Zhong, Ruofei

2017-10-01

Robust image matching is crucial for many applications of remote sensing and photogrammetry, such as image fusion, image registration, and change detection. In this paper, we propose a robust feature matching method based on support-line voting and affine-invariant ratios. We first use popular feature matching algorithms, such as SIFT, to obtain a set of initial matches. A support-line descriptor based on multiple adaptive binning gradient histograms is subsequently applied in the support-line voting stage to filter outliers. In addition, we use affine-invariant ratios computed by a two-line structure to refine the matching results and estimate the local affine transformation. The local affine model is more robust to distortions caused by elevation differences than the global affine transformation, especially for high-resolution remote sensing images and UAV images. Thus, the proposed method is suitable for both rigid and non-rigid image matching problems. Finally, we extract as many high-precision correspondences as possible based on the local affine extension and build a grid-wise affine model for remote sensing image registration. We compare the proposed method with six state-of-the-art algorithms on several data sets and show that our method significantly outperforms the other methods. The proposed method achieves 94.46% average precision on 15 challenging remote sensing image pairs, while the second-best method, RANSAC, only achieves 70.3%. In addition, the number of detected correct matches of the proposed method is approximately four times the number of initial SIFT matches.

Binding of carbonyl flavours to canola, pea and wheat proteins using GC/MS approach.

PubMed

Wang, Kun; Arntfield, Susan D

2014-08-15

Interactions of homologous aldehydes (hexanal, heptanal, and octanal) and ketones (2-hexanone, 2-heptanone, and 2-octanone) to salt and alkaline-extracted canola and pea proteins and commercial wheat gluten were studied using GC/MS. Long-chain aldehyde flavours exhibited higher binding affinity, regardless of protein type and isolation method. Salt-extracted canola protein isolates (CPIs) revealed the highest binding capacity to all aldehydes followed by wheat gluten and salt-extracted pea protein isolates (PPIs), while binding of ketone flavours decreased in the order: PPIs>wheat gluten>CPIs. Two aldolisation products, 2-butyl-2-octenal and 2-pentyl-2-nonenal, were detected from the interactions between CPIs with hexanal and heptanal, respectively. Protein thermal behaviour in the presence of these compounds was analysed by differential scanning calorimeter, where decreased ΔH inferred potential conformational changes due to partial denaturation of PPIs. Compared to ketones, aldehyde flavours possessed much higher "unfolding capacity" (lower ΔH), which accounted for their higher binding affinities. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ.

PubMed

Baptist, Matilda; Panagabko, Candace; Cockcroft, Shamshad; Atkinson, Jeffrey

2016-12-01

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

PubMed

Naimuddin, Mohammed; Kubo, Tai

2011-12-01

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery. Copyright © 2011 Elsevier Inc. All rights reserved.

A structure-function study of PACAP using conformationally-restricted analogs: identification of PAC1 receptor-selective PACAP agonists

PubMed Central

Ramos-Álvarez, Irene; Mantey, Samuel A.; Nakamura, Taichi; Nuche-Berenguer, Bernardo; Moreno, Paola; Moody, Terry W.; Maderdrut, Jerome L.; Coy, David H.; Jensen, Robert T.

2015-01-01

Pituitary adenylate-cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure-function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally-restricted PACAP -analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22 ,28,34,38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to-103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases. PMID:25698233

Pharmacological characterization of the human histamine H2 receptor stably expressed in Chinese hamster ovary cells.

PubMed Central

Leurs, R.; Smit, M. J.; Menge, W. M.; Timmerman, H.

1994-01-01

1. The gene for the human histamine H2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [125I]-iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2. After cotransfection of CHO cells with pCMVhumH2 and pUT626, a phleomycine-resistant clonal cell line (CHOhumH2) was isolated that expressed 565 +/- 35 fmol kg-1 protein binding sites with high affinity (0.21 +/- 0.02 nM) for the H2 antagonist, [125I]-iodoaminopotentidine. 3. Displacement studies with a variety of H2 antagonists indicated that the encoded protein was indistinguishable from the H2 receptor identified in human brain membranes and guinea-pig right atrium. The Ki-values observed in the various preparations correlated very well (r2 = 0.996-0.920). 4. Displacement studies with histamine showed that a limited fraction (32 +/- 6%) of the binding sites showed a high affinity for histamine (2 +/- 1.2 microM); the shallow displacement curves were reflected by a Hill-coefficient significantly different from unity (nH = 0.58 +/- 0.09). The addition of 100 microM Gpp(NH)p resulted in a steepening of the displacement curve (nH = 0.79 +/- 0.02) and a loss of high affinity sites for histamine. 5. Displacement studies with other agonists indicated that the recently developed specific H2 agonists, amthamine and amselamine, showed an approximately 4-5 fold higher affinity for the human H2 receptor than histamine. 6. Stimulation of CHOhumH2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7921611

Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

PubMed Central

Løset, Geir Åge; Roos, Norbert; Bogen, Bjarne; Sandlie, Inger

2011-01-01

Background Phage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display. Methodology/Principal Findings Here we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient. Conclusions/Significance Display through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before. PMID:21390283

The role of living/controlled radical polymerization in the formation of improved imprinted polymers.

PubMed

Salian, Vishal D; Vaughan, Asa D; Byrne, Mark E

2012-06-01

In this work, living/controlled radical polymerization (LRP) is compared with conventional free radical polymerization in the creation of highly and weakly cross-linked imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) networks. It elucidates, for the first time, the effect of LRP on the chain level and begins to explain why the efficiency of the imprinting process is improved using LRP. Imprinted polymers produced via LRP exhibited significantly higher template affinity and capacity compared with polymers prepared using conventional methods. The use of LRP in the creation of highly cross-linked imprinted polymers resulted in a fourfold increase in binding capacity without a decrease in affinity; whereas weakly cross-linked gels demonstrated a nearly threefold increase in binding capacity at equivalent affinity when LRP was used. In addition, by adjusting the double bond conversion, we can choose to increase either the capacity or the affinity in highly cross-linked imprinted polymers, thus allowing the creation of imprinted polymers with tailorable binding parameters. Using free radical polymerization in the creation of polymer chains, as the template-monomer ratio increased, the average molecular weight of the polymer chains decreased despite a slight increase in the double bond conversion. Thus, the polymer chains formed were shorter but greater in number. Using LRP neutralized the effect of the template. The addition of chain transfer agent resulted in slow, uniform, simultaneous chain growth, resulting in the formation of longer more monodisperse chains. Reaction analysis revealed that propagation time was extended threefold in the formation of highly cross-linked polymers when LRP techniques were used. This delayed the transition to the diffusion-controlled stage of the reaction, which in turn led to the observed enhanced binding properties, decreased polydispersity in the chains, and a more homogeneous macromolecular architecture. Copyright © 2012 John Wiley & Sons, Ltd.

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

PubMed

Wang, Juan; Shi, Yali; Cai, Yaqi

2018-04-06

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

Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

PubMed

Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

2012-05-07

The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

Synthesis and pharmacological evaluation of indole-based sigma receptor ligands

PubMed Central

Mésangeau, Christophe; Amata, Emanuele; Alsharif, Walid; Seminerio, Michael J.; Robson, Matthew J.; Matsumoto, Rae R.; Poupaert, Jacques H.; McCurdy, Christopher R.

2011-01-01

A series of novel indole-based analogues were prepared and their affinities for sigma receptors were determined using in vitro radioligand binding assays. The results of this study identified several compounds with nanomolar sigma-2 affinity and significant selectivity over sigma-1 receptors. In particular, 2-(4-(3-(4-fluorophenyl)indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (9f) was found to display high affinity at sigma-2 receptors with good selectivity (σ-1/σ-2 = 395). The pharmacological binding profile for this compound was established with other relevant nonsigma sites. PMID:21899931

Affinity of hydroxyapatite for furfural and a brown pigment formed by furfural and glycine.

PubMed

Nordbö, H; Eriksen, H M; Rölla, G

1979-10-01

The affinity of hydroxyapatite for furfural and a brown pigment formed by furfural and glycine was studied. A series of mixtures containing 1 M furfural and 0.25-2.0 M glycine were incubated at 37 degrees C and aliquots of hydroxyapatite added. The apatite showed a strong affinity for the brown pigment formed, and an excess of glycine in the mixtures appeared to enhance the binding. The adsorption of furfural to hydroxyapatite was estimated by a spectrophotometric method. The data revealed that pretreatment with CaCl2 and glycine significantly increased the adsorption of furfural.

Studies on Changes of β-Adrenergic Receptors in Polymorphonuclear Cell and Mononuclear Cell with the Changes of Thyroid Function

PubMed Central

Lee, Jong Do; You, Myung Hee; Kim, Young Seol; Kim, Jin Woo; Kim, Kwang Won; Kim, Sun Woo; Choi, Young Kil

1986-01-01

Although it has been well established that thyroid hormones increase β-adrenergic receptors of various tissues in the animal studies, there are controversies about the β-adrenergic receptor changes of human mononuclear cells and polymorphonuclear cells. The present study was performed to analyze the change of β-adrenergic receptor of those cells according to the thyroid functional status and to evaluate their usefulness in assessment of sympathetic hyperactivity. We measured [3H]-dihydroalprenolol binding to circulating mononuclear and polymorphonuclear cells from 18 patients with hyperthyrodism, 7 with hypothyroidism, 8 with euthyroid goiter and 21 normal controls. Only with polymorphonuclear cells the receptor concentration was significantly higher (P<0.01) in hyperthyroidism (46.07±4.78 fmol/mg protein) than in the normal control (28.42±2.06 fmol/mg protein) and the affinity constants of both cells were comparable to normal control values. And serum concentrations of T3 were not correlated well with the changes of receptor concentrations in hyperthyroidism. The patients with hypothyroidism and euthyroid goiter showed no significant difference in the receptor concentration and the affinity constants with both cell binding assays. These results indicate that thyroid hormones increase the receptor concentration in polymorphonuclear cells which might be responsible for the symptoms of sympathetic hyperactivity and the polymorphornuclear cells are useful for β-adrenergic receptor assay. PMID:15759381

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

NASA Astrophysics Data System (ADS)

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R.; Allen, Rosalind J.

2017-12-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

G2(+)M study on N-alkylamino cation affinities of neutral main-group element hydrides: trends across the periodic table.

PubMed

Geng, Song; Wu, Ding-Lu; Yang, Jing; Wei, Xi-Guang; Zhu, Jun; Zhang, Hai-Bo; Ren, Yi; Lau, Kai-Chung

2014-05-08

We have made an extensive theoretical exploration of gas-phase N-alkylamino cation affinities (NAAMCA), including amino cation affinities (AMCA) and N-dimethylamino cation affinities (NDMAMCA), of neutral main-group element hydrides of groups 15-17 and periods 2-4 in the periodic table by using the G2(+)M method. Some similarities and differences are found between NAAMCA and the corresponding alkyl cation affinities (ACA) of H(n)X. Our calculations show that the AMCA and NDMAMCA are systematically lower than the corresponding proton affinities (PA) for H(n)X. In general, there is no linear correlation between NAAMCA and PA of H(n)X. Instead, the correlations exist only within the central elements X in period 2, or periods 3-4, which is significantly different from the reasonable correlations between ACA and PA for all H(n)X. NAAMCA (H(n)X) are weaker than NAAMCA (H(n-1)X(-)) by more than 700 kJ/mol and generally stronger than ACA (H(n)X), with three exceptions: H2ONR2(+)(R = H, Me) and HFNH2(+). These new findings can be rationalized by the negative hyperconjugation and Pauli repulsion.

Solubilization and purification of melatonin receptors from lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

PubMed Central

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R; Allen, Rosalind J

2017-01-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance. PMID:28714461

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle.

PubMed Central

Godt, R E; Nosek, T M

1989-01-01

1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8-4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated). PMID:2600830

Serotonin and stress: protective or malevolent actions in the biobehavioral response to repeated trauma?

PubMed

Harvey, Brian H; Naciti, Carla; Brand, Linda; Stein, Dan J

2004-12-01

Structural hippocampus and prefrontal cortex changes occur in patients with posttraumatic stress disorder (PTSD) that appears correlated with cognitive dysfunction. In these brain regions, serotonin (5HT) plays a prominent role in symptom presentation and treatment of PTSD. However, 5HT is both anxiogenic and anxiolytic, and while 5HT reuptake inhibitors are effective in treatment, the role of 5HT in the development of PTSD remains uncertain. Using a model of repeated trauma in rats, we observed significant spatial memory impairment together with significantly increased 5HT(1A) receptor density (B(max)), decreased 5HT(1A) receptor affinity (K(d)), and significantly increased 5HT(2A) receptor affinity on day 7 poststress. The serotonergic agent fluoxetine (FLX; 10 mg/kg/d ip) administered 1 week before stress and continuing throughout the stress procedure, but not the 5HT depleter p-chloro-phenylalanine (PCPA; 300/100/50 mg/kg/d ip), prevented stress-induced cognitive dysfunction. PCPA, however, reversed stress-induced hippocampal 5HT(1A) receptor affinity changes, with FLX narrowly missing significance. Neither drug reversed stress effects on 5HT(2A) receptor affinity. Thus, 5HT plays an important part in the cognitive-behavioral changes evoked by repeated trauma. That raised 5HT activity may mediate hippocampal 5HT(1A) receptor changes evoked by stress suggests a bidirectional role for 5HT in the development of PTSD.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

PubMed

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

PubMed Central

Adler, Adam S.; Bedinger, Daniel; Adams, Matthew S.; Asensio, Michael A.; Edgar, Robert C.; Leong, Renee; Leong, Jackson; Mizrahi, Rena A.; Spindler, Matthew J.; Bandi, Srinivasa Rao; Huang, Haichun; Brams, Peter; Johnson, David S.

2018-01-01

ABSTRACT Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs. PMID:29376776

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

PubMed Central

Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

1996-01-01

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means. PMID:8937710

Semiconductor light source with electrically tunable emission wavelength

DOEpatents

Belenky, Gregory [Port Jefferson, NY; Bruno, John D [Bowie, MD; Kisin, Mikhail V [Centereach, NY; Luryi, Serge [Setauket, NY; Shterengas, Leon [Centereach, NY; Suchalkin, Sergey [Centereach, NY; Tober, Richard L [Elkridge, MD

2011-01-25

A semiconductor light source comprises a substrate, lower and upper claddings, a waveguide region with imbedded active area, and electrical contacts to provide voltage necessary for the wavelength tuning. The active region includes single or several heterojunction periods sandwiched between charge accumulation layers. Each of the active region periods comprises higher and lower affinity semiconductor layers with type-II band alignment. The charge carrier accumulation in the charge accumulation layers results in electric field build-up and leads to the formation of generally triangular electron and hole potential wells in the higher and lower affinity layers. Nonequillibrium carriers can be created in the active region by means of electrical injection or optical pumping. The ground state energy in the triangular wells and the radiation wavelength can be tuned by changing the voltage drop across the active region.

HLA-DRB1*07:01 is associated with a higher risk of asparaginase allergies

PubMed Central

Fernandez, Christian A.; Smith, Colton; Yang, Wenjian; Daté, Mihir; Bashford, Donald; Larsen, Eric; Bowman, W. Paul; Liu, Chengcheng; Ramsey, Laura B.; Chang, Tamara; Turner, Victoria; Loh, Mignon L.; Raetz, Elizabeth A.; Winick, Naomi J.; Hunger, Stephen P.; Carroll, William L.; Onengut-Gumuscu, Suna; Chen, Wei-Min; Concannon, Patrick; Rich, Stephen S.; Scheet, Paul; Jeha, Sima; Pui, Ching-Hon; Evans, William E.; Devidas, Meenakshi

2014-01-01

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children’s Research Hospital (n = 541) and the Children’s Oncology Group (n = 1329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P = 7.5 × 10−5, odds ratio [OR] = 1.64; P = 1.4 × 10−5, OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1 alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P = 3.3 × 10−4, OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1 alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. These trials were registered at www.clinicaltrials.gov as NCT00137111, NCT00549848, NCT00005603, and NCT00075725. PMID:24970932

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

Comparative serum albumin interactions and antitumor effects of Au(III) and Ga(III) ions.

PubMed

Sarioglu, Omer Faruk; Ozdemir, Ayse; Karaboduk, Kuddusi; Tekinay, Turgay

2015-01-01

In the present study, interactions of Au(III) and Ga(III) ions on human serum albumin (HSA) were studied comparatively via spectroscopic and thermal analysis methods: UV-vis absorbance spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and isothermal titration calorimetry (ITC). The potential antitumor effects of these ions were studied on MCF-7 cells via Alamar blue assay. It was found that both Au(III) and Ga(III) ions can interact with HSA, however; Au(III) ions interact with HSA more favorably and with a higher affinity. FT-IR second derivative analysis results demonstrated that, high concentrations of both metal ions led to a considerable decrease in the α-helix content of HSA; while Au(III) led to around 5% of decrease in the α-helix content at 200μM, it was around 1% for Ga(III) at the same concentration. Calorimetric analysis gave the binding kinetics of metal-HSA interactions; while the binding affinity (Ka) of Au(III)-HSA binding was around 3.87×10(5)M(-1), it was around 9.68×10(3)M(-1) for Ga(III)-HSA binding. Spectroscopy studies overall suggest that both metal ions have significant effects on the chemical structure of HSA, including the secondary structure alterations. Antitumor activity studies on MCF7 tumor cell line with both metal ions revealed that, Au(III) ions have a higher antiproliferative activity compared to Ga(III) ions. Copyright © 2014 Elsevier GmbH. All rights reserved.

Glass-eel-stage American Eels respond to conspecific odor as a function of concentration

USGS Publications Warehouse

Schmucker, Andrew K.; Johnson, Nicholas; Galbraith, Heather S.; Li, Weiming

2016-01-01

The American Eel Anguilla rostrata has experienced staggering population declines in recent decades and is now the focus of restoration efforts. Studies have demonstrated that olfaction is critical to anguillid behavior and that glass eels (the life stage which migrates inland from saltwater to freshwater) are attracted to conspecific washings. We evaluated conspecific cueing as a potential mechanism for American Eel inland migration coordination by assessing (1) the affinity of glass eels to conspecific washings, (2) the concentration–response relationships, and (3) changes in responsiveness to washings during the glass eel-to-elver transition. In two-choice maze assays, glass eels were attracted to glass eel washings over a wide range of concentrations (0.20–0.40 g of glass eels·L−1·h−1), and a logarithmic function provided the best fit to the concentration–response relationship. When given a choice between two conspecific washings of higher and lower concentrations, the glass eels generally preferred the higher concentration. Responses to undiluted glass eel washings did not significantly differ among stage-3–7 glass eels, although stage-7 eels were not attracted to the washings, whereas the other stages were. Washing affinity remained similar over the course of several weeks. These results support aspects of the conspecific cueing hypothesis at the glass eel life stage under laboratory conditions, suggesting that conspecific cueing is an important component of migration coordination among juvenile American Eels and warrants additional study.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

PubMed

Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

2017-03-28

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Development of a nonlinear model for the prediction of response times of glucose affinity sensors using concanavalin A and dextran and the development of a differential osmotic glucose affinity sensor

NASA Astrophysics Data System (ADS)

Reis, Louis G.

With the increasing prevalence of diabetes in the United States and worldwide, blood glucose monitoring must be accurate and reliable. Current enzymatic sensors have numerous disadvantages that make them unreliable and unfavorable among patients. Recent research in glucose affinity sensors correct some of the problems that enzymatic sensors experience. Dextran and concanavalin A are two of the more common components used in glucose affinity sensors. When these sensors were first explored, a model was derived to predict the response time of a glucose affinity sensor using concanavalin A and dextran. However, the model assumed the system was linear and fell short of calculating times representative of the response times determined through experimental tests with the sensors. In this work, a new model that uses the Stokes-Einstein Equation to demonstrate the nonlinear behavior of the glucose affinity assay was developed to predict the response times of similar glucose affinity sensors. In addition to the device tested by the original linear model, additional devices were identified and tested with the proposed model. The nonlinear model was designed to accommodate the many different variations between systems. The proposed model was able to accurately calculate response times for sensors using the concanavalin A-dextran affinity assay with respect to the experimentally reported times by the independent research groups. Parameter studies using the nonlinear model were able to identify possible setbacks that could compromise the response of thesystem. Specifically, the model showed that the improper use of asymmetrical membranes could increase the response time by as little as 20% or more as the device is miniaturized. The model also demonstrated that systems using the concanavalin Adextran assay would experience higher response times in the hypoglycemic range. This work attempted to replicate and improve an osmotic glucose affinity sensor. The system was designed to negate additional effects that could cause artifacts or irregular readings such as external osmotic differences and external pressure differences. However, the experimental setup and execution faced numerous setbacks that highlighted the additional difficulty that sensors using asymmetrical ceramic membranes and the concanavalin A-dextran affinity assay may experience.

Comment on: Negative ions, molecular electron affinity and orbital structure of cata-condensed polycyclic aromatic hydrocarbons by Rustem V. Khatymov, Mars V. Muftakhov and Pavel V. Shchukin.

PubMed

Chen, Edward S; Chen, Edward C M

2018-02-15

The anion mass spectral lifetimes for several aromatic hydrocarbons reported in the subject article were related to significantly different electron affinities. The different values are rationalized using negative ion mass spectral data. Electron affinities for polycyclic aromatic hydrocarbons are reported from the temperature dependence of unpublished electron capture detector data. These are compared with published values and the largest values are assigned to the ground state. The ground state adiabatic electron affinities: (eV) pentacene, 1.41 (3); tetracene, 1.058 (5); benz(a)pyrene, 0.82 (4); benz(a) anthracene, 0.69 (2) anthracene, 0.68 (2); and pyrene, 0.59 (1) are used to assign excited state adiabatic electron affinities: (eV) tetracene: 0.88 (4); anthracene 0.53 (1); pyrene, 0.41 (1); benz(a)anthracene, 0.39 (10); chrysene, 0.32 (1); and phenanthrene, 0.12 (2) and ground state adiabatic electron affinities: (eV) dibenz(a,j)anthracene, 0.69 (3); dibenz(a,h)anthracene, 0.68 (3); benz(e)pyrene, 0.60 (3); and picene, 0.59 (3) from experimental data. The lifetime of benz(a)pyrene is predicted to be larger than 150 μs and for benzo(c)phenanthrene and picene about 40 μs, from ground state adiabatic electron affinities. The assignments of adiabatic electron affinities of aromatic hydrocarbons determined from electron capture detector and mass spectrometric data to ground and excited states are supported by constant electronegativities. A set of consistent ground state adiabatic electron affinities for 15 polycyclic aromatic hydrocarbons is related to lifetimes from the subject article. Copyright © 2017 John Wiley & Sons, Ltd.

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

PubMed Central

Gopishetty, Bhaskar; Zhang, Suhong; Kharkar, Prashant S.; Antonio, Tamara; Reith, Maarten; Dutta, Aloke K.

2013-01-01

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (Ki) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization. PMID:23623679

Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

PubMed Central

Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

2013-01-01

When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

PubMed

Orwig, Kevin S; Lassetter, McKensie R; Hadden, M Kyle; Dix, Thomas A

2009-04-09

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

PubMed

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

2017-03-01

Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. Published by Elsevier Inc.

CoMFA analyses of C-2 position salvinorin A analogs at the kappa-opioid receptor provides insights into epimer selectivity.

PubMed

McGovern, Donna L; Mosier, Philip D; Roth, Bryan L; Westkaemper, Richard B

2010-04-01

The highly potent and kappa-opioid (KOP) receptor-selective hallucinogen Salvinorin A and selected analogs have been analyzed using the 3D quantitative structure-affinity relationship technique Comparative Molecular Field Analysis (CoMFA) in an effort to derive a statistically significant and predictive model of salvinorin affinity at the KOP receptor and to provide additional statistical support for the validity of previously proposed structure-based interaction models. Two CoMFA models of Salvinorin A analogs substituted at the C-2 position are presented. Separate models were developed based on the radioligand used in the kappa-opioid binding assay, [(3)H]diprenorphine or [(125)I]6 beta-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([(125)I]IOXY). For each dataset, three methods of alignment were employed: a receptor-docked alignment derived from the structure-based docking algorithm GOLD, another from the ligand-based alignment algorithm FlexS, and a rigid realignment of the poses from the receptor-docked alignment. The receptor-docked alignment produced statistically superior results compared to either the FlexS alignment or the realignment in both datasets. The [(125)I]IOXY set (Model 1) and [(3)H]diprenorphine set (Model 2) gave q(2) values of 0.592 and 0.620, respectively, using the receptor-docked alignment, and both models produced similar CoMFA contour maps that reflected the stereoelectronic features of the receptor model from which they were derived. Each model gave significantly predictive CoMFA statistics (Model 1 PSET r(2)=0.833; Model 2 PSET r(2)=0.813). Based on the CoMFA contour maps, a binding mode was proposed for amine-containing Salvinorin A analogs that provides a rationale for the observation that the beta-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding alpha-epimers (S-configuration). (c) 2010. Published by Elsevier Inc.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Specific interaction between negative atmospheric ions and organic compounds in atmospheric pressure corona discharge ionization mass spectrometry.

PubMed

Sekimoto, Kanako; Sakai, Mami; Takayama, Mitsuo

2012-06-01

The interaction between negative atmospheric ions and various types of organic compounds were investigated using atmospheric pressure corona discharge ionization (APCDI) mass spectrometry. Atmospheric negative ions such as O(2)(-), HCO(3)(-), COO(-)(COOH), NO(2)(-), NO(3)(-), and NO(3)(-)(HNO(3)) having different proton affinities served as the reactant ions for analyte ionization in APCDI in negative-ion mode. The individual atmospheric ions specifically ionized aliphatic and aromatic compounds with various functional groups as atmospheric ion adducts and deprotonated analytes. The formation of the atmospheric ion adducts under certain discharge conditions is most likely attributable to the affinity between the analyte and atmospheric ion and the concentration of the atmospheric ion produced under these conditions. The deprotonated analytes, in contrast, were generated from the adducts of the atmospheric ions with higher proton affinity attributable to efficient proton abstraction from the analyte by the atmospheric ion.

Differential niche dynamics among major marine invertebrate clades

PubMed Central

Hopkins, Melanie J; Simpson, Carl; Kiessling, Wolfgang

2014-01-01

The degree to which organisms retain their environmental preferences is of utmost importance in predicting their fate in a world of rapid climate change. Notably, marine invertebrates frequently show strong affinities for either carbonate or terrigenous clastic environments. This affinity is due to characteristics of the sediments as well as correlated environmental factors. We assessed the conservatism of substrate affinities of marine invertebrates over geological timescales, and found that niche conservatism is prevalent in the oceans, and largely determined by the strength of initial habitat preference. There is substantial variation in niche conservatism among major clades with corals and sponges being among the most conservative. Time-series analysis suggests that niche conservatism is enhanced during times of elevated nutrient flux, whereas niche evolution tends to occur after mass extinctions. Niche evolution is not necessarily elevated in genera exhibiting higher turnover in species composition. PMID:24313951

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction

PubMed Central

Xu, Xiaojuan; Yano, Masafumi; Uchinoumi, Hitoshi; Hino, Akihiro; Suetomi, Takeshi; Ono, Makoto; Tateishi, Hiroki; Oda, Tetsuro; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Yamamoto, Takeshi; Ikeda, Yasuhiro; Ikemoto, Noriaki; Matsuzaki, Masunori

2010-01-01

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca2+ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca2+ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca2+ leak, and then lethal arrhythmia. PMID:20226167

Pharmacokinetics of bisphenol A in neonatal and adult Sprague-Dawley rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doerge, Daniel R., E-mail: daniel.doerge@fda.hhs.go; Twaddle, Nathan C.; Vanlandingham, Michelle

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA in urine of > 90% of Americans aged 6-60 suggests ubiquitous and frequent exposure. The current study used LC/MS/MS to measure serum pharmacokinetics of aglycone (active) and conjugated (inactive) BPA in adult and neonatal Sprague-Dawley rats by oral and injection routes. Deuterated BPA was used to avoid issues of background contamination. Linear pharmacokinetics were observed in adult rats treated orally in the range of 0-200 {mu}g/kg bw. Evidence for enterohepatic recirculation of conjugated, butmore » not aglycone, BPA was observed in adult rats. Significant inverse relationships were observed between postnatal age and measures of internal exposures to aglycone BPA and its elimination. In neonatal rats treated orally, internal exposures to aglycone BPA were substantially lower than from subcutaneous injection. The results reinforce the critical role for first-pass Phase II metabolism of BPA in gut and liver after oral exposure that attenuates internal exposure to the aglycone form in rats of all ages. The internal exposures to aglycone BPA observed in adult and neonatal rats following a single oral dose of 100 {mu}g/kg bw are inconsistent with effects mediated by classical estrogen receptors based on binding affinities. However, an impact on alternative estrogen signaling pathways that have higher receptor affinity cannot be excluded in neonatal rats. These findings emphasize the importance of matching aglycone BPA internal dosimetry with receptor affinities in experimental animal studies reporting toxicity.« less

Psychological, situational and application-related determinants of the intention to self-test: a factorial survey among students.

PubMed

Kuecuekbalaban, Pinar; Rostalski, Tim; Schmidt, Silke; Muehlan, Holger

2017-07-10

The Internet enables an unprecedented opportunity to access a broad range of self-tests (e.g. testing for HIV, cancer, hepatitis B/C), which can be conducted by lay consumers without the help of a health professional. However, there is only little knowledge about the determinants of the use of self-tests. Thus, the aims of this study were (1) to experimentally investigate the impact of situational and application-related characteristics on the intention to use a self-test (ST), compared to being tested by a health professional at home (HPH) or at a doctor's office (HPD), (2) to examine the applicability of social-cognitive health behaviour theories on self-testing, and (3) to explore the advantages of integrating technological affinity into social-cognitive health behaviour models to predict self-testing. In a factorial survey, 1248 vignettes were rated by 208 students. The core concepts of social-cognitive health behaviour theories, technological affinity, and different situational and application-related characteristics were investigated. Intention to ST was only predicted by the medical expertise of the tested person, while HPH and HPD were also associated with the application purpose of the test and the presence of an emotionally supporting person. Perceived severity and outcome-expectancy significantly predicted intention to self-test. Technological enthusiastic people had a higher intention to use a self-test. Intention to ST, HPH and HPD were predicted by different situational and application-related characteristics. Social-cognitive health behaviour theories can be applied to predict self-testing and do not need to be extended by technological affinity.

Synthesis and biological evaluation of a series of aminoalkyl-tetralones and tetralols as dual dopamine/serotonin ligands.

PubMed

Carro, Laura; Torrado, María; Raviña, Enrique; Masaguer, Christian F; Lage, Sonia; Brea, José; Loza, María I

2014-01-01

A series of novel α-tetralone and α-tetralol derivatives was synthesized, and their binding affinities for 5-HT(2A) and D₂ receptors, the most important targets implicated in the anti-schizophrenia drug action, were evaluated to elucidate how substitutions in the aromatic ring of the pharmacophore affect to the affinity or selectivity for these receptors. The replacement of the H-7 in the tetrahydronaphthalene system by an amino group resulted in privileged 5-HT(2A) affinity of the 6-fluorobenzo[d]isoxazol derivative 36 and the alcohol 25 both showing a pK(i) value for 5-HT(2A) higher than 8.3 and good binding affinities for D₂ receptor leading to a Meltzer's ratio characteristic of an atypical antipsychotic profile. Additionally, a small collection of 3-aminomethyltetralone derivatives was prepared and examined here for their affinities and selectivities as 5-HT(2A)/D₂ dual ligands. Compound 11 shows the best profile with good pKi values for 5-HT(2A) and D₂ receptors leading to a Meltzer's ratio characteristic of a typical antipsychotic behaviour. These three compounds behaved as competitive antagonists of both 5-HT(2A) and D₂ receptors, and might be promising pharmacological tools for the investigation of the dual function of the 5HT(2A)-D₂ ligands. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

PubMed

Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

2003-12-01

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Differential effects of the steaming time and frequency for manufactured red Liriope platyphylla on nerve growth factor secretion ability, nerve growth factor receptor signaling pathway and regulation of calcium concentration.

PubMed

Choi, Sun Il; Goo, Jun Seo; Kim, Ji Eun; Nam, So Hee; Hwang, In Sik; Lee, Hye Ryun; Lee, Young Ju; Son, Hong Joo; Lee, Hee Seob; Lee, Jong Sup; Kim, Hak Jin; Hwang, Dae Youn

2012-11-01

The herb Liriope platyphylla (LP) has been considered to have curative properties for diabetes, asthma and neurodegenerative disorders. To examine the effects of steaming time and frequency of manufactured red LP (RLP) on the nerve growth factor (NGF) secretion ability and NGF receptor signaling pathway, the NGF concentration, cell differentiation, NGF signaling pathway and calcium concentration were analyzed in neuronal cells treated with several types of LPs manufactured under different conditions. The maximum NGF secretion was observed in B35 cells treated with 50 µg/ml LP extract steamed for 9 h (9-SLP) and with two repeated steps (3 h steaming and 24 h air-dried) carried out 7 times (7-SALP). No significant changes in viability were detected in any of the cells treated with the various LPs, with the exception of 0-SLP and 0-SALP. In addition, PC12 cell differentiation was induced by treatment with the NGF-containing conditional medium (CM) collected from the RLP-treated cells. The levels of TrkA and extracellular signal-regulated kinase (ERK) phosphorylation in the high affinity NGF receptor signaling pathway were significantly higher in the cells treated with 3-SLP or 1-SALP/3-SALP CM compared with those treated with the vehicle CM. In the low affinity NGF receptor pathway, the expression levels of most components were higher in the 9-, 15- and 24-SALP CM-treated cells compared with the vehicle CM-treated cells. However, this level was significantly altered in cells treated with 3-SALP CM. Furthermore, an examination of the RLP function on calcium regulation revealed that only the LP- or RLP-treated cells exhibited changes in intracellular and extracellular calcium levels. RLP induced a significant decrease in the intracellular calcium levels and an increase in the extracellular calcium levels. These results suggest the possibility that steaming-processed LP may aid in the relief of neurodegenerative diseases through the NGF secretion ability and NGF signaling pathway.

Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation.

PubMed

Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette

2016-02-11

To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

Bacteria-Affinity 3D Macroporous Graphene/MWCNTs/Fe3O4 Foams for High-Performance Microbial Fuel Cells.

PubMed

Song, Rong-Bin; Zhao, Cui-E; Jiang, Li-Ping; Abdel-Halim, Essam Sayed; Zhang, Jian-Rong; Zhu, Jun-Jie

2016-06-29

Promoting the performance of microbial fuel cells (MFCs) relies heavily on the structure design and composition tailoring of electrode materials. In this work, three-dimensional (3D) macroporous graphene foams incorporated with intercalated spacer of multiwalled carbon nanotubes (MWCNTs) and bacterial anchor of Fe3O4 nanospheres (named as G/MWCNTs/Fe3O4 foams) were first synthesized and used as anodes for Shewanella-inoculated microbial fuel cells (MFCs). Thanks to the macroporous structure of 3D graphene foams, the expanded electrode surface by MWCNTs spacing, as well as the high affinity of Fe3O4 nanospheres toward Shewanella oneidensis MR-1, the anode exhibited high bacterial loading capability. In addition to spacing graphene nanosheets for accommodating bacterial cells, MWCNTs paved a smoother way for electron transport in the electrode substrate of MFCs. Meanwhile, the embedded bioaffinity Fe3O4 nanospheres capable of preserving the bacterial metabolic activity provided guarantee for the long-term durability of the MFCs. With these merits, the constructed MFC possessed significantly higher power output and stronger stability than that with conventional graphite rod anode.

In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis.

PubMed

Savory, Nasa; Lednor, Danielle; Tsukakoshi, Kaori; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Jones, Brian V; Ikebukuro, Kazunori

2013-10-01

Proteus mirabilis is a prominent cause of catheter-associated urinary tract infections (CAUTIs) among patients undergoing long-term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane-associated proteins in this organism. Copyright © 2013 Wiley Periodicals, Inc.

Tailoring charge density and hydrogen bonding of imidazolium copolymers for efficient gene delivery.

PubMed

Allen, Michael H; Green, Matthew D; Getaneh, Hiwote K; Miller, Kevin M; Long, Timothy E

2011-06-13

Conventional free radical polymerization with subsequent postpolymerization modification afforded imidazolium copolymers with controlled charge density and side chain hydroxyl number. Novel imidazolium-containing copolymers where each permanent cation contained one or two adjacent hydroxyls allowed precise structure-transfection efficiency studies. The degree of polymerization was identical for all copolymers to eliminate the influence of molecular weight on transfection efficiency. DNA binding, cytotoxicity, and in vitro gene transfection in African green monkey COS-7 cells revealed structure-property-transfection relationships for the copolymers. DNA gel shift assays indicated that higher charge densities and hydroxyl concentrations increased DNA binding. As the charge density of the copolymers increased, toxicity of the copolymers also increased; however, as hydroxyl concentration increased, cytotoxicity remained constant. Changing both charge density and hydroxyl levels in a systematic fashion revealed a dramatic influence on transfection efficiency. Dynamic light scattering of the polyplexes, which were composed of copolymer concentrations required for the highest luciferase expression, showed an intermediate DNA-copolymer binding affinity. Our studies supported the conclusion that cationic copolymer binding affinity significantly impacts overall transfection efficiency of DNA delivery vehicles, and the incorporation of hydroxyl sites offers a less toxic and effective alternative to more conventional highly charged copolymers.

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

PubMed

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

2013-07-05

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

Quantum-Mechanics Methodologies in Drug Discovery: Applications of Docking and Scoring in Lead Optimization.

PubMed

Crespo, Alejandro; Rodriguez-Granillo, Agustina; Lim, Victoria T

2017-01-01

The development and application of quantum mechanics (QM) methodologies in computer- aided drug design have flourished in the last 10 years. Despite the natural advantage of QM methods to predict binding affinities with a higher level of theory than those methods based on molecular mechanics (MM), there are only a few examples where diverse sets of protein-ligand targets have been evaluated simultaneously. In this work, we review recent advances in QM docking and scoring for those cases in which a systematic analysis has been performed. In addition, we introduce and validate a simplified QM/MM expression to compute protein-ligand binding energies. Overall, QMbased scoring functions are generally better to predict ligand affinities than those based on classical mechanics. However, the agreement between experimental activities and calculated binding energies is highly dependent on the specific chemical series considered. The advantage of more accurate QM methods is evident in cases where charge transfer and polarization effects are important, for example when metals are involved in the binding process or when dispersion forces play a significant role as in the case of hydrophobic or stacking interactions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cloning and characterization of giant panda (Ailuropoda melanoleuca) IL-18 binding protein.

PubMed

Yan, Yue; Deng, Jiabo; Niu, Lili; Wang, Qiang; Yu, Jianqiu; Shao, Huanhuan; Cao, Qinghua; Zhang, Yizheng; Tan, Xuemei

2016-06-01

The giant panda (Ailuropoda melanoleuca) is an endangered species. Interleukin-18 (IL-18) plays an important role in the innate and adaptive immune responses by inducing IFN-γ. IL-18 has been implicated in the pathogenesis of various diseases. IL-18 binding protein (IL-18BP) is an intrinsic inhibitor of IL-18 that possesses higher affinity to IL-18. In this study, we cloned and characterized IL-18BP in giant panda (AmIL-18BP) from the spleen. The amino acid sequence of giant panda IL-18BP ORF shared about 65% identities with other species. To evaluate the effects of AmIL-18BP on the immune responses, we expressed the recombinant AmIL-18BP in Escherichia coli BL21 (DE3).The fusing protein PET-AmIL-18BP was purified by nickel affinity column chromatography. The biological function of purified PET-AmIL-18BP was determined on mice splenocyte by qRT-PCR. The results showed that AmIL-18BP was functional and could significantly reduce IFN-γ production in murine splenocytes. These results will facilitate the study of protecting giant panda on etiology and immunology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distribution of trace metals in surface seawater and zooplankton of the Bay of Bengal, off Rushikulya estuary, East Coast of India.

PubMed

Srichandan, Suchismita; Panigrahy, R C; Baliarsingh, S K; Rao B, Srinivasa; Pati, Premalata; Sahu, Biraja K; Sahu, K C

2016-10-15

Concentrations of trace metals such as iron (Fe), copper (Cu), zinc (Zn), cobalt (Co), nickel (Ni), manganese (Mn), lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), vanadium (V), and selenium (Se) were determined in seawater and zooplankton from the surface waters off Rushikulya estuary, north-western Bay of Bengal. During the study period, the concentration of trace metals in seawater and zooplankton showed significant spatio-temporal variation. Cu and Co levels in seawater mostly remained non-detectable. Other elements were found at higher concentrations and exhibited marked variations. The rank order distribution of trace metals in terms of their average concentration in seawater was observed as Fe>Ni>Mn>Pb>As>Zn>Cr>V>Se>Cd while in zooplankton it was Fe>Mn>Cd>As>Pb>Ni>Cr>Zn>V>Se. The bioaccumulation factor (BAF) of Fe was highest followed by Zn and the lowest value was observed with Ni. Results of correlation analysis discerned positive affinity and good relationship among the majority of the trace metals, both in seawater and zooplankton suggesting their strong affinity and coexistence. Copyright © 2016 Elsevier Ltd. All rights reserved.

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

PubMed

Ferreira, Iêda Mendes; de Souza Lacerda, Camila Maria; de Faria, Lígia Santana; Corrêa, Cristiane Rodrigues; de Andrade, Antero Silva Ribeiro

2014-12-01

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 μM and for Antibac2 was 1.261 + 0.280 μM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marks, J.L.; Ax, R.L.

1985-08-01

The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bullsmore » of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.« less

Hemoglobin–oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed Central

2016-01-01

ABSTRACT In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)–O2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb–O2 affinity should be expected to improve tissue O2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O2 loading and peripheral O2 unloading. Theoretical results indicate that the optimal Hb–O2 affinity varies as a non-linear function of environmental O2 availability, and the threshold elevation at which an increased Hb–O2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O2 equilibration at the blood–gas interface is limited by the kinetics of O2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb–O2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb–O2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb–O2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. PMID:27802149

Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

PubMed

Storz, Jay F

2016-10-15

In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)-O 2 affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb-O 2 affinity should be expected to improve tissue O 2 delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O 2 loading and peripheral O 2 unloading. Theoretical results indicate that the optimal Hb-O 2 affinity varies as a non-linear function of environmental O 2 availability, and the threshold elevation at which an increased Hb-O 2 affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O 2 equilibration at the blood-gas interface is limited by the kinetics of O 2 exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb-O 2 affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb-O 2 affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb-O 2 affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates. © 2016. Published by The Company of Biologists Ltd.

[Adsorption of phenol chemicals by surfactant-modified zeolites].

PubMed

Xie, Jie; Wang, Zhe; Wu, De-Yi; Li, Chun-Jie

2012-12-01

Two kinds of zeolites were prepared from fly ash and modified by surfactant subsequently. Surfactant-modified zeolites were studied for adsorption of phenol chemicals (phenol, p-chlorphenol, bisphenol A). It showed that the adsorption affinity of zeolite to phenol chemicals was significantly improved after surfactant modification. The adsorption isotherms of phenol chemicals were well fitted by the Langmuir isotherm. For the two surfactant-surfactant modified zeolites, the maximum adsorption amounts of phenol, p-chlorphenol, and bisphenol A calculated from the Langmuir equation were 37.7, 52.36, 90.9 mg x g(-1) and 10.7, 22.83, 56.8 mg x g(-1), respectively. When pH values of solutions were higher than the pK(a) values of phenol chemicals, the removal efficiencies were getting higher with the increase of pH values. The octanol/water partition coefficient (K(ow)) was also found to be an important factor affecting adsorption of phenol chemicals by the modified zeolites. Higher K(ow) value, which means the greater hydrophobicity of the chemicals, resulted in a higher removal.

Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

PubMed Central

KASAI, Kenichi

2014-01-01

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

Demonstration of muscarinic and nicotinic receptor binding activities of distigmine to treat detrusor underactivity.

PubMed

Harada, Taketsugu; Fushimi, Kazumi; Kato, Aya; Ito, Yoshihiko; Nishijima, Saori; Sugaya, Kimio; Yamada, Shizuo

2010-01-01

The present study was undertaken to examine whether distigmine, a therapeutic agent used to treat detrusor underactivity, binds directly to muscarinic and nicotinic receptors. We used radioreceptor binding assays and compared the effects of distigmine with those of neostigmine and donepedil. The inhibitory effect of distigmine on the blood acetylcholinesterase (AChE) activity was significantly weaker than that of neostigmine. Distigmine, neostigmine, and donepezil competed for specific binding sites of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS ) and [(3)H]oxotremorine-M in the bladder, submaxillary gland and cerebral cortex of rats in a concentration-dependent manner, indicating significant binding activity of muscarinic receptors. Distigmine displayed significantly higher affinity for binding sites of [(3)H]oxotremorine-M compared with those of [(3)H]NMS as revealed by large ratios of its K(i) value for [(3)H]NMS to that for [(3)H]oxotremorine-M, suggesting that it has preferential affinity for agonist sites of muscarinic receptors. Distigmine seemed to bind to the agonist sites of muscarinic receptors in a competitive manner. Repeated oral administration of distigmine caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]NMS in the bladder and submaxillary gland but not cerebral cortex. Distigmine also bound to nicotinic receptors in the rat cerebral cortex. In conclusion, distigmine shows direct binding to muscarinic receptors in the rat bladder, and repeated oral administration of distigmine causes downregulation of muscarinic receptors in the rat bladder. The observed direct interaction of distigmine with the bladder muscarinic receptors may partly contribute to the therapeutic and/or side effects seen in the treatment of detrusor underactivity.

Neurochemical binding profiles of novel indole and benzofuran MDMA analogues.

PubMed

Shimshoni, Jakob A; Winkler, Ilan; Golan, Ezekiel; Nutt, David

2017-01-01

3,4-Methylenedioxy-N-methylamphetamine (MDMA) has been shown to be effective in the treatment of post-traumatic stress disorder (PTSD) in numerous clinical trials. In the present study, we have characterized the neurochemical binding profiles of three MDMA-benzofuran analogues (1-(benzofuran-5-yl)-propan-2-amine, 5-APB; 1-(benzofuran-6-yl)-N-methylpropan-2-amine, 6-MAPB; 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 5-MAPB) and one MDMA-indole analogue (1-(1H-indol-5-yl)-2-methylamino-propan-1-ol, 5-IT). These compounds were screened as potential second-generation anti-PTSD drugs, against a battery of human and non-human receptors, transporters, and enzymes, and their potencies as 5-HT 2 receptor agonist and monoamine uptake inhibitors determined. All MDMA analogues displayed high binding affinities for 5-HT 2a,b,c and NE α2 receptors, as well as significant 5-HT, DA, and NE uptake inhibition. 5-APB revealed significant agonist activity at the 5-HT 2a,b,c receptors, while 6-MAPB, 5-MAPB, and 5-IT exhibited significant agonist activity at the 5-HT 2c receptor. There was a lack of correlation between the results of functional uptake and the monoamine transporter binding assay. MDMA analogues emerged as potent and selective monoamine oxidase A inhibitors. Based on 6-MAPB favorable pharmacological profile, it was further subjected to IC 50 determination for monoamine transporters. Overall, all MDMA analogues displayed higher monoamine receptor/transporter binding affinities and agonist activity at the 5-HT 2a,c receptors as compared to MDMA.

Visualising neuroinflammation in post-stroke patients: a comparative PET study with the TSPO molecular imaging biomarkers [11C]PK11195 and [11C]vinpocetine.

PubMed

Gulyas, Balazs; Toth, Miklos; Vas, Adam; Shchukin, Evgeni; Kostulas, Konstantinos; Hillert, Jan; Halldin, Christer

2012-01-01

With the main objective of comparing the prospective diagnostic power of two 11C-labelled molecular imaging biomarkers with affinity for TSPO and used for the visualisation of activated microglia after a stroke, we measured with positron emission tomography (PET) in four post-stroke patients the regional brain uptake and binding potential of [11C]vinpocetine and [11C]PK11195. Percentage standard uptake values (%SUV) and binding potential (BPND) were used as outcome measures. The total peak brain uptake value and average global brain uptake value were higher for [11C]vinpocetine than for [11C]PK11195. The regional %SUV values were significantly higher for [11C]vinpocetine than for [11C]PK11195 in the hemispheres as well as in almost all standard brain regions. The %SUV values of [11C]vinpocetine were higher in the peri-infarct zone than in the ischaemic core, however, the difference did not prove to be significant. There was basically no difference in %SUV values between the ischaemic core and the peri-infarct zone for [11C]PK11195. The BPND values for [11C]vinpocetine were higher in all standard regions than those for [11C]PK11195, but the difference was not significant between them. The BPND values of [11C]vinpocetine were higher in the peri-infarct zone than in the ischaemic core, however, the difference did not prove to be significant. A comparative analysis of the two ligands indicates that [11C]vinpocetine shows a number of favourable characteristics over [11C]PK11195, but to demonstrate that it may serve as a prospective molecular imaging biomarker of microglia activation in post-stroke patients, further studies are required.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-05-01

Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

PubMed Central

Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

2016-01-01

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

PubMed

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K uptake.

PubMed

Rubio, Francisco; Alemán, Fernando; Nieves-Cordones, Manuel; Martínez, Vicente

2010-06-01

The high-affinity K(+) transporter AtHAK5 and the inward-rectifier K(+) channel AtAKT1 have been described to contribute to K(+) uptake in Arabidopsis thaliana. Studies with T-DNA insertion lines showed that both systems participate in the high-affinity range of concentrations and only AtAKT1 in the low-affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock-out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K(+) uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K(+) AtHAK5 and AtAKT1 are the only contributors to K(+) acquisition. At higher K(+) concentrations, unknown systems come into operation and participate together with AtAKT1 in low-affinity K(+) uptake. These systems can supply sufficient K(+) to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K(+) where AtAKT1 is not essential.

Self-Assembled N-Heterocyclic Carbene-Based Carboxymethylated Dextran Monolayers on Gold as a Tunable Platform for Designing Affinity-Capture Biosensor Surfaces.

PubMed

Li, Zhijun; Munro, Kim; Narouz, Mina R; Lau, Andrew; Hao, Hongxia; Crudden, Cathleen M; Horton, J Hugh

2018-05-30

Sensor surfaces play a predominant role in the development of optical biosensor technologies for the analysis of biomolecular interactions. Thiol-based self-assembled monolayers (SAMs) on gold have been widely used as linker layers for sensor surfaces. However, the degradation of the thiol-gold bond can limit the performance and durability of such surfaces, directly impacting their performance and cost-effectiveness. To this end, a new family of materials based on N-heterocyclic carbenes (NHCs) has emerged as an alternative for surface modification, capable of self-assembling onto a gold surface with higher affinity and superior stability as compared to the thiol-based systems. Here we demonstrate three applications of NHC SAMs supporting a dextran layer as a tunable platform for developing various affinity-capture biosensor surfaces. We describe the development and testing of NHC-based dextran biosensor surfaces modified with each of streptavidin, nitrilotriacetic acid, and recombinant Protein A. These affinity-capture sensor surfaces enable oriented binding of ligands for optimal performance in biomolecular assays. Together, the intrinsic high stability and flexible design of the NHC biosensing platforms show great promise and open up exciting possibilities for future biosensing applications.

Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands.

PubMed Central

Kinnally, K W; Zorov, D B; Antonenko, Y N; Snyder, S H; McEnery, M W; Tedeschi, H

1993-01-01

The mitochrondrial benzodiazepine receptor (mBzR) binds a subset of benzodiazepines and isoquinoline carboxamides with nanomolar affinity and consists of the voltage-dependent anion channel, the adenine nucleotide translocator, and an 18-kDa protein. The effect of ligands of the mBzR on two inner mitochondrial membrane channel activities was determined with patch-clamp techniques. The relative inhibitory potencies of the drugs resemble their binding affinities for the mBzR. Ro5-4864 and protoporphyrin IX inhibit activity of the multiple conductance channel (MCC) and the mitochondrial centum-picosiemen (mCtS) channel activities at nanomolar concentrations. PK11195 inhibits mCtS activity at similar levels. Higher concentrations of protoporphyrin IX induce MCC but possibly not mCtS activity. Clonazepam, which has low affinity for mBzR, is at least 500 times less potent at both channel activities. Ro15-1788, which also has a low mBzR affinity, inhibits MCC at very high concentrations (16 microM). The findings indicate an association of these two channel activities with the proteins forming the mBzR complex and are consistent with an interaction of inner and outer membrane channels. PMID:7679505

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

PubMed

Zhang, Zijie; Liu, Juewen

2016-03-01

Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

Polypharmacotherapy in rheumatology: 1H NMR analysis of binding of phenylbutazone and methotrexate to serum albumin

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Równicka-Zubik, J.; Bojko, B.; Szkudlarek-Haśnik, A.; Knopik, M.; Sułkowski, W. W.

2011-05-01

The influence of phenylbutazone (Phe) and methotrexate (MTX) on binding of MTX and Phe to human (HSA) and bovine (BSA) serum albumin in the low-affinity binding sites is investigated. The strength and kind of interactions between serum albumin (SA) and drugs used in combination therapy were found using 1H NMR spectroscopy. A stoichiometric molar ratios for Phe-SA and MTX-SA complexes are 36:1 and 31:1, respectively. It appeared these molar ratios are higher for the ternary systems than it were in the binary ones. The presence of the additional drug (MTX or Phe) causes the increase of an affinity of albumin towards Phe and MTX. It was found that the aliphatic groups of MTX are more resistant to the influence of Phe on the MTX-SA complex than the aromatic rings. The results showed the important impact of another drug (MTX or Phe) on the affinity of SA towards Phe and MTX in the low-affinity binding sites. This work is a subsequent part of the spectroscopic study on Phe-MTX-SA interactions (Maciążek-Jurczyk, 2009 [1]).

From Biomass-Derived Furans to Aromatics with Ethanol over Zeolite.

PubMed

Teixeira, Ivo F; Lo, Benedict T W; Kostetskyy, Pavlo; Stamatakis, Michail; Ye, Lin; Tang, Chiu C; Mpourmpakis, Giannis; Tsang, Shik Chi Edman

2016-10-10

We report a novel catalytic conversion of biomass-derived furans and alcohols to aromatics over zeolite catalysts. Aromatics are formed via Diels-Alder cycloaddition with ethylene, which is produced in situ from ethanol dehydration. The use of liquid ethanol instead of gaseous ethylene, as the source of dienophile in this one-pot synthesis, makes the aromatics production much simpler and renewable, circumventing the use of ethylene at high pressure. More importantly, both our experiments and theoretical studies demonstrate that the use of ethanol instead of ethylene, results in significantly higher rates and higher selectivity to aromatics, due to lower activation barriers over the solid acid sites. Synchrotron-diffraction experiments and proton-affinity calculations clearly suggest that a preferred protonation of ethanol over the furan is a key step facilitating the Diels-Alder and dehydration reactions in the acid sites of the zeolite. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of granule swelling on starch saccharification by granular starch hydrolyzing enzyme.

PubMed

Li, Zhaofeng; Cai, Liming; Gu, Zhengbiao; Shi, Yong-Cheng

2014-08-13

The effects of granule swelling on enzymatic saccharification of normal corn starch by granular starch hydrolyzing enzyme were investigated. After swelling, Km values for the saccharification of granular starch decreased compared with native granular starch, indicating that granule swelling caused granular starch hydrolyzing enzyme to have higher affinity for starch granules. The partial swelling of starch granules enhanced starch saccharification. Furthermore, the enhancement at an earlier stage of enzymatic reaction was much more significant than that at later stages. For granular starch pretreated at 67.5 °C for 30 min, conversions to glucose after incubation with the enzyme at 32 °C for 4 and 24 h were approximately 3-fold and 26% higher than for native granular starch, respectively. As a result, proper heat pretreatment of granular starch before simultaneous saccharification and fermentation has great potential to facilitate industrial production of ethanol by use of granular starch hydrolyzing enzyme.

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

Modeling of non-ideal hard permanent magnets with an affine-linear model, illustrated for a bar and a horseshoe magnet

NASA Astrophysics Data System (ADS)

Glane, Sebastian; Reich, Felix A.; Müller, Wolfgang H.

2017-11-01

This study is dedicated to continuum-scale material modeling of isotropic permanent magnets. An affine-linear extension to the commonly used ideal hard model for permanent magnets is proposed, motivated, and detailed. In order to demonstrate the differences between these models, bar and horseshoe magnets are considered. The structure of the boundary value problem for the magnetic field and related solution techniques are discussed. For the ideal model, closed-form analytical solutions were obtained for both geometries. Magnetic fields of the boundary value problems for both models and differently shaped magnets were computed numerically by using the boundary element method. The results show that the character of the magnetic field is strongly influenced by the model that is used. Furthermore, it can be observed that the shape of an affine-linear magnet influences the near-field significantly. Qualitative comparisons with experiments suggest that both the ideal and the affine-linear models are relevant in practice, depending on the magnetic material employed. Mathematically speaking, the ideal magnetic model is a special case of the affine-linear one. Therefore, in applications where knowledge of the near-field is important, the affine-linear model can yield more accurate results—depending on the magnetic material.

Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model.

PubMed

Muddana, Hari S; Gilson, Michael K

2012-06-12

The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy function, PM6-DH+, coupled with the COSMO solvation model, to 29 host-guest systems with a wide range of measured binding affinities. After correction for a systematic error, which appears to derive from the treatment of polar solvation, the computed absolute binding affinities agree well with experimental measurements, with a mean error 1.6 kcal/mol and a correlation coefficient of 0.91. These calculations also delineate the contributions of various energy components, including solute energy, configurational entropy, and solvation free energy, to the binding free energies of these host-guest complexes. Comparison with our previous calculations, which used empirical force fields, point to significant differences in both the energetic and entropic components of the binding free energy. The present study demonstrates successful combination of a quantum mechanical Hamiltonian with the M2 affinity method.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

PubMed

Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

1998-07-03

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

OER Awareness and Use: The Affinity between Higher Education and K-12

ERIC Educational Resources Information Center

Blomgren, Constance

2018-01-01

Educators within Higher Education (HE) and K-12 share in the need for high quality educational resources to assist in the pursuit of teaching and learning. Although there are numerous differences between the two levels of education, there are commonalties in the perceptions of the purpose, practical uses, and challenges that abide in the use of…

Independent-particle models for light negative atomic ions

NASA Technical Reports Server (NTRS)

Ganas, P. S.; Talman, J. D.; Green, A. E. S.

1980-01-01

For the purposes of astrophysical, aeronomical, and laboratory application, a precise independent-particle model for electrons in negative atomic ions of the second and third period is discussed. The optimum-potential model (OPM) of Talman et al. (1979) is first used to generate numerical potentials for eight of these ions. Results for total energies and electron affinities are found to be very close to Hartree-Fock solutions. However, the OPM and HF electron affinities both depart significantly from experimental affinities. For this reason, two analytic potentials are developed whose inner energy levels are very close to the OPM and HF levels but whose last electron eigenvalues are adjusted precisely with the magnitudes of experimental affinities. These models are: (1) a four-parameter analytic characterization of the OPM potential and (2) a two-parameter potential model of the Green, Sellin, Zachor type. The system O(-) or e-O, which is important in upper atmospheric physics is examined in some detail.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

PubMed

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

PubMed Central

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

PubMed

Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

2006-06-01

Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

An affinity chromatography-gel filtration device for preparing thyroid microsomal antigen.

PubMed

Wang, L; Zheng, W F

1987-09-24

On the basis of conventional differential centrifugation for preparing crude thyroid microsomal antigen (TMAg), we have employed Sepharose 4B gel filtration and affinity chromatography separately to study the elution pattern in terms of absorbance and antigenic activity. The result indicates that thyroglobulin (TG) exists in two forms in crude TMAg, i.e., 'free TG' and 'membrane-bound TG'. TMAg is present in two forms in the eluate: (1) the TM fragment or TMAg polymer, which is produced at a higher rate and has greater antigenic activity, but which is less pure; (2) soluble TMAg, which is produced at a lower rate and has less antigenic activity, but which is more pure. We have developed an affinity chromatography-gel filtration (AC-GF) device which is a combination of affinity chromatography and a Sepharose 4B column. Sephadex G-50 is placed between the rubber stopper and Sepharose 4B in the GF column to ensure intactness of the entire system. With such a device, the AC removes the contaminated TG from TM homogenate, and allows the latter to pass directly from AC to GF for rechromatography. This device extracts the full advantages of both methods and each compensates for any deficiency of the other. Using this one-step procedure, one has the greatest chance of removing TG and obtaining TM fragments of TMAg polymers of higher antigenic activity, as well as separating small amounts of more purified soluble TMAg. Thus, the newly developed method meets the need of large quantities of TMAg for practical application, and at the same time the more purified preparations can be used for analytical purposes.

Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

PubMed Central

Wang, Sho-Ya; Mitchell, Jane; Moczydlowski, Edward; Wang, Ging Kuo

2004-01-01

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding. PMID:15545401

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Aptamers that bind to the hemagglutinin of the recent pandemic influenza virus H1N1 and efficiently inhibit agglutination.

PubMed

Gopinath, Subash C B; Kumar, Penmetcha K R

2013-11-01

Influenza virus hemagglutinin (HA) mediates both receptor (glycan) binding and membrane fusion for cell entry and has been the basis for typing influenza A viruses. In this study we have selected RNA aptamers (D-12 and D-26) that specifically target the HA protein of the recent pandemic influenza virus pdmH1N1 (A/California/07/2009). Among the selected aptamers the D-26 aptamer showed higher affinity for the HA of pdmH1N1 and was able to distinguish HA derived from other sub-types of influenza A viruses. The affinity of the D-26 aptamer was further improved upon incorporation of 2'-fluoropyrimidines to a level of 67 fM. Furthermore, the high affinity D-12 and D-26 aptamers were tested for their ability to interfere with HA-glycan interactions using a chicken red blood cell (RBC) agglutination assay. At a concentration of 200 nM the D-26 aptamer completely abolished the agglutination of RBCs, whereas D-12 only did so at 400 nM. These studies suggest that the selected aptamer D-26 not only has a higher affinity and specificity for the HA of pdmH1N1 but also has a better ability to efficiently interfere with HA-glycan interactions compared with the D-12 aptamer. The D-26 aptamer warrants further study regarding its application in developing topical virucidal products against the pdmH1N1 virus and also in surveillance of the pdmH1N1 influenza virus. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Affine invariants of convex polygons.

PubMed

Flusser, Jan

2002-01-01

In this correspondence, we prove that the affine invariants, for image registration and object recognition, proposed recently by Yang and Cohen (see ibid., vol.8, no.7, p.934-46, July 1999) are algebraically dependent. We show how to select an independent and complete set of the invariants. The use of this new set leads to a significant reduction of the computing complexity without decreasing the discrimination power.

Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.

An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented.more » The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.« less

Crystal structures of thrombin in complex with chemically modified thrombin DNA aptamers reveal the origins of enhanced affinity.

PubMed

Dolot, Rafal; Lam, Curtis H; Sierant, Malgorzata; Zhao, Qiang; Liu, Feng-Wu; Nawrot, Barbara; Egli, Martin; Yang, Xianbin

2018-05-18

Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.

Progesterone receptors in the female lower urinary tract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Batra, S.C.; Iosif, C.S.

1987-11-01

When female estrogenized rabbits were injected i.v. with /sup 3/H-progesterone, the tritium concentration determined after one hour was about two to three times higher in urethra, urinary bladder and vagina than in the heart. High affinity progesterone receptors (KD = 1-2 nM) could be demonstrated in both cytoplasmic and nuclear fractions prepared from estrogenized rabbit urethra, bladder and vagina. The cytosolic receptor concentration in both urethra and bladder was about half of that in the vagina. The concentration of nuclear receptors in urethra was not significantly different from that in the vagina, but in the bladder the concentration was onlymore » about one fourth of that in the vagina or urethra. The mean KD of cytosolic receptors from bladder was significantly higher than the corresponding values in urethra and vagina. Progesterone binding sites in the bladder had a broader hormonal specificity than those in the urethra or vagina. The present demonstration of specific progesterone receptors in the female urethra might provide a possible link between estrogen progesterone interaction and the appearance of urinary incontinence during pregnancy in women.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Linlin; Sun, Xiaodong; Xie, Songbo

Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreaticmore » and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.« less

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Predicting the Effects of Sugar-Sweetened Beverage Taxes on Food and Beverage Demand in a Large Demand System

PubMed Central

Zhen, Chen; Finkelstein, Eric A.; Nonnemaker, James; Karns, Shawn; Todd, Jessica E.

2013-01-01

A censored Exact Affine Stone Index incomplete demand system is estimated for 23 packaged foods and beverages and a numéraire good. Instrumental variables are used to control for endogenous prices. A half-cent per ounce increase in sugar-sweetened beverage prices is predicted to reduce total calories from the 23 foods and beverages but increase sodium and fat intakes as a result of product substitution. The predicted decline in calories is larger for low-income households than for high-income households, although welfare loss is also higher for low-income households. Neglecting price endogeneity or estimating a conditional demand model significantly overestimates the calorie reduction. PMID:24839299

Predicting the Effects of Sugar-Sweetened Beverage Taxes on Food and Beverage Demand in a Large Demand System.

PubMed

Zhen, Chen; Finkelstein, Eric A; Nonnemaker, James; Karns, Shawn; Todd, Jessica E

2014-01-01

A censored Exact Affine Stone Index incomplete demand system is estimated for 23 packaged foods and beverages and a numéraire good. Instrumental variables are used to control for endogenous prices. A half-cent per ounce increase in sugar-sweetened beverage prices is predicted to reduce total calories from the 23 foods and beverages but increase sodium and fat intakes as a result of product substitution. The predicted decline in calories is larger for low-income households than for high-income households, although welfare loss is also higher for low-income households. Neglecting price endogeneity or estimating a conditional demand model significantly overestimates the calorie reduction.

A Virtual Screening Approach for the Identification of High Affinity Small Molecules Targeting BCR-ABL1 Inhibitors for the Treatment of Chronic Myeloid Leukemia.

PubMed

Sharda, Saphy; Sarmandal, Palash; Cherukommu, Shirisha; Dindhoria, Kiran; Yadav, Manisha; Bandaru, Srinivas; Sharma, Anudeep; Sakhi, Aditi; Vyas, Tanmay; Hussain, Tajamul; Nayarisseri, Anuraj; Singh, Sanjeev Kumar

2017-01-01

CML originates due to reciprocal translocation in Philadelphia chromosome leading to the formation of fusion product BCR-ABL which constitutively activates tyrosine kinase signaling pathways eventually leading to abnormal proliferation of granulocytic cells. As a therapeutic strategy, BCR-ABL inhibitors have been clinically approved which terminates its phosphorylation activity and retards cancer progression. However, a number of patients develop resistance to inhibitors which demand for the discovery of new inhibitors. Given the drawbacks of present inhibitors, by high throughput virtual screening approaches, present study pursues to identify high affinity compounds targeting BCR-ABL1 anticipated to have safer pharmacological profiles. Five established BCR-ABL inhibitors formed the query compounds for identification of structurally similar compounds by Tanimoto coefficient based linear fingerprint search with a threshold of 95% against PubChemdatabase. Assisted by MolDock algorithm all compounds were docked against BCR-ABL protein in order to retrieve high affinity compounds. The parents and similars were further tested for their ADMET propertiesand bioactivity. Rebastinib formed higher affinity inhibitor than rest of the four established compound investigated in the study. Interestingly, Rebastinib similar compound with Pubchem ID: 67254402 was also shown to have highest affinity than other similars including the similars of respective five parents. In terms of ADMET properties Pubchem ID: 67254402 had appreciable ADMET profile and bioactivity. However, Rebastinib still stood as the best inhibitor in terms of binding affinity and ADMET properties than Pubchem ID: 67254402. Nevertheless, owing to the similar pharmacological properties with Rebastinib, Pubchem ID: 67254402 can be expected to form potential BCR-ABL inhibitor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

PubMed

Malm, Magdalena; Kronqvist, Nina; Lindberg, Hanna; Gudmundsdotter, Lindvi; Bass, Tarek; Frejd, Fredrik Y; Höidén-Guthenberg, Ingmarie; Varasteh, Zohreh; Orlova, Anna; Tolmachev, Vladimir; Ståhl, Stefan; Löfblom, John

2013-01-01

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

PubMed

Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

2010-01-01

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

PubMed

Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

1999-09-17

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Genetic affinities among the lower castes and tribal groups of India: inference from Y chromosome and mitochondrial DNA.

PubMed

Thanseem, Ismail; Thangaraj, Kumarasamy; Chaubey, Gyaneshwer; Singh, Vijay Kumar; Bhaskar, Lakkakula V K S; Reddy, B Mohan; Reddy, Alla G; Singh, Lalji

2006-08-07

India is a country with enormous social and cultural diversity due to its positioning on the crossroads of many historic and pre-historic human migrations. The hierarchical caste system in the Hindu society dominates the social structure of the Indian populations. The origin of the caste system in India is a matter of debate with many linguists and anthropologists suggesting that it began with the arrival of Indo-European speakers from Central Asia about 3500 years ago. Previous genetic studies based on Indian populations failed to achieve a consensus in this regard. We analysed the Y-chromosome and mitochondrial DNA of three tribal populations of southern India, compared the results with available data from the Indian subcontinent and tried to reconstruct the evolutionary history of Indian caste and tribal populations. No significant difference was observed in the mitochondrial DNA between Indian tribal and caste populations, except for the presence of a higher frequency of west Eurasian-specific haplogroups in the higher castes, mostly in the north western part of India. On the other hand, the study of the Indian Y lineages revealed distinct distribution patterns among caste and tribal populations. The paternal lineages of Indian lower castes showed significantly closer affinity to the tribal populations than to the upper castes. The frequencies of deep-rooted Y haplogroups such as M89, M52, and M95 were higher in the lower castes and tribes, compared to the upper castes. The present study suggests that the vast majority (> 98%) of the Indian maternal gene pool, consisting of Indio-European and Dravidian speakers, is genetically more or less uniform. Invasions after the late Pleistocene settlement might have been mostly male-mediated. However, Y-SNP data provides compelling genetic evidence for a tribal origin of the lower caste populations in the subcontinent. Lower caste groups might have originated with the hierarchical divisions that arose within the tribal groups with the spread of Neolithic agriculturalists, much earlier than the arrival of Aryan speakers. The Indo-Europeans established themselves as upper castes among this already developed caste-like class structure within the tribes.

Genetic affinities among the lower castes and tribal groups of India: inference from Y chromosome and mitochondrial DNA

PubMed Central

Thanseem, Ismail; Thangaraj, Kumarasamy; Chaubey, Gyaneshwer; Singh, Vijay Kumar; Bhaskar, Lakkakula VKS; Reddy, B Mohan; Reddy, Alla G; Singh, Lalji

2006-01-01

Background India is a country with enormous social and cultural diversity due to its positioning on the crossroads of many historic and pre-historic human migrations. The hierarchical caste system in the Hindu society dominates the social structure of the Indian populations. The origin of the caste system in India is a matter of debate with many linguists and anthropologists suggesting that it began with the arrival of Indo-European speakers from Central Asia about 3500 years ago. Previous genetic studies based on Indian populations failed to achieve a consensus in this regard. We analysed the Y-chromosome and mitochondrial DNA of three tribal populations of southern India, compared the results with available data from the Indian subcontinent and tried to reconstruct the evolutionary history of Indian caste and tribal populations. Results No significant difference was observed in the mitochondrial DNA between Indian tribal and caste populations, except for the presence of a higher frequency of west Eurasian-specific haplogroups in the higher castes, mostly in the north western part of India. On the other hand, the study of the Indian Y lineages revealed distinct distribution patterns among caste and tribal populations. The paternal lineages of Indian lower castes showed significantly closer affinity to the tribal populations than to the upper castes. The frequencies of deep-rooted Y haplogroups such as M89, M52, and M95 were higher in the lower castes and tribes, compared to the upper castes. Conclusion The present study suggests that the vast majority (>98%) of the Indian maternal gene pool, consisting of Indio-European and Dravidian speakers, is genetically more or less uniform. Invasions after the late Pleistocene settlement might have been mostly male-mediated. However, Y-SNP data provides compelling genetic evidence for a tribal origin of the lower caste populations in the subcontinent. Lower caste groups might have originated with the hierarchical divisions that arose within the tribal groups with the spread of Neolithic agriculturalists, much earlier than the arrival of Aryan speakers. The Indo-Europeans established themselves as upper castes among this already developed caste-like class structure within the tribes. PMID:16893451

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Differences in adsorption mechanisms of heavy metal by two different plant biomasses: reed and brown seaweed.

PubMed

Southichak, B; Nakano, K; Nomura, M; Chiba, N; Nishimura, O

2009-01-01

The adsorption of Pb(II) by two different biomaterials, reed (Phragmites australis) and brown seaweed (Sargassum horneri) biomass pretreated with CaCl(2), were compared in an attempt to explain the differences in adsorption performance between the two biosorbents. A very interesting characteristic was found in their individual adsorption performances; the Pb(II) adsorption capacity of brown seaweed (Q(max)=0.45 mmol/g) was much higher than that of reed (Q(max)=0.05 mmol/g), but its adsorption affinity (b=112 L/mmol) was much lower compared with that of reed (b=471 L/mmol). To elucidate the mechanism, the elemental components, ion exchange phenomenon and roles of functional groups of these two biosorbents were compared. The higher Pb(II) adsorption by brown seaweed could be due to its richness in total functional groups and calcium contents on its surface. In contrast, the functional complexity, higher zeta potential and pK(a) value (deprotonation state) of reed are believed to lead to its high adsorption affinity.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.

Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1more » vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion trapping. • Human peripheral blood leukocytes capture and concentrate quinacrine. • Polymorphonuclear leukocytes do so with higher apparent affinity. • Polymorphonuclear are also more competent than lymphocytes for pinocytosis.« less

Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg(2+), and Trimethylamine N-Oxide.

PubMed

McPhie, Peter; Brown, Patrick; Chen, Bin; Dayie, Theodore K; Minton, Allen P

2016-09-13

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg(2+) concentrations, Mg(2+) binds preferentially to the open state, favoring its formation, but at high concentrations, Mg(2+) binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg(2+) to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg(2+), high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

PubMed

Miyanokoshi, Miki; Yokosawa, Takumi; Wakasugi, Keisuke

2018-06-01

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Formation of Carbamate Anions by the Gas-phase Reaction of Anilide Ions with CO2

NASA Astrophysics Data System (ADS)

Liu, Chongming; Nishshanka, Upul; Attygalle, Athula B.

2016-05-01

The anilide anion ( m/z 92) generated directly from aniline, or indirectly as a fragmentation product of deprotonated acetanilide, captures CO2 readily to form the carbamate anion ( m/z 136) in the collision cell, when CO2 is used as the collision gas in a tandem-quadrupole mass spectrometer. The gas-phase affinity of the anilide ion to CO2 is significantly higher than that of the phenoxide anion ( m/z 93), which adds to CO2 only very sluggishly. Our results suggest that the efficacy of CO2 capture depends on the natural charge density on the nitrogen atom, and relative nucleophilicity of the anilide anion. Generally, conjugate bases generated from aniline derivatives with proton affinities (PA) less than 350 kcal/mol do not tend to add CO2 to form gaseous carbamate ions. For example, the anion generated from p-methoxyaniline (PA = 367 kcal/mol) reacts significantly faster than that obtained from p-nitroaniline (PA = 343 kcal/mol). Although deprotonated p-aminobenzoic acid adds very poorly because the negative charge is now located primarily on the carboxylate group, it reacts more efficiently with CO2 if the carboxyl group is esterified. Moreover, mixture of CO2 and He as the collision gas was found to afford more efficient adduct formation than CO2 alone, or as mixtures made with nitrogen or argon, because helium acts as an effective "cooling" gas and reduces the internal energy of reactant ions.

Integration of biotic ligand models (BLM) and bioaccumulation kinetics into a mechanistic framework for metal uptake in aquatic organisms.

PubMed

Veltman, Karin; Huijbregts, Mark A J; Hendriks, A Jan

2010-07-01

Both biotic ligand models (BLM) and bioaccumulation models aim to quantify metal exposure based on mechanistic knowledge, but key factors included in the description of metal uptake differ between the two approaches. Here, we present a quantitative comparison of both approaches and show that BLM and bioaccumulation kinetics can be merged into a common mechanistic framework for metal uptake in aquatic organisms. Our results show that metal-specific absorption efficiencies calculated from BLM-parameters for freshwater fish are highly comparable, i.e. within a factor of 2.4 for silver, cadmium, copper, and zinc, to bioaccumulation-absorption efficiencies for predominantly marine fish. Conditional affinity constants are significantly related to the metal-specific covalent index. Additionally, the affinity constants of calcium, cadmium, copper, sodium, and zinc are significantly comparable across aquatic species, including molluscs, daphnids, and fish. This suggests that affinity constants can be estimated from the covalent index, and constants can be extrapolated across species. A new model is proposed that integrates the combined effect of metal chemodynamics, as speciation, competition, and ligand affinity, and species characteristics, as size, on metal uptake by aquatic organisms. An important direction for further research is the quantitative comparison of the proposed model with acute toxicity values for organisms belonging to different size classes.

Receptor interactrions of imidazolines. VI. Significance of carbon bridge separating phenyl and imidazoline rings of tolazoline-like alpha adrenergic imidazolines.

PubMed

Ruffolo, R R; Yaden, E L; Waddell, J E; Dillard, R D

1980-09-01

The pharmacological significance of the carbon bridge separating the imidazoline and phenyl rings of tolazoline-like alpha adrenergic imidazolines has been investigated. Extending the carbon bridge to two carbon atoms, or deleting the carbon bridge, lowers affinity of the imidazolines for the alpha receptor and markedly decreases or abolishes efficacy (i.e., agonist activity), suggesting that a single carbon atome optimallyu separates the phenyl and imidazoline rings. Although one carbon is optimal for alpha adrenergic activity, this particular atom does not appear to be essential since nitrogen may substitute for carbon with no marked or consistent changes observed in affinity or efficacy. Hydroxylation of the carbon bridge decreases affinity for the receptor approximately 10-fold but does not alter efficacy, whereas a similar substitution made in the norepinephrine-series of phenethylamines markedly increases affinity (Patil et al., 1974). With both the imidazolines and phenethylamines, this carbon atom may stereoselectively influence binding to the receptor. These results suggest that the carbon atom bridging the phenyl and imidazoline rings of tolazoline-like imidazolines serves only to provide optimal separation between these rings and does not contribute directly to the binding process. It is proposed that alpha adrenergic imidazolines interact differently with the alpha adrenergic receptor than the norepinephrine-like phenethylamines.

Low density and high affinity of platelet [3H]paroxetine binding in women with bulimia nervosa.

PubMed

Ekman, Agneta; Sundblad-Elverfors, Charlotta; Landén, Mikael; Eriksson, Tomas; Eriksson, Elias

2006-06-15

Impaired serotonin transmission has been suggested to be implicated in the pathophysiology of bulimia nervosa. As an indirect measure of brain serotonergic activity, the binding of tritiated ligands to platelet serotonin transporters has been studied in bulimia nervosa as well as in other putatively serotonin-related psychiatric disorders. In this study, the density and affinity of platelet serotonin transporters were assessed in 20 women meeting the DSM-IV criteria for bulimia nervosa and in 14 controls without previous or ongoing eating disorder using [(3)H]paroxetine as a ligand. In comparison to controls, women with bulimia nervosa had a significantly reduced number of platelet binding sites (B(max) = 721 +/- 313 vs. 1145 +/- 293 fmol/mg protein) and an increase in the affinity for the ligand demonstrated by a lower dissociaton constant (K(d) = 33 +/- 10 vs. 44 +/- 10 pM). A significant correlation between B(max) and K(d) values was found in patients but not in controls. Our results support the notion that bulimia nervosa is associated with a reduction in platelet serotonin transporter density. In addition, our study is the first to report that this reduced transporter density in women with bulimia nervosa is accompanied by an increase in the affinity of the transporter for the ligand.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

The tumor affinity of chlorin e6 and its sonodynamic effects on non-small cell lung cancer.

PubMed

Chen, Bei; Zheng, Ruinian; Liu, Duan; Li, Baofeng; Lin, Jinrong; Zhang, Weimin

2013-03-01

Sonodynamic therapy (SDT) is a promising new approach for cancer therapy. The aim of this study was to investigate the tumor affinity of chlorin e6, a photosensitizer, and its sonodynamic effects on NSCLC. Human lung adenocarcinoma cells SPCA-1 and mice bearing SPCA-1 tumor xenograft were exposed to ultrasound in the presence or absence of chlorin e6. Chlorin e6 distribution was detected by laser scan confocal microscope. Cell apoptosis and necrosis were studied by flow cytometry analysis. Tumor size and weight were measured after different treatments. The concentration of chlorin e6 in tumor tissue was remarkably higher than that in normal muscle near tumor, and the difference was greatest at 18h (the fluorescence intensity was 5.38-fold higher in tumor than in muscle, P<0.05). In vivo, ultrasound (0.4-1.6W/cm(2)) or chlorin e6 (10-40mg/kg) alone had no remarkable anti-tumor effects, but the combination of ultrasound (1.6W/cm(2)) with chlorin e6 (SDT) hampered tumor growth significantly (P<0.05). Intraperitoneal injection of 40mg/kg chlorin e6 exerted no notable side effect on blood, liver and kidney function. Flow cytometry analysis showed that chlorin e6-mediated sonodynamic effect was mainly through the induction of cell necrosis. Chlorin e6 is a promising sonosensitizer and chlorin e6-mediated SDT may provide a new approach for NSCLC therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Changes in avidity and level of immunoglobulin G antibodies to Mycobacterium tuberculosis in sera of patients undergoing treatment for pulmonary tuberculosis.

PubMed

Arias-Bouda, Lenka M Pereira; Kuijper, Sjoukje; Van der Werf, Anouk; Nguyen, Lan N; Jansen, Henk M; Kolk, Arend H J

2003-07-01

Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens.

Changes in Avidity and Level of Immunoglobulin G Antibodies to Mycobacterium tuberculosis in Sera of Patients Undergoing Treatment for Pulmonary Tuberculosis

PubMed Central

Pereira Arias-Bouda, Lenka M.; Kuijper, Sjoukje; Van Der Werf, Anouk; Nguyen, Lan N.; Jansen, Henk M.; Kolk, Arend H. J.

2003-01-01

Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens. PMID:12853408

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

PubMed Central

2013-01-01

Background Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth. PMID:23621895

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

PubMed

Zádor, Ferenc; Király, Kornél; Váradi, András; Balogh, Mihály; Fehér, Ágnes; Kocsis, Dóra; Erdei, Anna I; Lackó, Erzsébet; Zádori, Zoltán S; Hosztafi, Sándor; Noszál, Béla; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

2017-08-15

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the μ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [ 35 S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile 5,6 deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

Antibiotics in surface water and sediments from Hanjiang River, Central China: Occurrence, behavior and risk assessment.

PubMed

Hu, Ying; Yan, Xue; Shen, Yun; Di, Mingxiao; Wang, Jun

2018-08-15

Thirteen antibiotics including sulfonamides (SAs), tetracyclines (TETs) and fluoroquinolones (FQs) were measured in Hanjiang River (HR) during two periods. The total concentrations of 13 antibiotics in surface water and sediments ranged from 3.1 to 109 ng/l and from 10 to 45 ng/g dry weight, respectively. SAs were dominant in water while the concentrations of TETs were the highest in sediments in two seasons. For their spatial distribution, total concentrations of 13 antibiotics in both matrices were significantly higher in the lower section of HR (p < 0.02, F > 5.15) due to wastewater release, agricultural activities and water transfer project. Obvious seasonal variations of sulfadiazine, sulfameter, trimethoprim and oxytetracycline in water were observed (p < 0.05, F > 4.62). Phase partition of antibiotics between water and sediments suggested a greater affinity of TETs and FQs to sediments. In addition, significantly positive relationships were found between SAs (sulfameter, sulfamethoxazole and trimethoprim) and sediment TOC (p < 0.05). Risk assessment indicated that the hazard quotients of antibiotics were higher in the sediment than those in the water. Moreover, antibiotic mixtures posed higher ecological risks to aquatic organisms. Copyright © 2018 Elsevier Inc. All rights reserved.

Efficacy of novel acridine derivatives in the inhibition of hPrP90-231 prion protein fragment toxicity.

PubMed

Villa, Valentina; Tonelli, Michele; Thellung, Stefano; Corsaro, Alessandro; Tasso, Bruno; Novelli, Federica; Canu, Caterina; Pino, Albiana; Chiovitti, Katia; Paludi, Domenico; Russo, Claudio; Sparatore, Anna; Aceto, Antonio; Boido, Vito; Sparatore, Fabio; Florio, Tullio

2011-05-01

Quinacrine is one of the few molecules tested to treat patients affected by prion diseases, although the clinical outcome is largely unsatisfactory. To identify novel derivatives with higher neuroprotective activity, we evaluated the effects of a small library of acridine derivatives. The 6-chloro-2-methoxyacridine derivatives bearing on position 9 a quinolizidin-1-ylamino (Q1, Q2) or a quinolizidin-1-ylalkylamino residue (Q3, Q4, Q6, Q7), the thio-bioisoster of Q3 (Q5), the 9-(N-lupinylthiopropyl)amino derivative (Q8) and simple acridines (Q9 and Q10) were considered. We compared the effects of quinacrine and these novel analogues in the inhibition of the cytotoxic activity and protease K (PK) resistance of the human prion protein fragment 90-231 (hPrP90-231). We demonstrate that quinacrine caused a significant reduction of hPrP90-231 toxicity due to its binding to the fragment and the prevention of its conversion in a toxic isoform. All acridine derivatives analyzed showed high affinity binding for hPrP90-231, but only Q3 and Q10, caused a significant reduction of hPrP90-231 cytotoxicity, with higher efficacy than quinacrine. We attempted to correlate the cytoprotective effects of the new compounds with some biochemical parameters (binding affinity to hPrP90-231, intrinsic fluorescence quenching, hydrophobic amino acid exposure), but a direct relationship occurred only with the reduction of PK resistance, likely due to the prevention of the acquisition of the β-sheet-rich toxic conformation. These data represent interesting leads for further modifications of the basic side chain and the substituent pattern of the acridine nucleus to develop novel compounds with improved antiprion activity to be tested in in vivo experimental setting.

In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe

PubMed Central

Zhao, Menglong; Dong, Lili; Liu, Zhuang; Yang, Shuohui

2018-01-01

Background Glypican-3 (GPC3) is highly expressed in most of the hepatocellular carcinomas (HCCs), even in small HCCs. It may be used as a potential biomarker for early detection of HCC. The aptamer is a promising targeting agent with unique advantages over antibody. This study was to introduce a novel GPC3 specific aptamer (AP613-1), to verify its specific binding property in vitro, and to evaluate its targeting efficiency in vivo by performing near-infrared (NIR) fluorescence imaging on an HCC xenograft model. Methods AP613-1 was generated from the systematic evolution of ligands by exponential enrichment. Flow cytometry and aptamer-based immunofluorescence imaging were performed to verify the binding affinity of AP613-1 to GPC3 in vitro. NIR Fluorescence images of nude mice with unilateral (n=12) and bilateral (n=4) subcutaneous xenograft tumors were obtained. Correlation between the tumor fluorescence intensities in vivo and ex vivo was analyzed. Results AP613-1 could specifically bind to GPC3 in vitro. In vivo and ex vivo tumors, fluorescence intensities were in excellent correlation (P<0.001, r=0.968). The fluorescence intensity is significantly higher in tumors given Alexa Fluor 750 (AF750) labeled AP613-1 than in those given AF750 labeled initial ssDNA library both in vivo (P<0.001) and ex vivo (P=0.022). In the mice with bilateral subcutaneous tumors injected with AF750 labeled AP613-1, Huh-7 tumors showed significantly higher fluorescence intensities than A549 tumors both in vivo (P=0.016) and ex vivo (P=0.004). Conclusions AP613-1 displays a specific binding affinity to GPC3 positive HCC. Fluorescently labeled AP613-1 could be used as an imaging probe to subcutaneous HCC in xenograft models. PMID:29675356

Cyclic versus Noncyclic Chelating Scaffold for 89Zr-Labeled ZEGFR:2377 Affibody Bioconjugates Targeting Epidermal Growth Factor Receptor Overexpression

PubMed Central

2017-01-01

Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates. FSC-ZEGFR:2377 and DFO-ZEGFR:2377 were evaluated regarding radiolabeling, in vitro stability, specificity, cell uptake, receptor affinity, biodistribution, and microPET-CT imaging. Both conjugates were efficiently labeled with zirconium-89 at room temperature but radiochemical yields increased substantially at elevated temperature, 85 °C. Both 89Zr-FSC-ZEGFR:2377 and 89Zr-DFO-ZEGFR:2377 revealed remarkable specificity, affinity and slow cell-line dependent internalization. Radiolabeling at 85 °C showed comparable results in A431 tumor xenografted mice with minor differences regarding blood clearance, tumor and liver uptake. In comparison 89Zr-DFO-ZEGFR:2377, radiolabeled at room temperature, showed a significant difference regarding tumor-to-organ ratios. MicroPET-CT imaging studies of 89Zr-FSC-ZEGFR:2377 as well as 89Zr-DFO-ZEGFR:2377 confirmed these findings. In summary we were able to show that FSC is a suitable alternative to DFO for radiolabeling of biomolecules with zirconium-89. Furthermore, our findings indicate that 89Zr-radiolabeling of DFO conjugates at higher temperature reduces off-chelate binding leading to significantly improved tumor-to-organ ratios and therefore enhancing image contrast. PMID:29160082

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

Binding of [3H] SR 49059, a potent nonpeptide vasopressin V1a antagonist, to rat and human liver membranes.

PubMed

Serradeil-Le Gal, C; Raufaste, D; Marty, E; Garcia, C; Maffrand, J P; Le Fur, G

1994-02-28

The new potent and selective nonpeptide vasopressin V1a antagonist, SR 49059, was tritiated and used for the characterization of rat and human liver AVP V1a receptors. Binding of [3H] SR 49059 was time-dependent, reversible and saturable. A single class of high affinity binding sites was identified with Kd values of 0.63 +/- 0.13 and 2.95 +/- 0.64 nM, in rat and human liver membranes, respectively. The maximal binding capacity (Bmax) was about 7 times higher in rat than in human liver preparations. The relative potencies of several AVP/oxytocin agonists or antagonists to inhibit [3H] SR 49059 binding confirmed that this ligand labeled a homogeneous population of sites with the expected AVP V1a profile. Furthermore, [3H] SR 49059 or unlabeled SR 49059 displayed only slight species differences between rat and human V1a receptors, whereas OPC-21268, another nonpeptide V1a antagonist, exhibited a high species-related potency with more than 500 fold higher affinity for rat than for human liver V1a receptors. Thus, [3H] SR 49059 is the first nonpeptide AVP V1a ligand reported having highly specific activity, stability, specificity and affinity. This makes it a suitable probe for labeling AVP V1a receptors in rat and also in human tissues.

Investigating the Affinities and Persistence of VX Nerve Agent in Environmental Matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Love, A H; Vance, A L; Reynolds, J G

2004-03-09

Laboratory experiments were conducted to determine environmental variables that affect the affinities and persistence of the nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) at dilute concentrations in environmental matrices. Quantitative analyses of VX and its degradation products were performed using LC-MS. Batch hydrolysis experiments demonstrated an increasing hydrolysis rate as pH increased, as shown in previous studies, but also indicated that dissolved aqueous constituents can cause significant differences in the absolute hydrolysis rate. Adsorption isotherms from batch aqueous experiments revealed that VX has a high affinity for hydrophobic organics, a moderate affinity for montmorillonite clay, and a very low affinity formore » an iron-oxyhydroxide soil mineral, goethite. The adsorption on goethite was increased with the presence of dissolved organic matter in solution. VX degraded rapidly when dried onto goethite, when an inner-sphere complex was forced. No enhanced degradation occurred with goethite in small amounts water. These results suggest that aqueous conditions have important controls on VX adsorption and degradation in the environment and a more mechanistic understanding of these controls is needed in order to enable accurate predictions of its long-term fate and persistence.« less

Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

NASA Astrophysics Data System (ADS)

Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

2008-05-01

Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

Treating Diabetes Mellitus: Pharmacophore Based Designing of Potential Drugs from Gymnema sylvestre against Insulin Receptor Protein

PubMed Central

Hossain, Mohammad Uzzal; Khan, Md. Arif; Rakib-Uz-Zaman, S. M.; Ali, Mohammad Tuhin; Islam, Md. Saidul; Keya, Chaman Ara; Salimullah, Md.

2016-01-01

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders which can affect the quality of life severely. Injectable insulin is currently being used to treat DM which is mainly associated with patient inconvenience. Small molecules that can act as insulin receptor (IR) agonist would be better alternatives to insulin injection. Herein, ten bioactive small compounds derived from Gymnema sylvestre (G. sylvestre) were chosen to determine their IR binding affinity and ADMET properties using a combined approach of molecular docking study and computational pharmacokinetic elucidation. Designing structural analogues were also performed for the compounds associated with toxicity and less IR affinity. Among the ten parent compounds, six were found to have significant pharmacokinetic properties with considerable binding affinity towards IR while four compounds were associated with toxicity and less IR affinity. Among the forty structural analogues, four compounds demonstrated considerably increased binding affinity towards IR and less toxicity compared with parent compounds. Finally, molecular interaction analysis revealed that six parent compounds and four analogues interact with the active site amino acids of IR. So this study would be a way to identify new therapeutics and alternatives to insulin for diabetic patients. PMID:27034931

Treating Diabetes Mellitus: Pharmacophore Based Designing of Potential Drugs from Gymnema sylvestre against Insulin Receptor Protein.

PubMed

Hossain, Mohammad Uzzal; Khan, Md Arif; Rakib-Uz-Zaman, S M; Ali, Mohammad Tuhin; Islam, Md Saidul; Keya, Chaman Ara; Salimullah, Md

2016-01-01

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders which can affect the quality of life severely. Injectable insulin is currently being used to treat DM which is mainly associated with patient inconvenience. Small molecules that can act as insulin receptor (IR) agonist would be better alternatives to insulin injection. Herein, ten bioactive small compounds derived from Gymnema sylvestre (G. sylvestre) were chosen to determine their IR binding affinity and ADMET properties using a combined approach of molecular docking study and computational pharmacokinetic elucidation. Designing structural analogues were also performed for the compounds associated with toxicity and less IR affinity. Among the ten parent compounds, six were found to have significant pharmacokinetic properties with considerable binding affinity towards IR while four compounds were associated with toxicity and less IR affinity. Among the forty structural analogues, four compounds demonstrated considerably increased binding affinity towards IR and less toxicity compared with parent compounds. Finally, molecular interaction analysis revealed that six parent compounds and four analogues interact with the active site amino acids of IR. So this study would be a way to identify new therapeutics and alternatives to insulin for diabetic patients.

Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giannopoulos, G.; Jackson, K.; Kredentser, J.

The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

Vakonomic Constraints in Higher-Order Classical Field Theory

NASA Astrophysics Data System (ADS)

Campos, Cédric M.

2010-07-01

We propose a differential-geometric setting for the dynamics of a higher-order field theory, based on the Skinner and Rusk formalism for mechanics. This approach incorporates aspects of both, the Lagrangian and the Hamiltonian description, since the field equations are formulated using the Lagrangian on a higher-order jet bundle and the canonical multisymplectic form on its affine dual. The result is that we obtain a unique and global intrinsic description of the dynamics. The case of vakonomic constraints is also studied within this formalism.

Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation.

PubMed

Zhang, Peng; Xu, Xiao-Yan; Chen, You-Peng; Xiao, Meng-Qian; Feng, Bo; Tian, Kai-Xun; Chen, Yue-Hui; Dai, You-Zhi

2018-02-01

In this work, the protein coronas of activated sludge proteins on TiO 2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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-.

Improvement of Aptamer Affinity by Dimerization

PubMed Central

Hasegawa, Hijiri; Taira, Ken-ichi; Sode, Koji; Ikebukuro, Kazunori

2008-01-01

To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets. PMID:27879754

Effects of TNF-alpha on Endothelial Cell Collective Migration

NASA Astrophysics Data System (ADS)

Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

2013-03-01

Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

A mechanism for the activation of the Na/H exchanger NHE-1 by cytoplasmic acidification and mitogens

PubMed Central

Lacroix, Jérôme; Poët, Mallorie; Maehrel, Céline; Counillon, Laurent

2004-01-01

Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na+/H+ exchanger NHE-1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton-sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE-1 activation is best described by a Monod–Wyman–Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low-affinity form of NHE-1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton-sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high-affinity form. PMID:14710192

Effect of ZnO#ZnS QDs heterojunctures on the stilbenes-plasma proteins interactions.

PubMed

Xiao, Jianbo; Wang, Feijiu; Liu, Jie; Wang, Litong; Kai, Guoyin; Yu, Xibin

2011-08-01

Zero-dimensional nanostructures such as ZnO#ZnS QDs heterojunctures (QDHJs) are green nanoparticles and have gained a tremendous amount of attention. However, very little information is available on the effects of these heterojunctures on the transportation of drugs in blood. Herein, stilbenes were studied for their affinities for common bovine plasma proteins (CBPP) in the presence and absence of QDHJs with different diameters. The affinities of QDHJs for CBPP improved with increasing QDHJs size. QDHJs improved the affinities of resveratrol and polydatin for CBPP by 14.74% to 22.36% and 12.56% to 21.34% depending on the size of QDHJs. The number of binding sites (n) between resveratrol and polydatin for CBPP in the presence of QDHJs were 1.04 ± 0.03 and 1.06 ± 0.04, which were obviously higher than those in the absence of QDHJs (n = 0.89 and 0.92). QDHJs in blood will decrease the free concentration of stilbenes and weaken their pharmacological effects.

Novel Carbonyl Analogues of Tamoxifen: Design, Synthesis, and Biological Evaluation

NASA Astrophysics Data System (ADS)

Kasiotis, Konstantinos M.; Lambrinidis, George; Fokialakis, Nikolas; Tzanetou, Evangelia N.; Mikros, Emmanuel; Haroutounian, Serkos A.

2017-09-01

Aim of this work was to provide tamoxifen analogues with enhanced estrogen receptor binding affinity. Hence, several derivatives were prepared using an efficient triarylethylenes synthetic protocol. The novel compounds bioactivity was evaluated through the determination of their receptor binding affinity and their agonist/antagonist activity against breast cancer tissue using a MCF-7 cell-based assay. Phenyl esters 6a,b and 8a,b exhibited binding affinity to both ERα and ERβ higher than 4-hydroxytamoxifen while compounds 13 and 14 have shown cellular antiestrogenic activity similar to 4-hydroxytamoxifen and the known estrogen receptor inhibitor ICI182,780. Theoretical calculations and molecular modelling were applied to investigate, support and explain the biological profile of the new compounds. The relevant data indicated an agreement between calculations and demonstrated biological activity allowing to extract useful structure-activity relationships. Results herein underline that modifications of tamoxifen structure still provide molecules with substantial activity, as portrayed in the inhibition of MCF-7 cells proliferation.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

Synthesis and receptor binding studies of novel 4,4-disubstituted arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as a new class of σ1 ligands.

PubMed

Sadeghzadeh, Masoud; Sheibani, Shahab; Ghandi, Mehdi; Daha, Fariba Johari; Amanlou, Massoud; Arjmand, Mohammad; Hasani Bozcheloie, Abolfazl

2013-06-01

This study presents the synthesis and biological evaluation of a new series of arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as sigma receptor ligands. It was found that a number of halogen substituted sulfonamides display relatively high and low affinities to σ1 and σ2 receptors, respectively. The σ1 affinities and subtype selectivities of four piperidine derivatives were also found to be generally comparable to those of piperazine analogues. Compared to σ1-Rs compounds with n = 0 and 2, those with n = 1 proved to have optimal length of carbon chain by exhibiting higher affinities. Within this series, the 4-benzyl-1-(3-iodobenzylsulfonyl)piperidine sigma ligand was identified with 96-fold σ1/σ2 selectivity ratio (Kiσ1 = 0.96 ± 0.05 nM and Kiσ2 = 91.8 ± 8.1 nM). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

PubMed

Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

2011-05-01

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

Ligand Binding Analysis and Screening by Chemical Denaturation Shift

PubMed Central

Sch n, Arne; Brown, Richard K.; Hutchins, Burleigh M.; Freire, Ernesto

2013-01-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities and the ligand rank order obtained at denaturation temperatures (60°C or higher) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations in which binding changes the cooperativity of the unfolding transition. In this paper we develop the basic analytical equations and provide several experimental examples. PMID:23994566

Selection of affinity peptides for interference-free detection of cholera toxin.

PubMed

Lim, Jong Min; Heo, Nam Su; Oh, Seo Yeong; Ryu, Myung Yi; Seo, Jeong Hyun; Park, Tae Jung; Huh, Yun Suk; Park, Jong Pil

2018-01-15

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB 5 . Copyright © 2017 Elsevier B.V. All rights reserved.

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

PubMed

Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

2012-06-01

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

Availability of phosphate for phytoplankton and bacteria and of glucose for bacteria at different pCO2 levels in a mesocosm study

NASA Astrophysics Data System (ADS)

Tanaka, T.; Thingstad, T. F.; Løvdal, T.; Grossart, H.-P.; Larsen, A.; Allgaier, M.; Meyerhöfer, M.; Schulz, K. G.; Wohlers, J.; Zöllner, E.; Riebesell, U.

2008-05-01

Availability of phosphate for phytoplankton and bacteria and of glucose for bacteria at different pCO2 levels were studied in a mesocosm experiment (PeECE III). Using nutrient-depleted SW Norwegian fjord waters, three different levels of pCO2 (350 μatm: 1×CO2; 700 μatm: 2×CO2; 1050 μatm: 3×CO2) were set up, and nitrate and phosphate were added at the start of the experiment in order to induce a phytoplankton bloom. Despite similar responses of total particulate P concentration and phosphate turnover time at the three different pCO2 levels, the size distribution of particulate P and 33PO4 uptake suggested that phosphate transferred to the >10 μm fraction was greater in the 3×CO2 mesocosm during the first 6-10 days when phosphate concentration was high. During the period of phosphate depletion (after Day 12), specific phosphate affinity and specific alkaline phosphatase activity (APA) suggested a P-deficiency (i.e. suboptimal phosphate supply) rather than a P-limitation for the phytoplankton and bacterial community at the three different pCO2 levels. Specific phosphate affinity and specific APA tended to be higher in the 3×CO2 than in the 2×CO2 and 1×CO2 mesocosms during the phosphate depletion period, although no statistical differences were found. Glucose turnover time was correlated significantly and negatively with bacterial abundance and production but not with the bulk DOC concentration. This suggests that even though constituting a small fraction of the bulk DOC, glucose was an important component of labile DOC for bacteria. Specific glucose affinity of bacteria behaved similarly at the three different pCO2 levels with measured specific glucose affinities being consistently much lower than the theoretical maximum predicted from the diffusion-limited model. This suggests that bacterial growth was not severely limited by the glucose availability. Hence, it seems that the lower availability of inorganic nutrients after the phytoplankton bloom reduced the bacterial capacity to consume labile DOC in the upper mixed layer of the stratified mesocosms.

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

PubMed

Tahara, A; Tsukada, J; Ishii, N; Tomura, Y; Wada, K; Kusayama, T; Yatsu, T; Uchida, W; Tanaka, A

1999-10-22

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

Electron affinity of liquid water

DOE PAGES

Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; ...

2018-01-16

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