A multi-spectroscopic and molecular docking approach to investigate the interaction of antiviral drug oseltamivir with ct-DNA.

PubMed

Moghadam, Neda Hosseinpour; Salehzadeh, Sadegh; Shahabadi, Nahid; Golbedaghi, Reza

2017-07-03

The possible interaction between the antiviral drug oseltamivir and calf thymus DNA at physiological pH was studied by spectrophotometry, competitive spectrofluorimetry, differential pulse voltammogram (DPV), circular dichroism spectroscopy (CD), viscosity measurements, salt effect, and computational studies. Intercalation of oseltamivir between the base pairs of DNA was shown by a sharp increase in specific viscosity of DNA and a decrease of the peak current and a positive shift in differential pulse voltammogram. Competitive fluorescence experiments were performed using neutral red (NR) as a probe for the intercalation binding mode. The studies showed that oseltamivir is able to release the NR.

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

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

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

PubMed

Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

1997-06-05

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Application of Sargassum biomass to remove heavy metal ions from synthetic multi-metal solutions and urban storm water runoff.

PubMed

Vijayaraghavan, K; Teo, Ting Ting; Balasubramanian, R; Joshi, Umid Man

2009-05-30

The ability of Sargassum sp. to biosorb four metal ions, namely lead, copper, zinc, and manganese from a synthetic multi-solute system and real storm water runoff has been investigated for the first time. Experiments on synthetic multi-solute systems revealed that Sargassum performed well in the biosorption of all four metal ions, with preference towards Pb, followed by Cu, Zn, and Mn. The solution pH strongly affected the metal biosorption, with pH 6 being identified as the optimal condition for achieving maximum biosorption. Experiments at different biosorbent dosages revealed that good biosorption capacity as well as high metal removal efficiency was observed at 3g/L. The biosorption kinetics was found to be fast with equilibrium being attained within 50 min. According to the Langmuir isotherm model, Sargassum exhibited maximum uptakes of 214, 67.5, 24.2 and 20.2mg/g for lead, copper, zinc, and manganese, respectively in single-solute systems. In multi-metal systems, strong competition between four metal ions in terms of occupancy binding sites was observed, and Sargassum showed preference in the order of Pb>Cu>Zn>Mn. The application of Sargassum to remove four heavy metal ions in real storm water runoff revealed that the biomass was capable of removing the heavy metal ions. However, the biosorption performance was slightly lower compared to that of synthetic metal solutions. Several factors were responsible for this difference, and the most important factor is the presence of other contaminants such as anions, organics, and other trace metals in the runoff.

Evaluation of the multiple-ion competition in the adsorption of As(V) onto reclaimed iron-oxide coated sands by fractional factorial design.

PubMed

Hsu, Jia-Chin; Lin, Chien-Jung; Liao, Chih-Hsiang; Chen, Shyi-Tien

2008-07-01

This study describes the competitive effects of selected ions and natural organic matter on As(V) removal using reclaimed iron-oxide coated sands (RIOCS) in the single- and multi-ion systems. A 2(7-3) factional factorial experimental design (FFD) was employed for screening main competitive factors in this adsorption process. As a result, the inhibitive competition effects of the anions on As(V) removal in the single ion system were in the following sequence: PO(4)(3-)>SiO(3)(2-)>HCO(3)(-)>humic acid (HA)>SO(4)(2-)>Cl(-), whereas the cation Ca(2+) was observed to enhance the As(V) removal. In addition, the optimum initial pH for As(V) removal in single-ion system was 5. Based on the estimates of major effects and interactions from the FFD, PO(4)(3-), SiO(3)(2-), Ca(2+) and HA were important factors on As(V) removal in the multi-ion system. The promoters for the As(V) removal were found to be Ca(2+) and, to a lesser extent, SO(4)(2-). The competitive effects of these ions on As(V) removal were in the order of PO(4)(3-), SiO(3)(2-), HA, HCO(3)(-), and Cl(-). In the single ion system, the efficiencies of As(V) removal range from 75% to 96%, much higher than those in the multi-ion system (44%) at the initial pH 5. Clearly, there were some complex anion interactions in the multi-ion system. To promote the removal of As(V) by RIOCS, it is proposed to lower the pH in the single-ion system, while in the multi-ion system, the increase of the Ca(2+) concentration, or decreases of PO(4)(3-), SiO(3)(2-) and HA concentrations is suggested.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence*

PubMed Central

Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

2015-01-01

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence. PMID:26491019

Discovering transcription factor binding sites in highly repetitive regions of genomes with multi-read analysis of ChIP-Seq data.

PubMed

Chung, Dongjun; Kuan, Pei Fen; Li, Bo; Sanalkumar, Rajendran; Liang, Kun; Bresnick, Emery H; Dewey, Colin; Keleş, Sündüz

2011-07-01

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.

A Unique Human Chorionic Gonadotropin Antagonist Suppresses Ovarian Hyperstimulation Syndrome in Rats

PubMed Central

Vardhana, Pratibhasri A.; Julius, Martin A.; Pollak, Susan V.; Lustbader, Evan G.; Trousdale, Rhonda K.; Lustbader, Joyce W.

2009-01-01

Ovarian hyperstimulation syndrome (OHSS) is a complication of in vitro fertilization associated with physiological changes after hCG administration to induce final oocyte maturation. It presents as widespread increases in vascular permeability and, in rare cases, results in cycle cancellation, multi-organ dysfunction, and pregnancy termination. These physiological changes are due primarily to activation of the vascular endothelial growth factor (VEGF) system in response to exogenous human chorionic gonadotropin (hCG). An hCG antagonist (hCG-Ant) could attenuate these effects by competitively binding to the LH/CG receptor, thereby blocking LH activity in vivo. We expressed a form of hCG that lacks three of its four N-linked glycosylation sites and tested its efficacy as an antagonist. The hCG-Ant binds the LH receptor with an affinity similar to native hCG and inhibits cAMP response in vitro. In a rat model for ovarian stimulation, hCG-Ant dramatically reduces ovulation and steroid hormone production. In a well-established rat OHSS model, vascular permeability and vascular endothelial growth factor (VEGF) expression are dramatically reduced after hCG-Ant treatment. Finally, hCG-Ant does not appear to alter blastocyst development when given after hCG in mice. These studies demonstrate that removing specific glycosylation sites on native hCG can produce an hCG-Ant that is capable of binding without activating the LH receptor and blocking the actions of hCG. Thus hCG-Ant will be investigated as a potential therapy for OHSS. PMID:19443574

Incorporating evolution of transcription factor binding sites into annotated alignments.

PubMed

Bais, Abha S; Grossmann, Stefen; Vingron, Martin

2007-08-01

Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield "conserved TFBSs". Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits)are generated. Moreover,the pair- profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions,as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs,we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification methods do not explicitly consider this.Additionally, prediction of conserved binding sites is carried out in a multi-step approach that segregates alignment from TFBS annotation. In this paper, we demonstrate how the simultaneous alignment and annotation approach of SimAnn can be further extended to incorporate TFBS evolutionary relationships. We study how alignments and binding site predictions interplay at varying evolutionary distances and for various profile qualities.

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds

PubMed Central

Rahman, Mona N.; Vukomanovic, Dragic; Vlahakis, Jason Z.; Szarek, Walter A.; Nakatsu, Kanji; Jia, Zongchao

2013-01-01

The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties and may play a role in several disease states, making it an enticing therapeutic target. Traditionally, the metalloporphyrins have been used as competitive HO inhibitors owing to their structural similarity with the substrate, heme. However, given heme's important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non-selectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated, which provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. PMID:23097500

Quantifying the Effect of DNA Packaging on Gene Expression Level

NASA Astrophysics Data System (ADS)

Kim, Harold

2010-10-01

Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.

Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

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

Ayyagari, R.R.; Khan-Dawood, F.S.

Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2more » hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol /sup 125/I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function.« less

Competition effects in cation binding to humic acid: Conditional affinity spectra for fixed total metal concentration conditions

NASA Astrophysics Data System (ADS)

David, Calin; Mongin, Sandrine; Rey-Castro, Carlos; Galceran, Josep; Companys, Encarnació; Garcés, José Luis; Salvador, José; Puy, Jaume; Cecilia, Joan; Lodeiro, Pablo; Mas, Francesc

2010-09-01

Information on the Pb and Cd binding to a purified Aldrich humic acid (HA) is obtained from the influence of different fixed total metal concentrations on the acid-base titrations of this ligand. NICA (Non-Ideal Competitive Adsorption) isotherm has been used for a global quantitative description of the binding, which has then been interpreted by plotting the Conditional Affinity Spectra of the H + binding at fixed total metal concentrations (CAScTM). This new physicochemical tool, here introduced, allows the interpretation of binding results in terms of distributions of proton binding energies. A large increase in the acidity of the phenolic sites as the total metal concentration increases, especially in presence of Pb, is revealed from the shift of the CAScTM towards lower affinities. The variance of the CAScTM distribution, which can be used as a direct measure of the heterogeneity, also shows a significant dependence on the total metal concentration. A discussion of the factors that influence the heterogeneity of the HA under the conditions of each experiment is provided, so that the smoothed pattern exhibited by the titration curves can be justified.

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

Kinetic mechanism of the dimeric ATP sulfurylase from plants

PubMed Central

Ravilious, Geoffrey E.; Herrmann, Jonathan; Goo Lee, Soon; Westfall, Corey S.; Jez, Joseph M.

2013-01-01

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively. PMID:23789618

Crystal structure of the Escherichia coli regulator of sigma70, Rsd, in complex with sigma70 domain 4.

PubMed

Patikoglou, Georgia A; Westblade, Lars F; Campbell, Elizabeth A; Lamour, Valérie; Lane, William J; Darst, Seth A

2007-09-21

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

Crystal structure of the Escherichia coli regulator of σ70, Rsd, in complex with σ70 domain 4

PubMed Central

Patikoglou, Georgia A.; Westblade, Lars F.; Campbell, Elizabeth A.; Lamour, Valérie; Lane, William J.; Darst, Seth A.

2007-01-01

Summary The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) σ70 factor. Rsd plays a role in alternative σ factor-dependent transcription by biasing the competition between σ70 and alternative σ factors for the available core RNAP. Here, we determined the 2.6 Å-resolution X-ray crystal structure of Rsd bound to σ70 domain 4 (σ704), the primary determinant for Rsd binding within σ70. The structure reveals that Rsd binding interferes with the two primary functions of σ704, core RNAP binding and promoter –35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the σ704-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between σ704 binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation. PMID:17681541

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

Patikoglou,G.; Westblade, L.; Campbell, E.

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) {sigma}{sup 70} factor. Rsd plays a role in alternative {sigma} factor-dependent transcription by biasing the competition between {sigma}{sup 70} and alternative {sigma} factors for the available core RNAP. Here, we determined the 2.6 {angstrom}-resolution X-ray crystal structure of Rsd bound to {sigma}{sup 70} domain 4 ({sigma}{sup 70}{sub 4}), the primary determinant for Rsd binding within {sigma}{sup 70}. The structure reveals that Rsd binding interferes with the two primary functions of {sigma}{sup 70}{sub 4}, core RNAP binding and promoter -35 element binding. Interestingly, the most highly conservedmore » Rsd residues form a network of interactions through the middle of the Rsd structure that connect the {sigma}{sup 70}{sub 4}-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between {sigma}{sup 70}{sub 4} binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.« less

Multi-spectroscopic and molecular modeling approaches to elucidate the binding interaction between bovine serum albumin and darunavir, a HIV protease inhibitor

NASA Astrophysics Data System (ADS)

Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

2018-01-01

Darunavir (DRV), a second-generation HIV protease inhibitor, is widely used across the world as an important component of HIV therapy. The interaction of DRV with bovine serum albumin (BSA), a major carrier protein, has been studied under simulated physiological conditions (pH 7.4) by multi-spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the intrinsic fluorescence of BSA was quenched by DRV in terms of a static quenching procedure due to the formation of the DRV-BSA complex. The results indicated the presence of single weak affinity binding site ( 103 M- 1, 310 K) on protein. The thermodynamic parameters, namely enthalpy change (ΔH0), entropy change (ΔS0) and Gibbs free energy change (ΔG0) were calculated, which signified that the binding reaction was spontaneous, the main binding forces were hydrogen bonding and van der Waals forces. Importantly, competitive binding experiments with three site probes, phenylbutazone (in sub-domain IIA, site I), ibuprofen (in sub-domain IIIA, site II) and artemether (in the interface between sub-domain IIA and IIB, site II'), suggested that DRV was preferentially bound to the hydrophobic cavity in site II' of BSA, and this finding was validated by the docking results. Additionally, synchronous fluorescence, three-dimensional fluorescence and Resonance Rayleigh Scattering (RRS) spectroscopy gave qualitative information on the conformational changes of BSA upon adding DRV, while quantitative data were obtained with Fourier transform infrared spectroscopy (FT-IR).

Spectroscopic and molecular modeling approaches to investigate the interaction of bisphenol A, bisphenol F and their diglycidyl ethers with PPARα.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Ruan, Ping; Ren, Dayong; Dai, Weichang; Yu, Hansong; Li, Tiezhu

2017-08-01

A fluorescence polarization (FP) assay for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) was developed. The method was based on the competition between bisphenols (BPs) and fluorescein-labeled dexamethasone derivative (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD). A recombinant soluble protein derivative mPPARα-LBD* was prepared, then in vitro binding of 4 BPs to mPPARα-LBD* was investigated. Fluorescence polarization assay showed that these compounds exhibited different binding potencies with mPPARα-LBD*. Additionally, molecular dynamics simulations were performed to further understand the mechanism of BPs binding affinity for mPPARα-LBD*. Docking results elucidated that the driving forces for the binding of BPs to mPPARα-LBD* were predominantly dependent on hydrophobic and hydrogen-bonding interactions. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.7258). The proposed method has potential for multi-residue detection of BPA, BPF, BADGE, and BFDGE. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

PubMed

Zhou, Kai-Li; Pan, Dong-Qi; Lou, Yan-Yue; Shi, Jie-Hua

2018-04-16

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH 7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, K b , value was found to lie between 2.69 × 10 3 and 9.55 × 10 3  M -1 at experimental temperatures (293, 298, 303, and 308 K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH 0 ) and entropy change (ΔS 0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction. Copyright © 2018 John Wiley & Sons, Ltd.

Avoiding false positives and optimizing identification of true ...

EPA Pesticide Factsheets

The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented from screening 94 chemicals from 54 chemical groups for estrogen receptor (ER) activation in a competitive rainbow trout ER (rtER) binding assay and a trout liver slice vitellogenin mRNA expression assay. Results from true competitive agonists and antagonists, and inactive chemicals with little or no indication of ER binding or gene activation were easily interpreted. However, results for numerous industrial chemicals were more challenging to interpret, including chemicals with: (1) apparent competitive binding curves but no gene activation, (2) apparent binding and gene inhibition with evidence of either cytotoxicity or changes in assay media pH, (3) apparent binding but non-competitive gene inhibition of unknown cause, or (4) no rtER binding and gene inhibition not due to competitive ER interaction but due to toxicity, pH change, or some unknown cause. The use of endpoints such as toxicity, pH, precipitate formation, and determination of inhibitor dissociation constants (Ki) for interpreting the results of antagonism and binding assays for diverse chemicals is presented. Of the 94 chemicals tested for antagonism only two, tamoxifen and ICI-182,780, were found to be true competitive

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Non-competitive inhibition by active site binders.

PubMed

Blat, Yuval

2010-06-01

Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

HMG I(Y) interferes with the DNA binding of NF-AT factors and the induction of the interleukin 4 promoter in T cells

PubMed Central

Klein-Hessling, Stefan; Schneider, Günter; Heinfling, Annette; Chuvpilo, Sergei; Serfling, Edgar

1996-01-01

HMG I(Y) proteins bind to double-stranded A+T oligonucleotides longer than three base pairs. Such motifs form part of numerous NF-AT-binding sites of lymphokine promoters, including the interleukin 4 (IL-4) promoter. NF-AT factors share short homologous peptide sequences in their DNA-binding domain with NF-κB factors and bind to certain NF-κB sites. It has been shown that HMG I(Y) proteins enhance NF-κB binding to the interferon β promoter and virus-mediated interferon β promoter induction. We show that HMG I(Y) proteins exert an opposite effect on the DNA binding of NF-AT factors and the induction of the IL-4 promoter in T lymphocytes. Introduction of mutations into a high-affinity HMG I(Y)-binding site of the IL-4 promoter, which decreased HMG I(Y)-binding to a NF-AT-binding sequence, the Pu-bB (or P) site, distinctly increased the induction of the IL-4 promoter in Jurkat T leukemia cells. High concentrations of HMG I(Y) proteins are able to displace NF-ATp from its binding to the Pu-bB site. High HMG I(Y) concentrations are typical for Jurkat cells and peripheral blood T lymphocytes, whereas El4 T lymphoma cells and certain T helper type 2 cell clones contain relatively low HMG I(Y) concentrations. Our results indicate that HMG I(Y) proteins do not cooperate, but instead compete with NF-AT factors for the binding to DNA even though NF-AT factors share some DNA-binding properties with NF-kB factors. This competition between HMG I(Y) and NF-AT proteins for DNA binding might be due to common contacts with minor groove nucleotides of DNA and may be one mechanism contributing to the selective IL-4 expression in certain T lymphocyte populations, such as T helper type 2 cells. PMID:8986808

The Binding of Four Licorice Flavonoids to Bovine Serum Albumin by Multi-Spectroscopic and Molecular Docking Methods: Structure-Affinity Relationship

NASA Astrophysics Data System (ADS)

Hou, J.; Liang, Q.; Shao, S.

2017-03-01

Flavanones are the main compound of licorice, and the C'-4 position substitution is a significant structural feature for their biological activity. The ability of three selected flavanones (liquiritigenin, liquiritin, and liquiritin apioside) bearing different substituents (hydroxyl groups, glucose, and glucose-apiose sugar moiety) at the C'-4 position and a chalcone ( isoliquiritigenin, an isomer of liquiritigenin) to bind bovine serum albumin (BSA) was studied by multispectroscopic and molecular docking methods under physiological conditions. The binding mechanism of fl avonoids to BSA can be explained by the formation of a flavonoids-BSA complex, and the binding affinity is the strongest for isoliquiritigenin, followed by liquiritin apioside, liquiritin, and liquiritigenin. The thermodynamic analysis and the molecular docking indicated that the interaction between flavonoids and BSA was dominated by the hydrophobic force and hydrogen bonds. The competitive experiments as well as the molecular docking results suggested the most possible binding site of licorice flavonoids on BSA at subdomain IIA. These results revealed that the basic skeleton structure and the substituents at the C'-4 position of flavanones significantly affect the structure-affinity relationships of the licorice flavonoid binding to BSA.

Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase*

PubMed Central

Wu, Xiaobin; Kim, Heejeong; Seravalli, Javier; Barycki, Joseph J.; Hart, P. John; Gohara, David W.; Di Cera, Enrico; Jung, Won Hee; Kosman, Daniel J.; Lee, Jaekwon

2016-01-01

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K+) compartmentalization to the trans-Golgi network via Kha1p, a K+/H+ exchanger. K+ in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K+ is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K+ acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K+ compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K+ in the binding of copper to apoFet3p and identifies a K+/H+ exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast. PMID:26966178

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity*

PubMed Central

Cheng, Jiongjia; Goldstein, Rebecca; Stec, Boguslaw; Gershenson, Anne; Roberts, Mary F.

2012-01-01

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane. PMID:23038258

The IP vs. VDE competition as a key factor determining the stability of the MgBX 5 (X = F, Cl) compounds

NASA Astrophysics Data System (ADS)

Sikorska, Celina; Skurski, Piotr

2010-11-01

The explanation of the MgBX 5 (X = F, Cl) systems existence and stability is provided on the basis of theoretical considerations supported by ab initio calculations. The equilibrium structures and most probable fragmentation processes of 26 (including isomers) MgBX 5 molecules and six OBX 5 compounds (for comparison) were examined and discussed. The competition between the ionization potential (IP) of the MgX subunit and the electron binding energy (VDE) of the BX 4 fragment was found to be the key factor determining the ability to form a stable compound of MgBX 5 type.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors

PubMed Central

Lipzig, Rosalinde van; Montagu, Marc Van; Cornelissen, Marc; Meulewaeter, Frank

2001-01-01

The satellite tobacco necrosis virus RNA is uncapped and requires a 3′ translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30. PMID:11222757

Does multi-functionality affect technical efficiency? A non-parametric analysis of the Scottish dairy industry.

PubMed

Barnes, A P

2006-09-01

Recent policy changes within the Common Agricultural Policy have led to a shift from a solely production-led agriculture towards the promotion of multi-functionality. Conversely, the removal of production-led supports would indicate that an increased concentration on production efficiencies would seem a critical strategy for a country's future competitiveness. This paper explores the relationship between the 'multi-functional' farming attitude desired by policy makers and its effect on technical efficiency within Scottish dairy farming. Technical efficiency scores are calculated by applying the non-parametric data envelopment analysis technique and then measured against causes of inefficiency. Amongst these explanatory factors is a constructed score of multi-functionality. This research finds that, amongst other factors, a multi-functional attitude has a significant positive effect on technical efficiency. Consequently, this seems to validate the promotion of a multi-functional approach to farming currently being championed by policy-makers.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

PubMed

Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E; Elshan, N G R D; Tafreshi, Narges K; Brabez, Nabila; Weber, Craig S; Lynch, Ronald M; Hruby, Victor J; Gillies, Robert J; Morse, David L; Mash, Eugene A

2013-09-01

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. Copyright © 2013 Elsevier Ltd. All rights reserved.

Multi-spectroscopic and molecular modeling approaches to elucidate the binding interaction between bovine serum albumin and darunavir, a HIV protease inhibitor.

PubMed

Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

2018-01-05

Darunavir (DRV), a second-generation HIV protease inhibitor, is widely used across the world as an important component of HIV therapy. The interaction of DRV with bovine serum albumin (BSA), a major carrier protein, has been studied under simulated physiological conditions (pH7.4) by multi-spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the intrinsic fluorescence of BSA was quenched by DRV in terms of a static quenching procedure due to the formation of the DRV-BSA complex. The results indicated the presence of single weak affinity binding site (~10 3 M -1 , 310K) on protein. The thermodynamic parameters, namely enthalpy change (ΔH 0 ), entropy change (ΔS 0 ) and Gibbs free energy change (ΔG 0 ) were calculated, which signified that the binding reaction was spontaneous, the main binding forces were hydrogen bonding and van der Waals forces. Importantly, competitive binding experiments with three site probes, phenylbutazone (in sub-domain IIA, site I), ibuprofen (in sub-domain IIIA, site II) and artemether (in the interface between sub-domain IIA and IIB, site II'), suggested that DRV was preferentially bound to the hydrophobic cavity in site II' of BSA, and this finding was validated by the docking results. Additionally, synchronous fluorescence, three-dimensional fluorescence and Resonance Rayleigh Scattering (RRS) spectroscopy gave qualitative information on the conformational changes of BSA upon adding DRV, while quantitative data were obtained with Fourier transform infrared spectroscopy (FT-IR). Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Non-steroidal anti-inflammatory drug naproxen destabilizes Aβ amyloid fibrils: A molecular dynamics investigation

PubMed Central

Takeda, Takako; Kumar, Rashmi; Raman, E. Prabhu; Klimov, Dmitri K.

2010-01-01

Using implicit solvent model and replica exchange molecular dynamics we examine the propensity of non-steroidal anti-inflammatory drug, naproxen, to interfere with Aβ fibril growth. We also compare the anti-aggregation propensity of naproxen with that of ibuprofen. Naproxen anti-aggregation effect is influenced by two factors. Similar to ibuprofen, naproxen destabilizes binding of incoming Aβ peptides to the fibril due to direct competition between the ligands and the peptides for the same binding location on the fibril surface (the edge). However, in contrast to ibuprofen naproxen binding also alters the conformational ensemble of Aβ monomers by promoting β-structure. The second factor weakens naproxen anti-aggregation effect. These findings appear to explain the experimental observations, according to which naproxen binds to Aβ fibril with higher affinity than ibuprofen, yet produces weaker anti-aggregation action. PMID:20979356

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

PubMed

Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

1993-11-01

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques.

PubMed

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R; Abyar, Fatemeh

2018-06-05

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H 2 O] (1) and [Zn (L)] (2) [L=N-2-hydroxyacetophenon-N'-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1 H and 13 C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities. Copyright © 2018 Elsevier B.V. All rights reserved.

Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

2018-06-01

Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

Competitive tuning: Competition's role in setting the frequency-dependence of Ca2+-dependent proteins

PubMed Central

Patel, Neal M.; Kinzer-Ursem, Tamara L.

2017-01-01

A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca2+) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca2+/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca2+ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca2+/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca2+/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca2+, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca2+ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the in vivo frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca2+/CaM-dependent protein kinase II in knockout models of neurogranin. We conclude that competitive tuning could be an important dynamic process underlying synaptic plasticity. PMID:29107982

A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.

PubMed Central

Clos, J; Buttgereit, D; Grummt, I

1986-01-01

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I. Images PMID:3456157

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication.

PubMed

Peixoto, Paul; Liu, Yang; Depauw, Sabine; Hildebrand, Marie-Paule; Boykin, David W; Bailly, Christian; Wilson, W David; David-Cordonnier, Marie-Hélène

2008-06-01

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5'-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

Insights into the interaction of methotrexate and human serum albumin: A spectroscopic and molecular modeling approach.

PubMed

Cheng, Li-Yang; Fang, Min; Bai, Ai-Min; Ouyang, Yu; Hu, Yan-Jun

2017-08-01

In this study, fluorescence spectroscopy and molecular modeling approaches were employed to investigate the binding of methotrexate to human serum albumin (HSA) under physiological conditions. From the mechanism, it was demonstrated that fluorescence quenching of HSA by methotrexate results from the formation of a methotrexate/HSA complex. Binding parameters calculated using the Stern-Volmer method and the Scatchard method showed that methotrexate binds to HSA with binding affinities in the order 10 4  L·mol -1 . Thermodynamic parameter studies revealed that the binding reaction is spontaneous, and that hydrogen bonds and van der Waals interactions play a major role in the reaction. Site marker competitive displacement experiments and a molecular modeling approach demonstrated that methotrexate binds with appropriate affinity to site I (subdomain IIA) of HSA. Furthermore, we discuss some factors that influence methotrexate binding to HSA. Copyright © 2017 John Wiley & Sons, Ltd.

Automated data processing and radioassays.

PubMed

Samols, E; Barrows, G H

1978-04-01

Radioassays include (1) radioimmunoassays, (2) competitive protein-binding assays based on competition for limited antibody or specific binding protein, (3) immunoradiometric assay, based on competition for excess labeled antibody, and (4) radioreceptor assays. Most mathematical models describing the relationship between labeled ligand binding and unlabeled ligand concentration have been based on the law of mass action or the isotope dilution principle. These models provide useful data reduction programs, but are theoretically unfactory because competitive radioassay usually is not based on classical dilution principles, labeled and unlabeled ligand do not have to be identical, antibodies (or receptors) are frequently heterogenous, equilibrium usually is not reached, and there is probably steric and cooperative influence on binding. An alternative, more flexible mathematical model based on the probability or binding collisions being restricted by the surface area of reactive divalent sites on antibody and on univalent antigen has been derived. Application of these models to automated data reduction allows standard curves to be fitted by a mathematical expression, and unknown values are calculated from binding data. The vitrues and pitfalls are presented of point-to-point data reduction, linear transformations, and curvilinear fitting approaches. A third-order polynomial using the square root of concentration closely approximates the mathematical model based on probability, and in our experience this method provides the most acceptable results with all varieties of radioassays. With this curvilinear system, linear point connection should be used between the zero standard and the beginning of significant dose response, and also towards saturation. The importance is stressed of limiting the range of reported automated assay results to that portion of the standard curve that delivers optimal sensitivity. Published methods for automated data reduction of Scatchard plots for radioreceptor assay are limited by calculation of a single mean K value. The quality of the input data is generally the limiting factor in achieving good precision with automated as it is with manual data reduction. The major advantages of computerized curve fitting include: (1) handling large amounts of data rapidly and without computational error; (2) providing useful quality-control data; (3) indicating within-batch variance of the test results; (4) providing ongoing quality-control charts and between assay variance.

Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11

PubMed Central

Ngondo-Mbongo, Richard Patryk; Myslinski, Evelyne; Aster, Jon C.; Carbon, Philippe

2013-01-01

ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation. PMID:23408857

Clinical relevance of drug binding to plasma proteins

NASA Astrophysics Data System (ADS)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

Structural basis for the sequestration of the anti-σ(70) factor Rsd from σ(70) by the histidine-containing phosphocarrier protein HPr.

PubMed

Park, Young Ha; Um, Si Hyeon; Song, Saemee; Seok, Yeong Jae; Ha, Nam Chul

2015-10-01

Histidine-containing phosphocarrier protein (HPr) is a general component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) involved in the phosphorylation-coupled transport of numerous sugars called PTS sugars. HPr mainly exists in a dephosphorylated form in the presence of PTS sugars in the medium, while its phosphorylation increases in the absence of PTS sugars. A recent study revealed that the dephosphorylated form of HPr binds and antagonizes the function of the antisigma factor Rsd. This anti-sigma factor sequesters the housekeeping sigma factor σ(70) to facilitate switching of the sigma subunit on RNA polymerase from σ(70) to the stress-responsive sigma factor σ(S) in stationary-phase cells. In this study, the structure of the complex of Rsd and HPr was determined at 2.1 Å resolution and revealed that the binding site for HPr on the surface of Rsd partly overlaps with that for σ(70). The localization of the phosphorylation site on HPr at the binding interface for Rsd explains why phosphorylation of HPr abolishes its binding to Rsd. The mutation of crucial residues involved in the HPr-Rsd interaction significantly influenced the competition between HPr and σ(70) for binding to Rsd both in vitro and in vivo. The results provide a structural basis for the linkage of global gene regulation to nutrient availability in the external environment.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

PubMed Central

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Dual DNA binding property of ABA insensitive 3 like factors targeted to promoters responsive to ABA and auxin.

PubMed

Nag, Ronita; Maity, Manas Kanti; Dasgupta, Maitrayee

2005-11-01

The ABA responsive ABI3 and the auxin responsive ARF family of transcription factors bind the CATGCATG (Sph) and TGTCTC core motifs in ABA and auxin response elements (ABRE and AuxRE), respectively. Several evidences indicate ABI3s to act downstream to auxin too. Because DNA binding domain of ABI3s shows significant overlap with ARFs we enquired whether auxin responsiveness through ABI3s could be mediated by their binding to canonical AuxREs. Investigations were undertaken through in vitro gel mobility shift assays (GMSA) using the DNA binding domain B3 of PvAlf (Phaseolus vulgaris ABI3 like factor) and upstream regions of auxin responsive gene GH3 (-267 to -141) and ABA responsive gene Em (-316 to -146) harboring AuxRE and ABRE, respectively. We demonstrate that B3 domain of PvAlf could bind AuxRE only when B3 was associated with its flanking domain B2 (B2B3). Such strict requirement of B2 domain was not observed with ABRE, where B3 could bind with or without being associated with B2. This dual specificity in DNA binding of ABI3s was also demonstrated with nuclear extracts of cultured cells of Arachis hypogea. Supershift analysis of ABRE and AuxRE bound nuclear proteins with antibodies raised against B2B3 domains of PvAlf revealed that ABI3 associated complexes were detectable in association with both cis elements. Competition GMSA confirmed the same complexes to bind ABRE and AuxRE. This dual specificity of ABI3 like factors in DNA binding targeted to natural promoters responsive to ABA and auxin suggests them to have a potential role in conferring crosstalk between these two phytohormones.

Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.

PubMed Central

del Angel, R M; Papavassiliou, A G; Fernández-Tomás, C; Silverstein, S J; Racaniello, V R

1989-01-01

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody. Images PMID:2554308

Staufen-mediated mRNA decay

PubMed Central

Park, Eonyoung; Maquat, Lynne E.

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

The role of competitive binding to human serum albumin on efavirenz-warfarin interaction: a nuclear magnetic resonance study.

PubMed

Wanke, Riccardo; Harjivan, Shrika G; Pereira, Sofia A; Marques, M Matilde; Antunes, Alexandra M M

2013-11-01

The potential for co-prescription of the anti-human immunodeficiency virus (anti-HIV) drug efavirenz (EFV) and the oral anticoagulant warfarin (WAR) is currently high as EFV is a drug of choice for HIV type 1 infection and because cardiovascular disease is increasing among HIV-infected individuals. However, clinical reports of EFV-WAR interaction, leading to WAR overdosing, call for elucidation of the mechanisms involved in this drug-drug interaction. Here we present the first report demonstrating competition of the two drugs for the same binding site of human serum albumin. Using ligand-based nuclear magnetic resonance experiments, this study proves that EFV has an effect on the concentration of free WAR. This previously unidentified EFV-WAR interaction represents a potential risk factor that should be taken into account when considering treatment options. Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

PubMed

Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D; Megat, Salim; Burton, Michael D; Price, Theodore J; Campbell, Zachary T

2018-01-02

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Pheromone induction of agglutination in Saccharomyces cerevisiae a cells

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

Terrance, K.; Lipke, P.N.

1987-10-01

a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, ..cap alpha..-agglutinin. The specific binding of /sup 125/I-..cap alpha..-agglutinin to a cells treated with the sex pheromone ..cap alpha..-factor was 2 to 2.5 times that of binding to a cells not treated with ..cap alpha..-factor. Competition with unlabeled ..cap alpha..-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant. The increase in site number was similar to the increase in cellular agglutinability. Increased expression of a-agglutinin followedmore » the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time. Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle. Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.« less

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

PubMed Central

Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2013-01-01

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

Development of a Multi-Species Biotic Ligand Model Predicting the Toxicity of Trivalent Chromium to Barley Root Elongation in Solution Culture

PubMed Central

Song, Ningning; Zhong, Xu; Li, Bo; Li, Jumei; Wei, Dongpu; Ma, Yibing

2014-01-01

Little knowledge is available about the influence of cation competition and metal speciation on trivalent chromium (Cr(III)) toxicity. In the present study, the effects of pH and selected cations on the toxicity of trivalent chromium (Cr(III)) to barley (Hordeum vulgare) root elongation were investigated to develop an appropriate biotic ligand model (BLM). Results showed that the toxicity of Cr(III) decreased with increasing activity of Ca2+ and Mg2+ but not with K+ and Na+. The effect of pH on Cr(III) toxicity to barley root elongation could be explained by H+ competition with Cr3+ bound to a biotic ligand (BL) as well as by the concomitant toxicity of CrOH2+ in solution culture. Stability constants were obtained for the binding of Cr3+, CrOH2+, Ca2+, Mg2+ and H+ with binding ligand: log KCrBL 7.34, log KCrOHBL 5.35, log KCaBL 2.64, log KMgBL 2.98, and log KHBL 4.74. On the basis of those estimated parameters, a BLM was successfully developed to predict Cr(III) toxicity to barley root elongation as a function of solution characteristics. PMID:25119269

Development of a multi-species biotic ligand model predicting the toxicity of trivalent chromium to barley root elongation in solution culture.

PubMed

Song, Ningning; Zhong, Xu; Li, Bo; Li, Jumei; Wei, Dongpu; Ma, Yibing

2014-01-01

Little knowledge is available about the influence of cation competition and metal speciation on trivalent chromium (Cr(III)) toxicity. In the present study, the effects of pH and selected cations on the toxicity of trivalent chromium (Cr(III)) to barley (Hordeum vulgare) root elongation were investigated to develop an appropriate biotic ligand model (BLM). Results showed that the toxicity of Cr(III) decreased with increasing activity of Ca(2+) and Mg(2+) but not with K(+) and Na(+). The effect of pH on Cr(III) toxicity to barley root elongation could be explained by H(+) competition with Cr(3+) bound to a biotic ligand (BL) as well as by the concomitant toxicity of CrOH(2+) in solution culture. Stability constants were obtained for the binding of Cr(3+), CrOH(2+), Ca(2+), Mg(2+) and H(+) with binding ligand: log KCrBL 7.34, log KCrOHBL 5.35, log KCaBL 2.64, log KMgBL 2.98, and log KHBL 4.74. On the basis of those estimated parameters, a BLM was successfully developed to predict Cr(III) toxicity to barley root elongation as a function of solution characteristics.

TATA Binding Protein Discriminates between Different Lesions on DNA, Resulting in a Transcription Decrease

PubMed Central

Coin, Frédéric; Frit, Philippe; Viollet, Benoit; Salles, Bernard; Egly, Jean-Marc

1998-01-01

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position −29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage. PMID:9632775

Interaction of Diuron and Related Substituted Phenylureas with the Ah Receptor Pathway

PubMed Central

Zhao, Bin; Baston, David S.; Hammock, Bruce; Denison, Michael S.

2011-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals, including the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we have examined the ability of diuron, a widely used herbicide, and several structurally related substituted phenylureas to bind to and activate/inhibit the AhR and AhR signal transduction. Diuron induced CYP1A1 mRNA levels in mouse hepatoma (Hepa1c1c7) cells and AhR-dependent luciferase reporter gene expression in stably transfected mouse, rat, guinea pig, and human cell lines. In addition, ligand binding and gel retardation analysis demonstrated the ability of diuron to competitively bind to and stimulate AhR transformation and DNA binding in vitro and in intact cells. Several structurally related substituted phenylureas competitively bound to the guinea pig hepatic cytosolic AhR, inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR-dependent luciferase reporter gene expression in a species-specific manner and stimulated AhR transformation and DNA binding, consistent with their role as partial AhR agonists. These results demonstrate not only that diuron and related substituted phenylureas are AhR ligands but also that exposure to these chemicals could induce/inhibit AhR-dependent biological effects. PMID:16788953

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages

PubMed Central

Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.

2015-01-01

Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. PMID:25712094

Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2006-07-01

Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

Competitive counterion complexation allows the true host : guest binding constants from a single titration by ionic receptors.

PubMed

Pessêgo, Márcia; Basílio, Nuno; Muñiz, M Carmen; García-Río, Luis

2016-07-06

Counterion competitive complexation is a background process currently ignored by using ionic hosts. Consequently, guest binding constants are strongly affected by the design of the titration experiments in such a way that the results are dependent on the guest concentration and on the presence of added salts, usually buffers. In the present manuscript we show that these experimental difficulties can be overcome by just considering the counterion competitive complexation. Moreover a single titration allows us to obtain not only the true binding constants but also the stoichiometry of the complex showing the formation of 1 : 1 : 1 (host : guest : counterion) complexes. The detection of high stoichiometry complexes is not restricted to a single titration experiment but also to a displacement assay where both competitive and competitive-cooperative complexation models are taken into consideration.

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods.

PubMed

Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

2017-08-01

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10 10  L mol -1  s -1 , indicating forming QNPL-BSA complex through the intermolecular binding interaction. The binding constant for the QNPL-BSA complex is in the order of 10 5  M -1 , indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal's forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.

Pathogenic Leptospira Species Acquire Factor H and Vitronectin via the Surface Protein LcpA

PubMed Central

da Silva, Ludmila Bezerra; Miragaia, Lidia dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima

2014-01-01

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn2+-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. PMID:25534939

Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

PubMed

da Silva, Ludmila Bezerra; Miragaia, Lidia Dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima; Barbosa, Angela Silva

2015-03-01

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Fulvic acid-sulfide ion competition for mercury ion binding in the Florida everglades

USGS Publications Warehouse

Reddy, M.M.; Aiken, G.R.

2001-01-01

Negatively charged functional groups of fulvic acid compete with inorganic sulfide ion for mercury ion binding. This competition is evaluated here by using a discrete site-electrostatic model to calculate mercury solution speciation in the presence of fulvic acid. Model calculated species distributions are used to estimate a mercury-fulvic acid apparent binding constant to quantify fulvic acid and sulfide ion competition for dissolved inorganic mercury (Hg(II)) ion binding. Speciation calculations done with PHREEQC, modified to use the estimated mercury-fulvic acid apparent binding constant, suggest that mercury-fulvic acid and mercury-sulfide complex concentrations are equivalent for very low sulfide ion concentrations (about 10-11 M) in Everglades' surface water. Where measurable total sulfide concentration (about 10-7 M or greater) is present in Everglades' surface water, mercury-sulfide complexes should dominate dissolved inorganic mercury solution speciation. In the absence of sulfide ion (for example, in oxygenated Everglades' surface water), fulvic acid binding should dominate Everglades' dissolved inorganic mercury speciation.

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding

PubMed Central

Gilad, Yoav; Pritchard, Jonathan K.; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede. PMID:26406244

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

PubMed

Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

2015-01-01

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

Identification of Location and Kinetically Defined Mechanism of Cofactors and Reporter Genes in the Cascade of Steroid-regulated Transactivation*

PubMed Central

Blackford, John A.; Guo, Chunhua; Zhu, Rong; Dougherty, Edward J.; Chow, Carson C.; Simons, S. Stoney

2012-01-01

A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes. PMID:23055525

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

PubMed

Tao, Pingyang; Poddar, Saumen; Sun, Zuchen; Hage, David S; Chen, Jianzhong

2018-02-02

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhancing the Teaching of Introductory Economics with a Team-Based, Multi-Section Competition

ERIC Educational Resources Information Center

Beaudin, Laura; Berdiev, Aziz N.; Kaminaga, Allison Shwachman; Mirmirani, Sam; Tebaldi, Edinaldo

2017-01-01

The authors describe a unique approach to enhancing student learning at the introductory economics level that utilizes a multi-section, team-based competition. The competition is structured to supplement learning throughout the entire introductory course. Student teams are presented with current economic issues, trends, or events, and use economic…

Single Image Super-Resolution Based on Multi-Scale Competitive Convolutional Neural Network

PubMed Central

Qu, Xiaobo; He, Yifan

2018-01-01

Deep convolutional neural networks (CNNs) are successful in single-image super-resolution. Traditional CNNs are limited to exploit multi-scale contextual information for image reconstruction due to the fixed convolutional kernel in their building modules. To restore various scales of image details, we enhance the multi-scale inference capability of CNNs by introducing competition among multi-scale convolutional filters, and build up a shallow network under limited computational resources. The proposed network has the following two advantages: (1) the multi-scale convolutional kernel provides the multi-context for image super-resolution, and (2) the maximum competitive strategy adaptively chooses the optimal scale of information for image reconstruction. Our experimental results on image super-resolution show that the performance of the proposed network outperforms the state-of-the-art methods. PMID:29509666

Single Image Super-Resolution Based on Multi-Scale Competitive Convolutional Neural Network.

PubMed

Du, Xiaofeng; Qu, Xiaobo; He, Yifan; Guo, Di

2018-03-06

Deep convolutional neural networks (CNNs) are successful in single-image super-resolution. Traditional CNNs are limited to exploit multi-scale contextual information for image reconstruction due to the fixed convolutional kernel in their building modules. To restore various scales of image details, we enhance the multi-scale inference capability of CNNs by introducing competition among multi-scale convolutional filters, and build up a shallow network under limited computational resources. The proposed network has the following two advantages: (1) the multi-scale convolutional kernel provides the multi-context for image super-resolution, and (2) the maximum competitive strategy adaptively chooses the optimal scale of information for image reconstruction. Our experimental results on image super-resolution show that the performance of the proposed network outperforms the state-of-the-art methods.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

PubMed

Doern, Adam; Cao, Xianjun; Sereno, Arlene; Reyes, Christopher L; Altshuler, Angelina; Huang, Flora; Hession, Cathy; Flavier, Albert; Favis, Michael; Tran, Hon; Ailor, Eric; Levesque, Melissa; Murphy, Tracey; Berquist, Lisa; Tamraz, Susan; Snipas, Tracey; Garber, Ellen; Shestowsky, William S; Rennard, Rachel; Graff, Christilyn P; Wu, Xiufeng; Snyder, William; Cole, Lindsay; Gregson, David; Shields, Michael; Ho, Steffan N; Reff, Mitchell E; Glaser, Scott M; Dong, Jianying; Demarest, Stephen J; Hariharan, Kandasamy

2009-04-10

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

PubMed

Pereira, L A; van der Knaap, J A; van den Boom, V; van den Heuvel, F A; Timmers, H T

2001-11-01

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

The Role of Competitive Inhibition and Top-Down Feedback in Binding during Object Recognition

PubMed Central

Wyatte, Dean; Herd, Seth; Mingus, Brian; O’Reilly, Randall

2012-01-01

How does the brain bind together visual features that are processed concurrently by different neurons into a unified percept suitable for processes such as object recognition? Here, we describe how simple, commonly accepted principles of neural processing can interact over time to solve the brain’s binding problem. We focus on mechanisms of neural inhibition and top-down feedback. Specifically, we describe how inhibition creates competition among neural populations that code different features, effectively suppressing irrelevant information, and thus minimizing illusory conjunctions. Top-down feedback contributes to binding in a similar manner, but by reinforcing relevant features. Together, inhibition and top-down feedback contribute to a competitive environment that ensures only the most appropriate features are bound together. We demonstrate this overall proposal using a biologically realistic neural model of vision that processes features across a hierarchy of interconnected brain areas. Finally, we argue that temporal synchrony plays only a limited role in binding – it does not simultaneously bind multiple objects, but does aid in creating additional contrast between relevant and irrelevant features. Thus, our overall theory constitutes a solution to the binding problem that relies only on simple neural principles without any binding-specific processes. PMID:22719733

Hierarchical competitions subserving multi-attribute choice

PubMed Central

Hunt, Laurence T; Dolan, Raymond J; Behrens, Timothy EJ

2015-01-01

Valuation is a key tenet of decision neuroscience, where it is generally assumed that different attributes of competing options are assimilated into unitary values. Such values are central to current neural models of choice. By contrast, psychological studies emphasize complex interactions between choice and valuation. Principles of neuronal selection also suggest competitive inhibition may occur in early valuation stages, before option selection. Here, we show behavior in multi-attribute choice is best explained by a model involving competition at multiple levels of representation. This hierarchical model also explains neural signals in human brain regions previously linked to valuation, including striatum, parietal and prefrontal cortex, where activity represents competition within-attribute, competition between attributes, and option selection. This multi-layered inhibition framework challenges the assumption that option values are computed before choice. Instead our results indicate a canonical competition mechanism throughout all stages of a processing hierarchy, not simply at a final choice stage. PMID:25306549

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms

PubMed Central

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako

2017-01-01

Abstract Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation. PMID:28180311

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms.

PubMed

Nazim, Mohammad; Masuda, Akio; Rahman, Mohammad Alinoor; Nasrin, Farhana; Takeda, Jun-Ichi; Ohe, Kenji; Ohkawara, Bisei; Ito, Mikako; Ohno, Kinji

2017-02-17

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

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

Staufen-mediated mRNA decay.

PubMed

Park, Eonyoung; Maquat, Lynne E

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing

PubMed Central

Gu, Xin; Yan, Yan; Novick, Scott J.; Kovach, Amanda; Goswami, Devrishi; Ke, Jiyuan; Tan, M. H. Eileen; Wang, Lili; Li, Xiaodan; de Waal, Parker W.; Webb, Martin R.; Griffin, Patrick R.; Xu, H. Eric

2017-01-01

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism. PMID:28615457

High-Affinity Quasi-Specific Sites in the Genome: How the DNA-Binding Proteins Cope with Them

PubMed Central

Chakrabarti, J.; Chandra, Navin; Raha, Paromita; Roy, Siddhartha

2011-01-01

Many prokaryotic transcription factors home in on one or a few target sites in the presence of a huge number of nonspecific sites. Our analysis of λ-repressor in the Escherichia coli genome based on single basepair substitution experiments shows the presence of hundreds of sites having binding energy within 3 Kcal/mole of the OR1 binding energy, and thousands of sites with binding energy above the nonspecific binding energy. The effect of such sites on DNA-based processes has not been fully explored. The presence of such sites dramatically lowers the occupation probability of the specific site far more than if the genome were composed of nonspecific sites only. Our Brownian dynamics studies show that the presence of quasi-specific sites results in very significant kinetic effects as well. In contrast to λ-repressor, the E. coli genome has orders of magnitude lower quasi-specific sites for GalR, an integral transcription factor, thus causing little competition for the specific site. We propose that GalR and perhaps repressors of the same family have evolved binding modes that lead to much smaller numbers of quasi-specific sites to remove the untoward effects of genomic DNA. PMID:21889449

In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia.

PubMed

Augustsson, Cecilia; Persson, Egon

2014-11-13

Successful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component. © 2014 by The American Society of Hematology.

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR†

PubMed Central

Golynskiy, Misha V.; Gunderson, William A.; Hendrich, Michael P.; Cohen, Seth M.

2007-01-01

Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10-7 to 10-4 M; D’Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn···Mn distance (4.4 Å) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (∼160 μM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor. PMID:17176058

Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

PubMed Central

Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

2013-01-01

Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

A Competitive Stapled Peptide Screen Identifies a Selective Small Molecule that Overcomes MCL-1-dependent Leukemia Cell Survival

PubMed Central

Cohen, Nicole A.; Stewart, Michelle L.; Gavathiotis, Evripidis; Tepper, Jared L.; Bruekner, Susanne R.; Koss, Brian; Opferman, Joseph T.; Walensky, Loren D.

2012-01-01

SUMMARY Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an anti-apoptotic surface groove that neutralizes the pro-apoptotic BH3 α-helix of death proteins. Anti-apoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Whereas targeting the BCL-2 anti-apoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lockhold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence. PMID:22999885

Building a maintenance policy through a multi-criterion decision-making model

NASA Astrophysics Data System (ADS)

Faghihinia, Elahe; Mollaverdi, Naser

2012-08-01

A major competitive advantage of production and service systems is establishing a proper maintenance policy. Therefore, maintenance managers should make maintenance decisions that best fit their systems. Multi-criterion decision-making methods can take into account a number of aspects associated with the competitiveness factors of a system. This paper presents a multi-criterion decision-aided maintenance model with three criteria that have more influence on decision making: reliability, maintenance cost, and maintenance downtime. The Bayesian approach has been applied to confront maintenance failure data shortage. Therefore, the model seeks to make the best compromise between these three criteria and establish replacement intervals using Preference Ranking Organization Method for Enrichment Evaluation (PROMETHEE II), integrating the Bayesian approach with regard to the preference of the decision maker to the problem. Finally, using a numerical application, the model has been illustrated, and for a visual realization and an illustrative sensitivity analysis, PROMETHEE GAIA (the visual interactive module) has been used. Use of PROMETHEE II and PROMETHEE GAIA has been made with Decision Lab software. A sensitivity analysis has been made to verify the robustness of certain parameters of the model.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

The transcription of the human fructose-bisphosphate aldolase C gene is activated by nerve-growth-factor-induced B factor in human neuroblastoma cells.

PubMed Central

Buono, P; Conciliis, L D; Izzo, P; Salvatore, F

1997-01-01

A DNA region located at around -200 bp in the 5' flanking region (region D) of the human brain-type fructose-bisphosphate aldolase (aldolase C) gene has been analysed. We show by transient transfection assay and electrophoretic-mobility-shift assay (EMSA) that the binding of transcriptional activators to region D is much more efficient (80% versus 30%) in human neuroblastoma cells (SKNBE) than in the non-neuronal cell line A1251, which contains low levels of aldolase C mRNA. The sequence of region D, CAAGGTCA, is very similar to the AAAGGTCA motif present in the mouse steroid 21-hydroxylase gene; the latter motif binds nerve-growth-factor-induced B factor (NGFI-B), which is a member of the thyroid/steroid/retinoid nuclear receptor gene family. Competition experiments in EMSA and antibody-directed supershift experiments showed that NGFI-B is involved in the binding to region D of the human aldolase C gene. Furthermore, the regulation of the aldolase C gene (which is the second known target of NGFI-B) expression during development parallels that of NGFI-B. PMID:9173889

Effects of an endurance cycling competition on resting serum insulin-like growth factor I (IGF-I) and its binding proteins IGFBP-1 and IGFBP-3

PubMed Central

Chicharro, J; Lopez-Calderon, A; Hoyos, J; Martin-Velasco, A; Villa, G; Villanua, M; Lucia, A

2001-01-01

Objectives—To determine whether consecutive bouts of intense endurance exercise over a three week period alters serum concentrations of insulin-like growth factor I (IGF-I) and/or its binding proteins. Methods—Seventeen professional cyclists (mean (SEM) VO2MAX, 74.7 (2.1) ml/kg/min; age, 27 (1) years) competing in a three week tour race were selected as subjects. Blood samples were collected at each of the following time points: t0 (control, before the start of competition), t1 (end of first week), and t3 (end of third week). Serum levels of both total and free IGF-I and IGF binding proteins 1 and 3 (IGFBP-1 and IGFBP-3) were measured in each of the samples. Cortisol levels were measured in nine subjects. Results—A significant (p<0.01) increase was found in total IGF-I and IGFBP-1 at both t1 and t3 compared with to (IGF-I: 110.9 (17.7), 186.8 (12.0), 196.9 (14.7) ng/ml at t0, t1, and t3 respectively; IGFBP-1: 54.6 (6.6), 80.6 (8.0), and 89.2 (7.9) ng/ml at t0, t1, and t3 respectively). A significant (p<0.01) decrease was noted in free IGF-I at t3 compared with both to and t1 (t0: 0.9 (0.1) ng/ml; t1: 0.9 (0.1) ng/ml; t3: 0.7 (0.1) ng/ml); in contrast, IGFBP-3 levels remained stable throughout the race. Conclusions—It would appear that the increase in circulating levels of both IGF-I and its binding protein IGFBP-1 is a short term (one week) endocrine adaptation to endurance exercise. After three weeks of training, total IGF-I and IGFBP-1 remained stable, whereas free IGF-I fell below starting levels. Key Words: cycling; insulin-like growth factor; exercise; endurance; binding proteins PMID:11579061

Determining ERβ Binding Affinity to Singly Mutant ERE Using Dual Polarization Interferometry

NASA Astrophysics Data System (ADS)

Song, Hong Yan; Su, Xiaodi

In a classic mode of estrogen action, estrogen receptors (ERs) bind to estrogen responsive element (ERE) to activate gene transcription. A perfect ERE contains a 13-base pair sequence of a palindromic repeat separated by a three-base spacer, 5‧-GGTCAnnnTGACC-3‧. In addition to the consensus or wild-type ERE (wtERE), naturally occurring EREs often have one or two base pairs’ alternation. Based on the newly constructed Thermodynamic Modeling of ChIP-seq (TherMos) model, binding energy between ERβ and a series of 34-bp mutant EREs (mutERE) was simulated to predict the binding affinity between ERs and EREs with single base pair deviation at different sites of the 13-bp inverted sequence. Experimentally, dual polarization interferometry (DPI) method was developed to measure ERβ-mutEREs binding affinity. On a biotin-NeutrAvidin (NA)-biotin treated DPI chip, wtERE is immobilized. In a direct binding assay, ERβ-wtERE binding affinity is determined. In a competition assay, ERβ was preincubated with mutant EREs before being added for competitive binding to the immobilized wtERE. This competition strategy provided a successful platform to evaluate the binding affinity variation among large number of ERE with different base mutations. The experimental result correlates well with the mathematically predicted binding energy with a Spearman correlation coefficient of 0.97.

Protein Surface Structural Recognition in Inactive Areas: A New Immobilization Strategy for Acetylcholinesterase.

PubMed

Diao, Jianxiong; Yu, Xiaolu; Ma, Lin; Li, Yuanqing; Sun, Ying

2018-05-16

This work reported a new method of design for the immobilization of acetylcholinesterase (AChE) based on its molecular structure to improve its sensitivity and stability. The immobilization binding site on the surface of AChE was determined using MOLCAD's multi-channel functionality. Then, 11 molecules ((+)-catechin, (-)-epicatechin, (-)-gallocatechin, hesperetin, naringenin, quercetin, taxifolin, (-)-epicatechin gallate, flupirtine, atropine, and hyoscyamine) were selected from the ZINC database (about 50 000 molecules) as candidate affinity ligands for AChE. The fluorescence results showed that the binding constant K b between AChE and the ligands ranged from 0.01344 × 10 4 to 4.689 × 10 4 M -1 and there was one independent class of binding site for the ligands on AChE. The AChE-ligand binding free energy ranged from -12.14 to -26.65 kJ mol -1 . Naringenin, hesperetin, and quercetin were the three most potent immobilized affinity ligands. In addition, it was confirmed that the binding between the immobilized ligands only occurred at a single site, located in an inactive area on the surface of AChE, and did not affect the enzymatic activity as shown through a competition experiment and enzyme assay. This method based on protein surface structural recognition with high sensitivity and stability can be used as a generic approach for design of the enzyme immobilization and biosensor development.

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.

PubMed

Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T

2015-03-11

Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB*

PubMed Central

Kensche, Tobias; Tokunaga, Fuminori; Ikeda, Fumiyo; Goto, Eiji; Iwai, Kazuhiro; Dikic, Ivan

2012-01-01

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli. PMID:22605335

Coaches’ Perceptions of Competence and Acknowledgement of Training Needs Related to Professional Competences

PubMed Central

Santos, Sofia; Mesquita, Isabel; GRAÇA, Amândio; Rosado, António

2010-01-01

The purpose of the present study was to examine coaches’ perceptions of competence and acknowledgement of training needs related to professional competences according to the professional experience and academic education. The participants were 343 coaches from several sports, who answered to a questionnaire that includes a scale focused on perceptions of competence and another scale on acknowledgment of training needs. An exploratory factor analysis with Maximum Likelihood Factoring was used with Oblimin rotation for the identification of emergent factors. Comparison on coaches’ perceptions in function of coaching experience and coaches’ academic background were made applying One-way ANOVA and Tukey’s post hoc multiple comparisons. Factor analysis on coaches’ perceptions of competence and acknowledgement of training needs made apparent three main areas of competences, i.e. competences related to annual and multi-annual planning; competences related to orientation towards practice and competition; and personal and coaching education competences. Coaches’ perceptions were influenced by their experience, as low experienced coaches rated themselves at lower levels of competence and with more training needs; also coaches with high education, in Physical Education or others, perceived themselves as more competent than coaches with no higher education. Finally, the majority of the coaches perceived themselves to be competent but, nevertheless, they indicated to have training needs, which brings an important feedback to coach education. This suggests that coaches are interested in increasing their knowledge and competence in a broad range of areas which should be considered in future coach education programs. Key points Coaches’ perceptions of competence and acknowledgement of training needs resulted in three main areas: competences related to annual and multi-annual planning, competences related to practice and competition orientation and, finally, personal and coaching education competences. The professional tasks that coaches had the most need in performing were related to the training orientation. Coaches with higher education degrees (P.E. or others) perceive themselves as more competent than coaches with no higher education. Low experienced coaches perceived themselves less competent than high experienced coaches. Also, they pointed out more training needs in issues related to practice and competition orientation, and annual and multi-annual planning. PMID:24149387

Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states

PubMed Central

2014-01-01

Background At the beginning of the transcription process, the RNA polymerase (RNAP) core enzyme requires a σ-factor to recognize the genomic location at which the process initiates. Although the crucial role of σ-factors has long been appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function. Results Using multiple genome-scale measurements, we elucidated the network of σ-factor and promoter interactions in Escherichia coli. The reconstructed network includes 4,724 σ-factor-specific promoters corresponding to transcription units (TUs), representing an increase of more than 300% over what has been previously reported. The reconstructed network was used to investigate competition between alternative σ-factors (the σ70 and σ38 regulons), confirming the competition model of σ substitution and negative regulation by alternative σ-factors. Comparison with σ-factor binding in Klebsiella pneumoniae showed that transcriptional regulation of conserved genes in closely related species is unexpectedly divergent. Conclusions The reconstructed network reveals the regulatory complexity of the promoter architecture in prokaryotic genomes, and opens a path to the direct determination of the systems biology of their transcriptional regulatory networks. PMID:24461193

Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2.

PubMed

Lo, Te-Wen; Bennett, Daniel C; Goodman, S Jay; Stern, Michael J

2010-06-01

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

Social structure affects mating competition in a damselfish

NASA Astrophysics Data System (ADS)

Wacker, Sebastian; Ness, Miriam Horstad; Östlund-Nilsson, Sara; Amundsen, Trond

2017-12-01

The strength of mating competition and sexual selection varies over space and time in many animals. Such variation is typically driven by ecological and demographic factors, including adult sex ratio and consequent availability of mates. The spatial scale at which demographic factors affect mating competition and sexual selection may vary but is not often investigated. Here, we analyse variation in size and sex ratio of social groups, and how group structure affects mating competition, in the site-attached damselfish Chrysiptera cyanea. Site-attached reef fishes are known to show extensive intraspecific variation in social structure. Previous work has focused on species for which the size and dynamics of social groups are constrained by habitat, whereas species with group structure unconstrained by habitat have received little attention. Chrysiptera cyanea is such a species, with individuals occurring in spatial clusters that varied widely in size and sex ratio. Typically, only one male defended a nest in multi-male groups. Nest-holding males were frequently visited by mate-searching females, with more visits in groups with more females, suggesting that courtship and mating mostly occur within groups and that male mating success depends on the number of females in the group. Male-male aggression was frequent in multi-male groups but absent in single-male groups. These findings demonstrate that groups are distinct social units. In consequence, the dynamics of mating and reproduction are mainly a result of group structure, largely unaffected short term by overall population demography which would be important in open social systems. Future studies of the C. cyanea model system should analyse longer-term dynamics, including how groups are formed, how they vary in relation to density and time of season and how social structure affects sexual selection.

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

PubMed

Hong, Kwang-Won; Kim, Chang-Goo; Lee, Seung-Hyun; Chang, Ki-Hwan; Shin, Yong Won; Ryoo, Kyung-Hwan; Kim, Se-Ho; Kim, Yong-Sung

2010-01-01

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5.

PubMed

Cai, Chang; Huo, Qiang; Wang, Xiaolong; Chen, Bing; Yang, Qifeng

2017-04-01

Long noncoding RNAs (lncRNAs) have been proved to play important roles in cellular processes of cancer, including the development, proliferation, and migration of cancer cells. In the present study, we demonstrated small nucleolar RNA host gene 16 (SNHG16) as an oncogene on cell migration in breast cancer. Expression levels of SNHG16 were found to be frequently higher in breast cancer tissues than in the paired noncancerous tissues. Gain- and loss-of-function studies proved that SNHG16 significantly promoted breast cancer cell migration. We predicted SNHG16 as a competitive endogenous RNA (ceRNA) of E2F transcription factor 5 protein (E2F5) via competition for the shared miR-98 through bioinformatics analysis, and proved this regulation using relative quantitative real-time PCR (qRT-PCR), western blot, RNA immunoprecipitation (RIP) assay and luciferase reporter assay. In addition, we identified a positive correlation between SNHG16 and E2F5 in breast cancer tissues. Furthermore, we demonstrated that forced expression of miR-98 could partially abrogate SNHG16-mediated increase of breast cancer cells migration, suggesting that SNHG16 promoted cell migration in a miR-98 dependent manner. Taken together, our findings indicated that SNHG16 induces breast cancer cell migration by competitively binding miR-98 with E2F5, and SNHG16 can serve as a potential therapeutic target for breast cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

The WRKY57 Transcription Factor Affects the Expression of Jasmonate ZIM-Domain Genes Transcriptionally to Compromise Botrytis cinerea Resistance.

PubMed

Jiang, Yanjuan; Yu, Diqiu

2016-08-01

Although necrotrophic pathogens cause many devastating plant diseases, our understanding of the plant defense response to them is limited. Here, we found that loss of function of WRKY57 enhanced the resistance of Arabidopsis (Arabidopsis thaliana) against Botrytis cinerea infection. Further investigation suggested that the negative regulation of WRKY57 against B cinerea depends on the jasmonic acid (JA) signaling pathway. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of JASMONATE ZIM-DOMAIN1 (JAZ1) and JAZ5, encoding two important repressors of the JA signaling pathway, and activates their transcription. In vivo and in vitro experiments demonstrated that WRKY57 interacts with nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2. Further experiments display that the same domain, the VQ motif, of SIB1 and SIB2 interact with WRKY33 and WRKY57. Moreover, transient transcriptional activity assays confirmed that WRKY57 and WRKY33 competitively regulate JAZ1 and JAZ5, SIB1 and SIB2 further enhance these competitions of WRKY57 to WRKY33. Therefore, coordinated regulation of Arabidopsis against B cinerea by transcription activators and repressors would benefit plants by allowing fine regulation of defense. © 2016 American Society of Plant Biologists. All Rights Reserved.

Cis-acting elements in the promoter region of the human aldolase C gene.

PubMed

Buono, P; de Conciliis, L; Olivetta, E; Izzo, P; Salvatore, F

1993-08-16

We investigated the cis-acting sequences involved in the expression of the human aldolase C gene by transient transfections into human neuroblastoma cells (SKNBE). We demonstrate that 420 bp of the 5'-flanking DNA direct at high efficiency the transcription of the CAT reporter gene. A deletion between -420 bp and -164 bp causes a 60% decrease of CAT activity. Gel shift and DNase I footprinting analyses revealed four protected elements: A, B, C and D. Competition analyses indicate that Sp1 or factors sharing a similar sequence specificity bind to elements A and B, but not to elements C and D. Sequence analysis shows a half palindromic ERE motif (GGTCA), in elements B and D. Region D binds a transactivating factor which appears also essential to stabilize the initiation complex.

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2009-04-01

Combination of several drugs is often necessary especially during long-them therapy. The competition between drugs can cause a decrease of the amount of a drug bound to albumin. This results in an increase of the free, biological active fraction of the drug. The aim of the presented study was to describe the competition between phenylbutazone (Phe) and methotrexate (MTX), two drugs recommended for the treatment of rheumatology in binding to bovine (BSA) and human (HSA) serum albumin in the high affinity binding site. Fluorescence analysis was used to estimate the effect of drugs on the protein fluorescence and to define the binding and quenching properties of drugs-serum albumin complexes. The effect of the displacement of one drug from the complex of the other with serum albumin has been described on the basis of the comparison of the quenching curves and binding constants for the binary and ternary systems. The conclusion that both Phe and MTX form a binding site in the same subdomain (IIA) points to the necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Multi-capillary based optical sensors for highly sensitive protein detection

NASA Astrophysics Data System (ADS)

Okuyama, Yasuhira; Katagiri, Takashi; Matsuura, Yuji

2017-04-01

A fluorescence measuring method based on glass multi-capillary for detecting trace amounts of proteins is proposed. It promises enhancement of sensitivity due to effects of the adsorption area expansion and the longitudinal excitation. The sensitivity behavior of this method was investigated by using biotin-streptavidin binding. According to experimental examinations, it was found that the sensitivity was improved by a factor of 70 from common glass wells. We also confirmed our measuring system could detect 1 pg/mL of streptavidin. These results suggest that multi-capillary has a potential as a high-sensitive biosensor.

Experiences with Interactive Multi-touch Tables

NASA Astrophysics Data System (ADS)

Fikkert, Wim; Hakvoort, Michiel; van der Vet, Paul; Nijholt, Anton

Interactive multi-touch tables can be a powerful means of communication for collaborative work as well as an engaging environment for competition. Through enticing gameplay we have evaluated user experience on competitive gameplay, collaborative work and musical expression. In addition, we report on our extensive experiences with two types of interactive multi-touch tables and we introduce a software framework that abstracts from their technical differences.

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils.

PubMed

Dagel, Daryl J; Liu, Yu-San; Zhong, Lanlan; Luo, Yonghua; Himmel, Michael E; Xu, Qi; Zeng, Yining; Ding, Shi-You; Smith, Steve

2011-02-03

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase--bacterial luciferase enzyme system.

PubMed

Huang, W; Feltus, A; Witkowski, A; Daunert, S

1996-05-01

A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

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

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

PubMed

Cao, Yanli; Zheng, Fanglin; Wang, Lei; Zhao, Guolei; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

2017-07-01

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. © 2017 John Wiley & Sons Ltd.

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

PubMed

Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K

2015-11-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics

PubMed Central

Corsino, Patrick E.; Narayan, Satya

2015-01-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non–ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non–ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. PMID:26018905

Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.

PubMed

Liu, Zheyi; Wang, Fangjun; Chen, Jin; Zhou, Ye; Zou, Hanfa

2016-08-26

Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252. Copyright © 2016 Elsevier B.V. All rights reserved.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

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

Crofton, K.M.; Reiter, L.W.; Mailman, R.B.

1987-01-01

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on /sup 3/H-flunitrazepam (FLU), /sup 3/H-muscimol (MUS), and (/sup 35/S-t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with /sup 3/H-FLU and /sup 3/H-MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of (/sup 35/S-TBPS at concentrations as high as 50 pM. Type II pyrethroids inhibited (/sup 35/S-TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 pM. The data presented suggest that the interaction of Type II pyrethroids with the GABA receptor-ionophore complex ismore » restricted to a site near the TBPS/picrotoxinin binding site.« less

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

PubMed Central

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

Why are Buckyonions Round?

NASA Technical Reports Server (NTRS)

Bates, Kevin R.; Scuseria, Gustavo E.

1998-01-01

Multi-layered round carbon particles (onions) containing tens to hundreds of thousands of atoms form during electron irradiation of graphite. However. theoretical models or large icosahedral fullerenes predict highly faceted shapes for molecules with more than a few hundred atoms. This discrepancy in shape may be explained by the presence of defects during the formation of carbon onions. Here, we use the semi-empirical tight-binding method for carbon to simulate the incorporation of pentagon-heptagon defects on to the surface of large icosahedral fullerenes. We show a simple mechanism that results in energetically competitive derivative structures and a global change in molecular shape from faceted to round. Our results provide a plausible explanation of the apparent discrepancy between experimental observations or round buckyonions and theoretical predictions of faceted icosahedral fullerenes.

78 FR 6120 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-29

..., including immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive... immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive Advantages: Binding of a new...

Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

USDA-ARS?s Scientific Manuscript database

This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

Binding interaction of ramipril with bovine serum albumin (BSA): Insights from multi-spectroscopy and molecular docking methods.

PubMed

Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

2016-11-01

The binding interaction between a typical angiotensin-converting enzyme inhibitor (ACEI), ramipril, and a transport protein, bovine serum albumin (BSA), was studied in vitro using UV-vis absorption spectroscopy, steady-state fluorescence spectroscopic titration, synchronous fluorescence spectroscopy, three dimensional fluorescence spectroscopy, circular dichroism and molecular docking under the imitated physiological conditions (pH=7.4). The experimental results suggested that the intrinsic fluorescence of BSA was quenched by ramipril thought a static quenching mechanism, indicating that the stable ramipril-BSA complex was formed by the intermolecular interaction. The number of binding sites (n) and binding constant of ramipril-BSA complex were about 1 and 3.50×10 4 M -1 at 298K, respectively, suggesting that there was stronger binding interaction of ramipril with BSA. The thermodynamic parameters together with molecular docking study revealed that both van der Waal's forces and hydrogen bonding interaction dominated the formation of the ramipril-BSA complex and the binding interaction of BSA with ramipril is enthalpy-driven processes due to |ΔH°|>|TΔS°| and ΔG°<0. The spatial distance between ramipril and BSA was calculated to be 3.56nm based on Förster's non-radiative energy transfer theory. The results of the competitive displacement experiments and molecular docking confirmed that ramipril inserted into the subdomain IIA (site I) of BSA, resulting in a slight change in the conformation of BSA but BSA still retained its secondary structure α-helicity. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

NASA Astrophysics Data System (ADS)

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-03-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

PubMed Central

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-01-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses. PMID:26928635

Analysis of Ligand Binding ErbB Receptor Tyrosine Kinases

DTIC Science & Technology

2000-07-01

epidermal growth factor receptor. Protein Sci 9, 310- 324 . 10 UNPUBLISHED DATA Principal Investigator (last, first, middle); Ferguson, Kathryn, M...and hetero-oligomerization. Purified Muaei- anto ane titse in tdi0ML protein (15 p.l) was loaded at a concentration of I mg/ml, and the gel Multi

The Impact of Large, Multi-Function/Multi-Site Competitions

DTIC Science & Technology

2003-08-01

this approach generates larger savings and improved service quality , and is less expensive to implement. Moreover, it is a way to meet the President s...of the study is to assess the degree to which large-scale competitions completed have resulted in increased savings and service quality and decreased

Complexes of lutein with bovine and caprine caseins and their impact on lutein chemical stability in emulsion systems: Effect of arabinogalactan.

PubMed

Mora-Gutierrez, A; Attaie, R; Núñez de González, M T; Jung, Y; Woldesenbet, S; Marquez, S A

2018-01-01

Lutein is an important xanthophyll carotenoid with many benefits to human health. Factors affecting the application of lutein as a functional ingredient in low-fat dairy-like beverages (pH 6.0-7.0) are not well understood. The interactions of bovine and caprine caseins with hydrophobic lutein were studied using UV/visible spectroscopy as well as fluorescence. Our studies confirmed that the aqueous solubility of lutein is improved after binding with bovine and caprine caseins. The rates of lutein solubilization by the binding to bovine and caprine caseins were as follows: caprine α S1 -II-casein 34%, caprine α S1 -I-casein 10%, and bovine casein 7% at 100 μM lutein. Fluorescence of the protein was quenched on binding supporting complex formation. The fluorescence experiments showed that the binding involves tryptophan residues and some nonspecific interactions. Scatchard plots of lutein binding to the caseins demonstrated competitive binding between the caseins and their sites of interaction with lutein. Competition experiments suggest that caprine α S1 -II casein will bind a larger number of lutein molecules with higher affinity than other caseins. The chemical stability of lutein was largely dependent on casein type and significant increases occurred in the chemical stability of lutein with the following pattern: caprine α S1 -II-casein > caprine α S1 -I-casein > bovine casein. Addition of arabinogalactan to lutein-enriched emulsions increases the chemical stability of lutein-casein complexes during storage under accelerated photo-oxidation conditions at 25°C. Therefore, caprine α S1 -II-casein alone and in combination with arabinogalactan can have important applications in the beverage industry as carrier of this xanthophyll carotenoid (lutein). Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

1988-07-01

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

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation

PubMed Central

Meeks, Shannon L.; Healey, John F.; Parker, Ernest T.; Barrow, Rachel T.

2007-01-01

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding β-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 β-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape. PMID:17848617

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations

NASA Astrophysics Data System (ADS)

Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

2016-09-01

We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations.

PubMed

Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

2016-09-01

We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

Competitive binding of (-)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

NASA Astrophysics Data System (ADS)

Yuan, Lixia; Liu, Min; Liu, Guiqin; Li, Dacheng; Wang, Zhengping; Wang, Bingquan; Han, Jun; Zhang, Min

2017-02-01

Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (-)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi.

PubMed

Monteil, Véronique; Kolb, Annie; Mayer, Claudine; Hoos, Sylviane; England, Patrick; Norel, Françoise

2010-12-01

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.

The design of a multi-harmonic step-tunable gyrotron

NASA Astrophysics Data System (ADS)

Qi, Xiang-Bo; Du, Chao-Hai; Zhu, Juan-Feng; Pan, Shi; Liu, Pu-Kun

2017-03-01

The theoretical study of a step-tunable gyrotron controlled by successive excitation of multi-harmonic modes is presented in this paper. An axis-encircling electron beam is employed to eliminate the harmonic mode competition. Physics images are depicted to elaborate the multi-harmonic interaction mechanism in determining the operating parameters at which arbitrary harmonic tuning can be realized by magnetic field sweeping to achieve controlled multiband frequencies' radiation. An important principle is revealed that a weak coupling coefficient under a high-harmonic interaction can be compensated by a high Q-factor. To some extent, the complementation between the high Q-factor and weak coupling coefficient makes the high-harmonic mode potential to achieve high efficiency. Based on a previous optimized magnetic cusp gun, the multi-harmonic step-tunable gyrotron is feasible by using harmonic tuning of first-to-fourth harmonic modes. Multimode simulation shows that the multi-harmonic gyrotron can operate on the 34 GHz first-harmonic TE11 mode, 54 GHz second-harmonic TE21 mode, 74 GHz third-harmonic TE31 mode, and 94 GHz fourth-harmonic TE41 mode, corresponding to peak efficiencies of 28.6%, 35.7%, 17.1%, and 11.4%, respectively. The multi-harmonic step-tunable gyrotron provides new possibilities in millimeter-terahertz source development especially for advanced terahertz applications.

Structural Basis for Antagonism by Suramin of Heparin Binding to Vaccinia Complement Protein

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

Ganesh, Vannakambadi K.; Muthuvel, Suresh Kumar; Smith, Scott A.

2010-07-19

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complexmore » with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.« less

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation

PubMed Central

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I.; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2016-01-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 when bound to yeast TBP, together with mutational data. The yTAF1-TAND1, which in itself acts as a transcriptional activator, binds into the DNA-binding TBP concave surface by presenting similar anchor residues to TBP as E. coli Mot1 but from a distinct structural scaffold. Furthermore, we show how yTAF1-TAND2 employs an aromatic and acidic anchoring pattern to bind a conserved yTBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides compelling insight into the competitive multiprotein TBP interplay critical to transcriptional regulation. PMID:23851461

High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation.

PubMed

Anandapadamanaban, Madhanagopal; Andresen, Cecilia; Helander, Sara; Ohyama, Yoshifumi; Siponen, Marina I; Lundström, Patrik; Kokubo, Tetsuro; Ikura, Mitsuhiko; Moche, Martin; Sunnerhagen, Maria

2013-08-01

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 Å) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBP's concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

Competitive folding of RNA structures at a termination–antitermination site

PubMed Central

Ait-Bara, Soraya; Clerté, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2017-01-01

Antitermination is a regulatory process based on the competitive folding of terminator–antiterminator structures that can form in the leader region of nascent transcripts. In the case of the Bacillus subtilis licS gene involved in β-glucosides utilization, the binding of the antitermination protein LicT to a short RNA hairpin (RAT) prevents the formation of an overlapping terminator and thereby allows transcription to proceed. Here, we monitored in vitro the competition between termination and antitermination by combining bulk and single-molecule fluorescence-based assays using labeled RNA oligonucleotide constructs of increasing length that mimic the progressive transcription of the terminator invading the antiterminator hairpin. Although high affinity binding is abolished as soon as the antiterminator basal stem is disrupted by the invading terminator, LicT can still bind and promote closing of the partially unfolded RAT hairpin. However, binding no longer occurs once the antiterminator structure has been disrupted by the full-length terminator. Based on these findings, we propose a kinetic competition model for the sequential events taking place at the termination–antitermination site, where LicT needs to capture its RAT target before completion of the terminator to remain tightly bound during RNAP pausing, before finally dissociating irreversibly from the elongated licS transcript. PMID:28235843

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

PubMed

Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

1992-08-01

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

Choline+ is a low-affinity ligand for alpha 1-adrenoceptors.

PubMed

Unelius, L; Cannon, B; Nedergaard, J

1994-10-07

The effect of choline+, a commonly used Na+ substitute, on ligand binding to alpha 1-adrenoceptors was investigated. It was found that replacement of 25% of the Na+ in a Krebs-Ringer bicarbonate buffer with choline+ led to a 3-fold decrease in the apparent affinity of [3H]prazosin for its binding site (i.e. the alpha 1-receptor) in a membrane preparation from brown adipose tissue, while no decrease in the total number of binding sites was observed. Similar effects were seen in membrane preparations from liver and brain. In competition experiments, it was found that choline+ could inhibit [3H]prazosin binding; from the inhibition curve, an affinity (Ki) of 31 mM choline+ for the [3H]prazosin-binding site could be calculated. In fully choline(+)-substituted buffers, where the level of [3H]prazosin binding was substantially reduced, both phentolamine and norepinephrine could still compete with [3H]prazosin for its binding site, with virtually unaltered affinity; thus choline+ did not substantially affect the characteristics of those receptors to which it did not bind. Choline+ did not affect the binding characteristics of the beta 1/beta 2 radioligand [3H]CGP-12177; thus, the effect on alpha 1-receptors was not due to general, unspecific effects on the membrane preparations. It is concluded that choline+ possesses characteristics similar to those of a competitive ligand for the alpha 1-adrenoceptor; it has a low affinity but the competitive type of interaction of choline may nonetheless under experimental conditions interfere with agonist interaction with the alpha 1-receptor.

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors.

PubMed

Halámek, Jan; Makower, Alexander; Knösche, Kristina; Skládal, Petr; Scheller, Frieder W

2005-01-30

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of cocaine was based on a competitive assay. The change of frequency measured after 300s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35Hz (with an imprecision 3%, n = 3) while the presence of 100pmoll(-1) cocaine decreased the binding by 11%. The limit of detection was consequently below 100pmoll(-1) for cocaine. The total time of one analysis was 15min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

Intrinsic mineralocorticoid agonist activity of some nonsteroidal anti-inflammatory drugs. A postulated mechanism for sodium retention.

PubMed Central

Feldman, D; Couropmitree, C

1976-01-01

Because some nonsteroidal anti-inflammatory drugs (NSAID) induce salt and water retention and exhibit other steroid-like actions, studies were performed to ascertain whether these drugs possess intrinsic mineralocorticoid agonist activity. In vitro competitive binding assays utilizing tissue from adrenalectomized rats demonstrated that some NSAID can displace [3H]-aldosterone from renal cytoplasmic mineralocorticoid receptors. Displacement potency for these sites was in the sequence: aldosterone greater than spironolactone greater than phenylbutazone (PBZ) greater than aspirin (ASA) greater than indomethacin (IDM). Concentration ratios required to obtain significant displacement of [3H]aldosterone were high but clearly within the therapeutic range for PBZ and ASA but not IDM. The analogues oxyphenbutazone (OBZ) and sodium salicylate (SS) were similar in binding activity to PBZ and ASA, respectively. Lineweaver-Burk analysis revealed that the inhibition of [3H]aldosterone binding was competitive in nature. In addition, PBZ was shown to prevent the nuclear binding of [3H]aldosterone. In vivo injection of PBZ and ASA resulted in competition for [3H]aldosterone renal binding comparable to the in vitro studies. Administration of PBZ and OBZ to adrenalectomized rats resulted in significant salt retention whereas ASA and SS did not differ significantly from controls. Salt retention elicited by PBZ and OBZ was inhibited by spironolactone, a competitive mineralocorticoid antagonist. These data suggest that, despite nonsteroidal structures, PBZ and OBZ induce salt retention via a receptor-mediated mineralocorticoid pathway analogous to aldosterone action. PMID:173739

Zn(II) stimulation of Fe(II)-activated repression in the iron-dependent repressor from Mycobacterium tuberculosis.

PubMed

Stapleton, Brian; Walker, Lawrence R; Logan, Timothy M

2013-03-19

Thermodynamic measurements of Fe(II) binding and activation of repressor function in the iron-dependent repressor from Mycobacterium tuberculosis (IdeR) are reported. IdeR, a member of the diphtheria toxin repressor family of proteins, regulates iron homeostasis and contributes to the virulence response in M. tuberculosis. Although iron is the physiological ligand, this is the first detailed analysis of iron binding and activation in this protein. The results showed that IdeR binds 2 equiv of Fe(II) with dissociation constants that differ by a factor of 25. The high- and low-affinity iron binding sites were assigned to physical binding sites I and II, respectively, using metal binding site mutants. IdeR was also found to contain a high-affinity Zn(II) binding site that was assigned to physical metal binding site II through the use of binding site mutants and metal competition assays. Fe(II) binding was modestly weaker in the presence of Zn(II), but the coupled metal binding-DNA binding affinity was significantly stronger, requiring 30-fold less Fe(II) to activate DNA binding compared to Fe(II) alone. Together, these results suggest that IdeR is a mixed-metal repressor, where Zn(II) acts as a structural metal and Fe(II) acts to trigger the physiologically relevant promoter binding. This new model for IdeR activation provides a better understanding of IdeR and the biology of iron homeostasis in M. tuberculosis.

A Comparative Study of the Application of Fluorescence Excitation-Emission Matrices Combined with Parallel Factor Analysis and Nonnegative Matrix Factorization in the Analysis of Zn Complexation by Humic Acids

PubMed Central

Boguta, Patrycja; Pieczywek, Piotr M.; Sokołowska, Zofia

2016-01-01

The main aim of this study was the application of excitation-emission fluorescence matrices (EEMs) combined with two decomposition methods: parallel factor analysis (PARAFAC) and nonnegative matrix factorization (NMF) to study the interaction mechanisms between humic acids (HAs) and Zn(II) over a wide concentration range (0–50 mg·dm−3). The influence of HA properties on Zn(II) complexation was also investigated. Stability constants, quenching degree and complexation capacity were estimated for binding sites found in raw EEM, EEM-PARAFAC and EEM-NMF data using mathematical models. A combination of EEM fluorescence analysis with one of the proposed decomposition methods enabled separation of overlapping binding sites and yielded more accurate calculations of the binding parameters. PARAFAC and NMF processing allowed finding binding sites invisible in a few raw EEM datasets as well as finding totally new maxima attributed to structures of the lowest humification. Decomposed data showed an increase in Zn complexation with an increase in humification, aromaticity and molecular weight of HAs. EEM-PARAFAC analysis also revealed that the most stable compounds were formed by structures containing the highest amounts of nitrogen. The content of oxygen-functional groups did not influence the binding parameters, mainly due to fact of higher competition of metal cation with protons. EEM spectra coupled with NMF and especially PARAFAC processing gave more adequate assessments of interactions as compared to raw EEM data and should be especially recommended for modeling of complexation processes where the fluorescence intensities (FI) changes are weak or where the processes are interfered with by the presence of other fluorophores. PMID:27782078

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

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

NASA Astrophysics Data System (ADS)

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-07-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

PubMed Central

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-01-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment. PMID:23881402

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation.

PubMed

Zgliczyński, J M; Stelmaszyńska, T; Olszowska, E; Krawczyk, A; Kwasnowska, E; Wróbel, J T

1983-01-01

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.

May the Best Molecule Win: Competition ESI Mass Spectrometry

PubMed Central

Laughlin, Sarah; Wilson, W. David

2015-01-01

Electrospray ionization mass spectrometry has become invaluable in the characterization of macromolecular biological systems such as nucleic acids and proteins. Recent advances in the field of mass spectrometry and the soft conditions characteristic of electrospray ionization allow for the investigation of non-covalent interactions among large biomolecules and ligands. Modulation of genetic processes through the use of small molecule inhibitors with the DNA minor groove is gaining attention as a potential therapeutic approach. In this review, we discuss the development of a competition method using electrospray ionization mass spectrometry to probe the interactions of multiple DNA sequences with libraries of minor groove binding molecules. Such an approach acts as a high-throughput screening method to determine important information including the stoichiometry, binding mode, cooperativity, and relative binding affinity. In addition to small molecule-DNA complexes, we highlight other applications in which competition mass spectrometry has been used. A competitive approach to simultaneously investigate complex interactions promises to be a powerful tool in the discovery of small molecule inhibitors with high specificity and for specific, important DNA sequences. PMID:26501262

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

PubMed

Selvakumar, Dakshnamurthy; Drescher, Marian J; Deckard, Nathan A; Ramakrishnan, Neeliyath A; Morley, Barbara J; Drescher, Dennis G

2017-01-01

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca 2+ ] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca 2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca 2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by K D values from SPR (+Ca 2+ ), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

In silico investigation into the interactions between murine 5-HT3 receptor and the principle active compounds of ginger (Zingiber officinale).

PubMed

Lohning, Anna E; Marx, Wolfgang; Isenring, Liz

2016-11-01

Gingerols and shogaols are the primary non-volatile actives within ginger (Zingiber officinale). These compounds have demonstrated in vitro to exert 5-HT 3 receptor antagonism which could benefit chemotherapy-induced nausea and vomiting (CINV). The site and mechanism of action by which these compounds interact with the 5-HT 3 receptor is not fully understood although research indicates they may bind to a currently unidentified allosteric binding site. Using in silico techniques, such as molecular docking and GRID analysis, we have characterized the recently available murine 5-HT 3 receptor by identifying sites of strong interaction with particular functional groups at both the orthogonal (serotonin) site and a proposed allosteric binding site situated at the interface between the transmembrane region and the extracellular domain. These were assessed concurrently with the top-scoring poses of the docked ligands and included key active gingerols, shogaols and dehydroshogaols as well as competitive antagonists (e.g. setron class of pharmacologically active drugs), serotonin and its structural analogues, curcumin and capsaicin, non-competitive antagonists and decoys. Unexpectedly, we found that the ginger compounds and their structural analogs generally outscored other ligands at both sites. Our results correlated well with previous site-directed mutagenesis studies in identifying key binding site residues. We have identified new residues important for binding the ginger compounds. Overall, the results suggest that the ginger compounds and their structural analogues possess a high binding affinity to both sites. Notwithstanding the limitations of such theoretical analyses, these results suggest that the ginger compounds could act both competitively or non-competitively as has been shown for palonosetron and other modulators of CYS loop receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-{alpha}-induced NF-{kappa}B activity by binding to TNFR1

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

Hwang, Jae Ryoung; Huh, Jae Ho; Lee, Yoonna

2011-02-25

Research highlights: {yields} Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. {yields} The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-{alpha} and was blocked TNF-{alpha}-activated NF-{kappa}B activity. {yields} IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-{alpha}. {yields} Competition between the L-domain of IGFBP-5 and TNF-{alpha} blocked TNF-{alpha}-induced NF-{kappa}B activity. {yields} This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor. -- Abstract: IGFBP-5 is known to be involved in various cell phenomena such as proliferation,more » differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-{alpha}, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-{kappa}B activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-{alpha} in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-{alpha} resulted in inhibition of TNF-{alpha}-induced NF-{kappa}{Beta} activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor.« less

Competition in human groups-Impact on group cohesion, perceived stress and outcome satisfaction.

PubMed

Boos, Margarete; Franiel, Xaver; Belz, Michael

2015-11-01

This study on competition in human groups was performed within the context of the competitive outcome interdependence concept: the degree to which personal outcomes among group members are affected by the consequences of task performance of others, e.g. when one group member gains a high reward for a task, this lowers the available reward for other group members. Our computer-based multi-participant game empirically assessed how competitive versus neutral conditions influenced the reward-maximising behaviour of 200 undergraduate students functioning in ten-person groups - each playing two games (1 neutral and 1 competitive), their perceived pay satisfaction as well as perceived stress levels and sense of calmness within the games' task to search for coins. Participants were represented by black dots moving on a virtual playground. Results showed that competition led to reward-maximising but fellow group member disadvantaging behaviour, and all participants experienced lower pay satisfaction, higher stress levels and less calmness. We conclude that short-term behavioural consequences of positive individual competitive behaviour were gained at the above-mentioned potential long-term negative costs for all group members. This implies group paradigms aimed at sustainability should avoid introducing competitive factors that at best result in short-lived gains and at worst cause widespread dissatisfaction, stress and a pervasive lack of calmness. Copyright © 2015. Published by Elsevier B.V.

Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes.

PubMed

Treuter, E; Johansson, L; Thomsen, J S; Wärnmark, A; Leers, J; Pelto-Huikko, M; Sjöberg, M; Wright, A P; Spyrou, G; Gustafsson, J A

1999-03-05

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

Artificial light-regulation of an allosteric bi-enzyme complex by a photosensitive ligand.

PubMed

Kneuttinger, Andrea C; Winter, Martin; Simeth, Nadja A; Heyn, Kristina; Merkl, Rainer; König, Burkhard; Sterner, Reinhard

2018-05-29

The artificial regulation of proteins by light is an emerging sub-discipline of synthetic biology. Here, we used this concept in order to photo-control both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). The ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradiation, the DTE-ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light-regulation of other multi-enzyme complexes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biotic ligand modeling approach: Synthesis of the effect of major cations on the toxicity of metals to soil and aquatic organisms.

PubMed

Ardestani, Masoud M; van Straalen, Nico M; van Gestel, Cornelis A M

2015-10-01

The biotic ligand model (BLM) approach is used to assess metal toxicity, taking into account the competition of other cations with the free metal ions for binding to the biotic ligand sites of aquatic and soil organisms. The bioavailable fraction of metals, represented by the free metal ion, is a better measure than the total concentration for assessing their potential risk to the environment. Because BLMs are relating toxicity to the fraction of biotic ligands occupied by the metal, they can be useful for investigating factors affecting metal bioaccumulation and toxicity. In the present review, the effects of major cations on the toxicity of metals to soil and aquatic organisms were comprehensively studied by performing a meta-analysis of BLM literature data. Interactions at the binding sites were shown to be species- and metal-specific. The main factors affecting the relationships between toxicity and conditional binding constants for metal binding at the biotic ligand appeared to be Ca(2+) , Mg(2+) , and protons. Other important characteristics of the exposure medium, such as levels of dissolved organic carbon and concentrations of other cations, should also be considered to obtain a proper assessment of metal toxicity to soil and aquatic organisms. © 2015 SETAC.

Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design.

PubMed

Faller, Christina E; Raman, E Prabhu; MacKerell, Alexander D; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules ("fragments") that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind nonoverlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy.The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is "soaked" in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called "FragMaps" can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine "Grid Free Energies (GFEs)," which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities.

Cultured bovine brain capillary endothelial cells (BBCEC) - a blood-brain barrier model for studying the binding and internalization of insulin and insulin-like growth factor 1

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

Keller, B.T.; Borchardt, R.T.

1987-05-01

Cultured bovine brain capillary endothelial cells (BBCEC) have previously been reported by their laboratory as a working model for studying nutrient and drug transport and metabolism at the blood-brain barrier. In the present study, they have utilized this culture system to investigate the binding and internalization of (/sup 125/I)-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23/sup 0/C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37/sup 0/C, the maximum specific binding was 0.9% for INS and 5.8% for IGF-1. Using an acid-wash technique to assess peptide internalization, itmore » was observed that, at 37/sup 0/C, approximately 60% of the bound INS rapidly became resistant to acid treatment, a value which was constant over 2 hr. With IGF-1, a similar proportion of the bound material, 62%, became resistant by 30 min, but subsequently decreased to 45% by 2 hr. Scatchard analysis of competitive binding studies indicated the presence of two binding sites for each protein, having K/sub d/'s of 0.82 nM and 19.2 nM for INS and 0.39 nM and 3.66 nM for IGF-1. Little change in the amount of INS binding was observed over a four-day interval as the cultures became a confluent monolayer. The present report of binding and internalization of these proteins suggests that the BBCEC may utilize a receptor-mediated process to internalize and/or transport (transcytosis) INS and IGF-1 from the circulation.« less

Why Are Buckyonions Round?

NASA Technical Reports Server (NTRS)

Bates, Kevin R.; Scuseria, Gustavo E.

1997-01-01

Multi-layered round carbon particles (onions) containing tens to hundreds of thousands of atoms form during electron irradiation of graphite carbon. However, theoretical models of large icosahedral fullerenes predict highly faceted shapes for molecules with more than a few hundred atoms. This discrepancy in shape may be explained by the presence of defects during the formation of carbon onions. Here, we use the semi-empirical tight-binding method for carbon to simulate the incorporation of pentagon-heptagon defects on to the surface of large icosahedral fullerenes. We show a simple mechanism that results in energetically competitive derivative structures and a global change in molecular shape from faceted to round. Our results provide a plausible explanation of the apparent discrepancy between experimental observations of round buckyonions and theoretical predictions of faceted icosahedral fullerenes.

A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

PubMed Central

Carlo, Troy; Sierra, Rebecca; Berget, Susan M.

2000-01-01

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon. PMID:10805741

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

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

Humphreys, C.J.

1989-01-01

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

Revealing multi-binding sites for taspine to VEGFR-2 by cell membrane chromatography zonal elution.

PubMed

Du, Hui; Wang, Sicen; Ren, Jing; Lv, Nan; He, Langchong

2012-03-01

A new high-expression vascular endothelial growth factor receptor-2 (VEGFR-2) cell membrane chromatography (CMC) method was developed to investigate the affinity of ligands for VEGFR-2. An HEK293 VEGFR-2/CMC system was applied to specifically recognize ligands acting on VEGFR-2. Sorafenib was used as a mobile phase additive to evaluate the effect of the marker's concentration on the retention of sorafenib and taspine, respectively. The relationship among the retention, the types of binding sites and the affinity of taspine binding to VEGFR-2 has also been concerned. The retention behavior indicated that sorafenib had two major binding regions on VEGFR-2, and that taspine might act as a multi-target VEGFR-2 inhibitor with similar biological activity to sorafenib. The equilibrium dissociation constants (K(D)) obtained from the model are (5.25 ± 0.31) × 10⁻⁷ and (9.88 ± 0.54) × 10⁻⁵ mol L⁻¹ for sorafenib at the high- and low-affinity sites, respectively, and the corresponding values for taspine are (3.88 ± 0.31) × 10⁻⁶ and (7.04 ± 0.49)×10⁻⁵ mol L⁻¹. The two types of binding sites contributed about a 1:2 ratio on the retention of taspine. Copyright © 2012 Elsevier B.V. All rights reserved.

A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases

PubMed Central

Guarnieri, Michael T.; Blagg, Brian S. J.

2011-01-01

Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069

Putative melatonin receptors in a human biological clock

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

Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.

In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completelymore » inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.« less

Photonic crystal borax competitive binding carbohydrate sensing motif†

PubMed Central

Cui, Qingzhou; Muscatello, Michelle M. Ward; Asher, Sanford A.

2009-01-01

We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols. PMID:19381378

Mechanistic Insights into Xenon Inhibition of NMDA Receptors from MD Simulations

PubMed Central

Liu, Lu Tian; Xu, Yan; Tang, Pei

2010-01-01

Inhibition of N-methyl-D-aspartate (NMDA) receptors has been viewed as a primary cause of xenon anesthesia, yet the mechanism is unclear. Here, we investigated interactions between xenon and the ligand-binding domain (LBD) of a NMDA receptor and examined xenon-induced structural and dynamical changes that are relevant to functional changes of the NMDA receptor. Several comparative molecular dynamics simulations were performed on two X-ray structures representing the open- and closed-cleft LBD of the NMDA receptor. We identified plausible xenon action sites in the LBD, including those nearby agonist sites, in the hinge region, and at the interface between two subunits. The xenon binding energy varies from −5.3 to −0.7 kcal/mol. Xenon's effect on the NMDA receptor is conformation-dependent and is produced through both competitive and non-competitive mechanisms. Xenon can promote cleft opening in the absence of agonists and consequently stabilizes the closed channel. Xenon can also bind at the interface of two subunits, alter the inter-subunit interaction, and lead to a reduction of the distance between GT-links. This reduction corresponds to a rearrangement of the channel toward a direction of pore size decreasing, implying a closed or desensitized channel. In addition to these non-competitive actions, xenon was found to weaken the glutamate binding, which could lead to low agonist efficacy and appear as competitive inhibition. PMID:20560662

Thermodynamics of binding interactions between extracellular polymeric substances and heavy metals by isothermal titration microcalorimetry.

PubMed

Yan, Peng; Xia, Jia-Shuai; Chen, You-Peng; Liu, Zhi-Ping; Guo, Jin-Song; Shen, Yu; Zhang, Cheng-Cheng; Wang, Jing

2017-05-01

Extracellular polymeric substances (EPS) play a crucial role in heavy metal bio-adsorption using activated sludge, but the interaction mechanism between heavy metals and EPS remains unclear. Isothermal titration calorimetry was employed to illuminate the mechanism in this study. The results indicate that binding between heavy metals and EPS is spontaneous and driven mainly by enthalpy change. Extracellular proteins in EPS are major participants in the binding process. Environmental conditions have significant impact on the adsorption performance. Divalent and trivalent cations severely impeded the binding of heavy metal ions to EPS. Electrostatic interaction mainly attributed to competition between divalent cations and heavy metal ions; trivalent cations directly competed with heavy metal ions for EPS binding sites. Trivalent cations were more competitive than divalent cations for heavy metal ion binding because they formed complexing bonds. This study facilitates a better understanding about the interaction between heavy metals and EPS in wastewater treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adherence of oral streptococci: evidence for nonspecific adsorption to saliva-coated hydroxylapatite surfaces.

PubMed Central

Staat, R H; Peyton, J C

1984-01-01

It is proposed that binding of oral streptococci to saliva-coated hydroxylapatite (SHA) surfaces is a multifactorial process involving both specific and nonspecific receptors. In this context, specific binding is described as a high-affinity, saturable interaction between the cell and binding surface. Conversely, nonspecific binding is considered to be a nonsaturable, generalized, low-affinity reaction. Experimental differentiation of specific binding from nonspecific binding was achieved with a competition assay which utilized a large excess of nonradiolabeled bacteria to compete with the 3H-labeled cells for attachment to receptors on 1.5 mg of SHA crystals. Competition assays of Streptococcus sanguis and Streptococcus mitis adhesion clearly demonstrated that the total binding isotherm was composed of a saturable specific binding reaction and a minor nonspecific binding component. This was further substantiated by analysis of nonlinear Scatchard plots of the total binding data. The competition data for Streptococcus mutans binding indicated that ca. 50% of the S. mutans binding appeared to be specific, although saturation of the SHA surfaces with bacterial cells could not be demonstrated. Experiments measuring desorption of radiolabeled cells from SHA crystals into buffer showed that ca. 50% of the bound S. mutans cells were removed after 4 h, whereas less than 5% of the S. sanguis cells were eluted from the SHA surfaces. The kinetics of attachment were studied by using an extract of Persea americana as a noncompetitive inhibitor of adherence. The total cell binding data for these experiments suggested a very rapid binding reaction followed by a slower rate of attachment. It was concluded from these three different experimental approaches that adherence of selected oral streptococci to SHA surfaces involves specific, high-affinity and nonspecific, low-affinity binding reactions. The concept is developed that in vitro streptococcal attachment to SHA can be described as a two-reaction process in which the low-affinity interaction of the cell with the SHA surface precedes the establishment of the stronger, specific bonds needed for the maintenance of streptococci in the oral cavity. PMID:6327530

Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

PubMed

Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

2004-04-01

The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. Copyright 2004 Wiley-Liss, Inc.

Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.

PubMed

Kibbey, Megan M; Jameson, Mark J; Eaton, Erin M; Rosenzweig, Steven A

2006-03-01

Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

Computational Identification of Diverse Mechanisms Underlying Transcription Factor-DNA Occupancy

PubMed Central

Cheng, Qiong; Kazemian, Majid; Pham, Hannah; Blatti, Charles; Celniker, Susan E.; Wolfe, Scot A.; Brodsky, Michael H.; Sinha, Saurabh

2013-01-01

ChIP-based genome-wide assays of transcription factor (TF) occupancy have emerged as a powerful, high-throughput method to understand transcriptional regulation, especially on a global scale. This has led to great interest in the underlying biochemical mechanisms that direct TF-DNA binding, with the ultimate goal of computationally predicting a TF's occupancy profile in any cellular condition. In this study, we examined the influence of various potential determinants of TF-DNA binding on a much larger scale than previously undertaken. We used a thermodynamics-based model of TF-DNA binding, called “STAP,” to analyze 45 TF-ChIP data sets from Drosophila embryonic development. We built a cross-validation framework that compares a baseline model, based on the ChIP'ed (“primary”) TF's motif, to more complex models where binding by secondary TFs is hypothesized to influence the primary TF's occupancy. Candidates interacting TFs were chosen based on RNA-SEQ expression data from the time point of the ChIP experiment. We found widespread evidence of both cooperative and antagonistic effects by secondary TFs, and explicitly quantified these effects. We were able to identify multiple classes of interactions, including (1) long-range interactions between primary and secondary motifs (separated by ≤150 bp), suggestive of indirect effects such as chromatin remodeling, (2) short-range interactions with specific inter-site spacing biases, suggestive of direct physical interactions, and (3) overlapping binding sites suggesting competitive binding. Furthermore, by factoring out the previously reported strong correlation between TF occupancy and DNA accessibility, we were able to categorize the effects into those that are likely to be mediated by the secondary TF's effect on local accessibility and those that utilize accessibility-independent mechanisms. Finally, we conducted in vitro pull-down assays to test model-based predictions of short-range cooperative interactions, and found that seven of the eight TF pairs tested physically interact and that some of these interactions mediate cooperative binding to DNA. PMID:23935523

Unraveling the binding interaction of a bioactive pyrazole-based probe with serum proteins: Relative concentration dependent 1:1 and 2:1 probe-protein stoichiometries.

PubMed

Kundu, Pronab; Chattopadhyay, Nitin

2018-06-15

Molecular interactions and binding of probes/drugs with biomacromolecular systems are of fundamental importance in understanding the mechanism of action and hence designing of proactive drugs. In the present study, binding interactions of a biologically potent fluorophore, (E)-1,5-diphenyl-3-styryl-4,5-dihydro-1H-pyrazole (DSDP) with two serum transport proteins, human serum albumin and bovine serum albumin, have been investigated exploiting multi-spectroscopic techniques. The spectrophotometric and fluorometric studies together with fluorescence quenching, fluorescence anisotropy, urea induced denaturation studies and fluorescence lifetime measurements reveal strong binding of DSDP with both the plasma proteins. Going beyond the vast literature data mostly providing 1:1 probe-protein complexation, the present investigation portrays 2:1 probe-protein complex formation at higher relative probe concentration. A newer approach has been developed to have an estimate of the binding constants varying the concentration of the protein, instead of the usual practice of varying the probe. The binding constants for the 2:1 DSDP-protein complexes are determined to be 1.37 × 10 10  M -2 and 1.47 × 10 10  M -2 for HSA and BSA respectively, while those for the 1:1 complexation process come out to be 1.85 × 10 5  M -1 and 1.73 × 10 5  M -1 for DSDP-HSA and DSDP-BSA systems respectively. Thermodynamic analysis at different temperatures implies that the forces primarily involved in the binding process are hydrogen bonding and hydrophobic interactions. Competitive replacement studies with known site markers and molecular docking simulations direct to the possible locations and binding energies of DSDP with the two serum proteins, corroborating well with the experimental results. Copyright © 2018 Elsevier B.V. All rights reserved.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1

NASA Astrophysics Data System (ADS)

Park, Hwangseo; Lee, Hye Seon; Ku, Bonsu; Lee, Sang-Rae; Kim, Seung Jun

2017-08-01

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

Molecular basis for TANK recognition by TRAF1 revealed by the crystal structure of TRAF1/TANK complex.

PubMed

Kim, Chang Min; Jeong, Jae-Hee; Son, Young-Jin; Choi, Jun-Hyuk; Kim, Sunghwan; Park, Hyun Ho

2017-03-01

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a multifunctional adaptor protein involved in important processes of cellular signaling, including innate immunity and apoptosis. TRAF family member-associated NF-kappaB activator (TANK) has been identified as a competitive intracellular inhibitor of TRAF2 function. Although TRAF recognition by various receptors has been studied extensively in the field of TRAF-mediated biology, molecular and functional details of TANK recognition and interaction with TRAF1 have not been studied. In this study, we report the crystal structure of the TRAF1/TANK peptide complex. Quantitative interaction experiments showed that TANK peptide interacts with both TRAF1 and TRAF2 with similar affinity in a micromolar range. Our structural study also reveals that TANK binds TRAF1 using a minor minimal consensus motif for TRAF binding, Px(Q/E)xT. Coordinate and structural factor were deposited in the Protein Data Bank under PDB ID code 5H10. © 2017 Federation of European Biochemical Societies.

Regulation of Global Transcription in Escherichia coli by Rsd and 6S RNA

PubMed Central

Lal, Avantika; Krishna, Sandeep; Seshasayee, Aswin Sai Narain

2018-01-01

In Escherichia coli, the sigma factor σ70 directs RNA polymerase to transcribe growth-related genes, while σ38 directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to σ70, and the non-coding 6S RNA which binds to the RNA polymerase-σ70 holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show that Rsd and 6S RNA facilitate σ38 activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression. PMID:29686109

Regulation of Global Transcription in Escherichia coli by Rsd and 6S RNA.

PubMed

Lal, Avantika; Krishna, Sandeep; Seshasayee, Aswin Sai Narain

2018-05-31

In Escherichia coli , the sigma factor σ 70 directs RNA polymerase to transcribe growth-related genes, while σ 38 directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to σ 70 , and the non-coding 6S RNA which binds to the RNA polymerase-σ 70 holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show that Rsd and 6S RNA facilitate σ 38 activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression. Copyright © 2018 Lal et al.

Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

PubMed Central

Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy. The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is “soaked” in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called “FragMaps” can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine “Grid Free Energies (GFEs),” which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

[Studying specific effects of nootropic drugs on glutamate receptors in the rat brain].

PubMed

Firstova, Iu Iu; Vasil'eva, E V; Kovalev, G I

2011-01-01

The influence of nootropic drugs of different groups (piracetam, phenotropil, nooglutil, noopept, semax, meclofenoxate, pantocalcine, and dimebon) on the binding of the corresponding ligands to AMPA, NMDA, and mGlu receptors of rat brain has been studied by the method of radio-ligand binding in vitro. It is established that nooglutil exhibits pharmacologically significant competition with a selective agonist of AMPA receptors ([G-3H]Ro 48-8587) for the receptor binding sites (with IC50 = 6.4 +/- 0.2 microM), while the competition of noopept for these receptor binding sites was lower by an order of magnitude (IC50 = 80 +/- 5.6 microM). The heptapeptide drug semax was moderately competitive with [G-3H]LY 354740 for mGlu receptor sites (IC50 = 33 +/- 2.4 microM). Dimebon moderately influenced the specific binding of the ligand of NMDA receptor channel ([G-3H]MK-801) at IC50 = 59 +/- 3.6 microM. Nootropic drugs of the pyrrolidone group (piracetam, phenotropil) as well as meclofenoxate, pantocalcine (pantogam) in a broad rage of concentrations (10(-4)-10(-10) M) did not affect the binding of the corresponding ligands to glutamate receptors (IC50 100 pM). Thus, the direct neurochemical investigation was used for the first time to qualitatively characterize the specific binding sites for nooglutil and (to a lower extent) noopept on AMPA receptors, for semax on metabotropic glutamate receptors, and for dimebon on the channel region of NMDA receptors. The results are indicative of a selective action of some nootropes on the glutamate family.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

76 FR 37191 - Notice of Competition for University Transportation Centers (UTC) Program Grants

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-24

... Competition for University Transportation Centers (UTC) Program Grants AGENCY: Research and Innovative... conduct a competition for University Transportation Centers (UTC) Program grants for the purpose of... of demonstrated ability, research, technology and education resources, leadership, multi-modal...

Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

PubMed

Stax, Martijn J; Mouser, Emily E I M; van Montfort, Thijs; Sanders, Rogier W; de Vries, Henry J C; Dekker, Henk L; Herrera, Carolina; Speijer, Dave; Pollakis, Georgios; Paxton, William A

2015-01-01

Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

The zinc fingers of YY1 bind single-stranded RNA with low sequence specificity.

PubMed

Wai, Dorothy C C; Shihab, Manar; Low, Jason K K; Mackay, Joel P

2016-11-02

Classical zinc fingers (ZFs) are traditionally considered to act as sequence-specific DNA-binding domains. More recently, classical ZFs have been recognised as potential RNA-binding modules, raising the intriguing possibility that classical-ZF transcription factors are involved in post-transcriptional gene regulation via direct RNA binding. To date, however, only one classical ZF-RNA complex, that involving TFIIIA, has been structurally characterised. Yin Yang-1 (YY1) is a multi-functional transcription factor involved in many regulatory processes, and binds DNA via four classical ZFs. Recent evidence suggests that YY1 also interacts with RNA, but the molecular nature of the interaction remains unknown. In the present work, we directly assess the ability of YY1 to bind RNA using in vitro assays. Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to identify preferred RNA sequences bound by the YY1 ZFs from a randomised library over multiple rounds of selection. However, a strong motif was not consistently recovered, suggesting that the RNA sequence selectivity of these domains is modest. YY1 ZF residues involved in binding to single-stranded RNA were identified by NMR spectroscopy and found to be largely distinct from the set of residues involved in DNA binding, suggesting that interactions between YY1 and ssRNA constitute a separate mode of nucleic acid binding. Our data are consistent with recent reports that YY1 can bind to RNA in a low-specificity, yet physiologically relevant manner. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Interaction of fenoterol stereoisomers with β2-adrenoceptor-G sα fusion proteins: antagonist and agonist competition binding.

PubMed

Reinartz, Michael T; Kälble, Solveig; Wainer, Irving W; Seifert, Roland

2015-05-01

The specific interaction between G-protein-coupled receptors and ligand is the starting point for downstream signaling. Fenoterol stereoisomers were successfully used to probe ligand-specific activation (functional selectivity) of the β2-adrenoceptor (β2AR) (Reinartz et al. 2015). In the present study, we extended the pharmacological profile of fenoterol stereoisomers using β2AR-Gsα fusion proteins in agonist and antagonist competition binding assays. Dissociations between binding affinities and effector potencies were found for (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol. Our data corroborate former studies on the importance of the aminoalkyl moiety of fenoterol derivatives for functional selectivity.

Measurement of monomolecular binding constants of neutral phenols into the beta-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay.

PubMed

Le Saux, Thomas; Hisamoto, Hideaki; Terabe, Shigeru

2006-02-03

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into beta-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

EPA Science Inventory

The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

PubMed Central

Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

2008-01-01

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

PubMed Central

Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

2016-01-01

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

PubMed

Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

2008-10-24

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

Model of transcriptional activation by MarA in Escherichia coli.

PubMed

Wall, Michael E; Markowitz, David A; Rosner, Judah L; Martin, Robert G

2009-12-01

The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

Effects of serum albumin on the degradation and cytotoxicity of single-walled carbon nanotubes.

PubMed

Ding, Yun; Tian, Rong; Yang, Zhen; Chen, Jianfa; Lu, Naihao

2017-03-01

Neutrophil myeloperoxidase (MPO) and peroxynitrite (ONOO - ) can oxidatively biodegrade carboxylated single-walled carbon nanotubes (SWCNTs). The protein-SWCNTs interactions will play an important role in the degradation and cytotoxicity of nanotubes. Here, we investigated the binding of bovine serum albumin (BSA, a common and well-characterized model blood serum protein) to SWCNTs, and found that the hydrophobic and electrostatic interactions might be crucial factors in stabilizing the binding of SWCNTs with BSA. The binding of BSA could impair SWCNTs biodegradation in vitro through the competitive adsorption to nanotube. Both SWCNTs and BSA-SWCNTs were significantly degraded in zymosan-stimulated macrophages, and the degradation degree was more for BSA-SWCNTs. The mechanism for SWCNTs degradation in activated macrophages was further investigated to demonstrate the dominant participation of MPO and ONOO - -driven pathways. Moreover, binding of BSA to SWCNTs reduced cytotoxicity and degraded nanotubes induced less cytotoxicity than non-degraded nanotubes. The binding of BSA may be an important determinant for the biodegradation and cytotoxicity of SWCNTs in inflammatory cells, and therefore, provide a new route to mitigate the potential toxicity of nanotubes in future biomedical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Synergistic Interactions of Sugars/Polyols and Monovalent Salts with Phospholipids Depend upon Sugar/Polyol Complexity and Anion Identity.

PubMed

Clark, Ginevra A; Henderson, J Michael; Heffern, Charles; Akgün, Bülent; Majewski, Jaroslaw; Lee, Ka Yee C

2015-11-24

We found that interactions of dipalmitoylphosphatidylcholine (DPPC) lipid monolayers with sugars are influenced by addition of NaCl. This work is of general importance in understanding how sugar-lipid-salt interactions impact biological systems. Using Langmuir isothermal compressions, fluorescence microscopy, atomic force microscopy, and neutron reflectometry, we examined DPPC monolayers upon addition of sugars/polyols and/or monovalent salts. Sugar-lipid interactions in the presence of NaCl increased with increasing complexity of the sugar/polyol in the order glycerol ≪ glucose < trehalose. When the anion was altered in the series NaF, NaCl, and NaBr, only minor differences were observed. When comparing LiCl, NaCl, and KCl, sodium chloride had the greatest influence on glucose and trehalose interactions with DPPC. We propose that heterogeneity created by cation binding allows for sugars to bind the lipid headgroups. While cation binding increases in the order K(+) < Na(+) < Li(+), lithium ions may also compete with glucose for binding sites. Thus, both cooperative and competitive factors contribute to the overall influence of salts on sugar-lipid interactions.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos

PubMed Central

Kim, Young Ah; Lee, Eun-Hye; Kim, Kwang-Ok; Lee, Yong Tae; Hammock, Bruce D.; Lee, Hye-Sung

2014-01-01

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL−1, respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos. PMID:21504817

High glucose disrupts oligosaccharide recognition function via competitive inhibition: a potential mechanism for immune dysregulation in diabetes mellitus.

PubMed

Ilyas, Rebecca; Wallis, Russell; Soilleux, Elizabeth J; Townsend, Paul; Zehnder, Daniel; Tan, Bee K; Sim, Robert B; Lehnert, Hendrik; Randeva, Harpal S; Mitchell, Daniel A

2011-01-01

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose - sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose-binding lectin, soluble DC-SIGN and DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins. Copyright © 2010 Elsevier GmbH. All rights reserved.

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

PubMed

Cuozzo, John W; Centrella, Paolo A; Gikunju, Diana; Habeshian, Sevan; Hupp, Christopher D; Keefe, Anthony D; Sigel, Eric A; Soutter, Holly H; Thomson, Heather A; Zhang, Ying; Clark, Matthew A

2017-05-04

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Apo-states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain

PubMed Central

Findeisen, Felix; Rumpf, Christine; Minor, Daniel L.

2013-01-01

In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. PMID:23811053

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

PubMed Central

Ullers, Ronald S.; Houben, Edith N.G.; Raine, Amanda; ten Hagen-Jongman, Corinne M.; Ehrenberg, Måns; Brunner, Joseph; Oudega, Bauke; Harms, Nellie; Luirink, Joen

2003-01-01

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain. PMID:12756233

Identification and quantification of human kidney atrial natriuretic peptide receptors.

PubMed

Kahana, L; Yechiely, H; Mecz, Y; Lurie, A

1995-04-01

The present study determined 125I-label atrial natriuretic peptide (ANP) binding sites in human kidney glomerular and papillary membranes. The membranes were prepared from non-malignant renal tissue obtained at nephrectomy of patients with renal carcinoma. To evaluate the proportion of ANP receptor classes ANP-R1 (ANPR-A, -B) versus ANP-R2 (ANPR-C), competitive binding studies were performed using [125I]-ANP in the presence of increasing concentrations of ANP or an internally ring-deleted analog, des(Gln116, Ser117, Gly118, Leu119, Gly120)ANP(102-121), called C-ANP, which binds selectively to ANPR-C receptors. Analysis of the competitive binding curve with ANP in glomerular membranes suggested the presence of one group of high-affinity receptors with dissociation constant Kd = 26 +/- 12 pmol/l and density Bmax = 101 +/- 47 nmol/kg protein. A decrease of 10-30% in Bmax with no change in Kd was obtained in the presence of excess (10(-6) mol/l) C-ANP, suggesting the existence of a small amount of a second class of receptors, the ANPR-C class. The densities of ANPR-A, -B versus ANPR-C receptors in human glomeruli, calculated from competitive inhibition experiments, were 75 +/- 42 and 22 +/- 16 nmol/kg protein (N = 8). Autoradiography of the sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions showed two bands: a highly labeled 130kD band and a weakly labeled 66 kD band, both displaced by ANP. Only the 66-kD band was displaced by the C-ANP analog. Human papilla membrane, as shown by competition binding studies and SDS gel electrophoresis, presented only one class of receptors with Kd = 40 +/- 23 pmol/l (mean +/- SD, N = 3) and Bmax = 17 +/- 6.3 nmol/kg protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Developing tools for risk assessment in protected species: relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse

PubMed Central

Jensen, Brenda A.; Reddy, Christopher M.; Nelson, Robert K.; Hahn, Mark E.

2011-01-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC50s for competitive binding to beluga AHR was: TCDD

Developing tools for risk assessment in protected species: Relative potencies inferred from competitive binding of halogenated aromatic hydrocarbons to aryl hydrocarbon receptors from beluga (Delphinapterus leucas) and mouse.

PubMed

Jensen, Brenda A; Reddy, Christopher M; Nelson, Robert K; Hahn, Mark E

2010-11-01

Persistent organic pollutants such as halogenated aromatic hydrocarbons (HAHs) biomagnify in food webs and accumulate to high concentrations in top predators like odontocete cetaceans (toothed whales). The most toxic HAHs are the 2,3,7,8-substituted halogenated dibenzo-p-dioxins and furans, and non-ortho-substituted polychlorinated biphenyls (PCBs), which exert their effects via the aryl hydrocarbon receptor (AHR). Understanding the impact of HAHs in wildlife is limited by the lack of taxon-specific information about the relative potencies of toxicologically important congeners. To assess whether Toxic Equivalency Factors (TEFs) determined in rodents are predictive of HAH relative potencies in a cetacean, we used beluga and mouse AHRs expressed in vitro from cloned cDNAs to measure the relative AHR-binding affinities of ten HAHs from five different structural classes. The rank order of mean IC(50)s for competitive binding to beluga AHR was: TCDD

The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

PubMed

Fong, Clifford W

2016-08-01

Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of Protein G Microcolumns in Chromatographic Immunoassays: A Comparison of Competitive Binding Formats

PubMed Central

Pfaunmiller, Erika L.; Anguizola, Jeanethe A.; Milanuk, Mitchell L.; Carter, NaTasha; Hage, David S.

2016-01-01

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limits of detection, and analysis time. All these methods gave detection limits in the range of 8–19 ng/mL and precisions ranging from ± 5% to ± 10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest. PMID:26777776

Absence of C-type natriuretic peptide receptors in hamster glomeruli.

PubMed

Luk, J K; Wong, E F; Wong, N L

1994-01-01

The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

Analysis of the interactions between host factor Sam68 and viral elements during foot-and-mouth disease virus infection

USDA-ARS?s Scientific Manuscript database

The nuclear protein Src-associated protein of 68 kDa in mitosis (Sam68) is known to bind RNA and be involved in cellular processes triggered in response to environmental stresses, including virus infection. Interestingly, Sam68, is a multi-functional protein implicated in the life cycle of retroviru...

Reciprocal inhibition of p53 and nuclear factor-kappaB transcriptional activities determines cell survival or death in neurons.

PubMed

Culmsee, Carsten; Siewe, Jan; Junker, Vera; Retiounskaia, Marina; Schwarz, Stephanie; Camandola, Simonetta; El-Metainy, Shahira; Behnke, Hagen; Mattson, Mark P; Krieglstein, Josef

2003-09-17

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 precedes apoptosis in many cell types. Controversial reports exist on the role of the transcription factor nuclear factor-kappaB (NF-kappaB) in p53-mediated apoptosis, depending on the cell type and experimental conditions. Therefore, we sought to elucidate the role of NF-kappaB in p53-mediated neuron death. In cultured neurons DNA damaging compounds induced activation of p53, whereas NF-kappaB activity declined significantly. The p53 inhibitor pifithrin-alpha (PFT) preserved NF-kappaB activity and protected neurons against apoptosis. Immunoprecipitation experiments revealed enhanced p53 binding to the transcriptional cofactor p300 after induction of DNA damage, whereas binding of p300 to NF-kappaB was reduced. In contrast, PFT blocked the interaction of p53 with the cofactor, whereas NF-kappaB binding to p300 was enhanced. Most interestingly, similar results were observed after oxygen glucose deprivation in cultured neurons and in ischemic brain tissue. Ischemia-induced repression of NF-kappaB activity was prevented and brain damage was reduced by the p53 inhibitor PFT in a dose-dependent manner. It is concluded that a balanced competitive interaction of p53 and NF-kappaB with the transcriptional cofactor p300 exists in neurons. Exposure of neurons to lethal stress activates p53 and disrupts NF-kappaB binding to p300, thereby blocking NF-kappaB-mediated survival signaling. Inhibitors of p53 provide pronounced neuroprotective effects because they block p53-mediated induction of cell death and concomitantly enhance NF-kappaB-induced survival signaling.

Coagulation factor VII variants resistant to inhibitory antibodies.

PubMed

Branchini, Alessio; Baroni, Marcello; Pfeiffer, Caroline; Batorova, Angelika; Giansily-Blaizot, Muriel; Schved, Jean F; Mariani, Guglielmo; Bernardi, Francesco; Pinotti, Mirko

2014-11-01

Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

Handball Coaches’ Perceptions About the Value of Working Competences According to Their Coaching Background

PubMed Central

Mesquita, Isabel; Borges, Mario; Rosado, Antonio; Souza, Adriano De

2011-01-01

The purpose of this study was to analyze the value attributed to given working competences, by Portuguese handball coaches according to their coaching background, certification level, coaching experience, and level of education. A sample of 207 handball coaches responded to a questionnaire which included demographic characteristics and a scale focused on perceptions of the level of importance attributed to working competences. Data analysis included an exploratory factorial analysis applying Maximum Likelihood Factoring (MLF) and Oblimin rotation. These factors were submitted to a One-way ANOVA and Tukey’s post hoc multiple comparisons to analyse coaches’ perceptions according to their coaching background. A six factor solution was found where three major domains of competences were highlighted; the first one related to training and competition (e.g. planning and conducting the training, team administration in competition, annual and multi-annual planning, and coaching methodology); the second one related to social and cultural issues and management (e.g. implementation of youth sport development projects, team leadership and coach education) and the third one related to the cognitive background (meta-cognitive competences). The importance ascribed to some working competences was influenced by their coaching experience and certification level. Highly experienced and qualified coaches perceived competences of everyday practice, social, cultural and management issues related to training and competition as more important than the other coaches. This study suggests the need to consider some working competences, until now not explicitly present in the Portuguese coaching education curriculum which could enable coaches to choose the best way to practice/work in a manner that will foster and support their professional development. Key points Three major domains of competences were highlighted by Portuguese handball coaches. The first one related to training and competition, the second one related to social and cultural issues and management and the third one related to the cognitive background. The importance ascribed by Portuguese handball coaches to some working competences was influenced by their coaching experience and certification level, as high experienced coaches and coaches with higher certification levels perceived competences related to training and competition of the everyday practice and social, cultural issues and management as more important. The value attributed by Portuguese handball coaches to working competences did not vary according to the coaches’ academic education level. Portuguese handball coaches valued the meta-cognitive competences, the competences to implement sport development project and related to annual and multi-annual planning independently of their coaching background. PMID:24149314

Handball coaches' perceptions about the value of working competences according to their coaching background.

PubMed

Mesquita, Isabel; Borges, Mario; Rosado, Antonio; Souza, Adriano De

2011-01-01

The purpose of this study was to analyze the value attributed to given working competences, by Portuguese handball coaches according to their coaching background, certification level, coaching experience, and level of education. A sample of 207 handball coaches responded to a questionnaire which included demographic characteristics and a scale focused on perceptions of the level of importance attributed to working competences. Data analysis included an exploratory factorial analysis applying Maximum Likelihood Factoring (MLF) and Oblimin rotation. These factors were submitted to a One-way ANOVA and Tukey's post hoc multiple comparisons to analyse coaches' perceptions according to their coaching background. A six factor solution was found where three major domains of competences were highlighted; the first one related to training and competition (e.g. planning and conducting the training, team administration in competition, annual and multi-annual planning, and coaching methodology); the second one related to social and cultural issues and management (e.g. implementation of youth sport development projects, team leadership and coach education) and the third one related to the cognitive background (meta-cognitive competences). The importance ascribed to some working competences was influenced by their coaching experience and certification level. Highly experienced and qualified coaches perceived competences of everyday practice, social, cultural and management issues related to training and competition as more important than the other coaches. This study suggests the need to consider some working competences, until now not explicitly present in the Portuguese coaching education curriculum which could enable coaches to choose the best way to practice/work in a manner that will foster and support their professional development. Key pointsThree major domains of competences were highlighted by Portuguese handball coaches. The first one related to training and competition, the second one related to social and cultural issues and management and the third one related to the cognitive background.The importance ascribed by Portuguese handball coaches to some working competences was influenced by their coaching experience and certification level, as high experienced coaches and coaches with higher certification levels perceived competences related to training and competition of the everyday practice and social, cultural issues and management as more important.The value attributed by Portuguese handball coaches to working competences did not vary according to the coaches' academic education level.Portuguese handball coaches valued the meta-cognitive competences, the competences to implement sport development project and related to annual and multi-annual planning independently of their coaching background.

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

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

Skafar, D.F.

1991-11-12

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with ({sup 3}H)progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020. This indicated that a difference in the binding mechanism of RU486more » and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable. These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.« less

Differential transcriptional regulation by alternatively designed mechanisms: A mathematical modeling approach.

PubMed

Yildirim, Necmettin; Aktas, Mehmet Emin; Ozcan, Seyma Nur; Akbas, Esra; Ay, Ahmet

2017-01-01

Cells maintain cellular homeostasis employing different regulatory mechanisms to respond external stimuli. We study two groups of signal-dependent transcriptional regulatory mechanisms. In the first group, we assume that repressor and activator proteins compete for binding to the same regulatory site on DNA (competitive mechanisms). In the second group, they can bind to different regulatory regions in a noncompetitive fashion (noncompetitive mechanisms). For both competitive and noncompetitive mechanisms, we studied the gene expression dynamics by increasing the repressor or decreasing the activator abundance (inhibition mechanisms), or by decreasing the repressor or increasing the activator abundance (activation mechanisms). We employed delay differential equation models. Our simulation results show that the competitive and noncompetitive inhibition mechanisms exhibit comparable repression effectiveness. However, response time is fastest in the noncompetitive inhibition mechanism due to increased repressor abundance, and slowest in the competitive inhibition mechanism by increased repressor level. The competitive and noncompetitive inhibition mechanisms through decreased activator abundance show comparable and moderate response times, while the competitive and noncompetitive activation mechanisms by increased activator protein level display more effective and faster response. Our study exemplifies the importance of mathematical modeling and computer simulation in the analysis of gene expression dynamics.

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

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

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Side-chain conformational space analysis (SCSA): A multi conformation-based QSAR approach for modeling and prediction of protein-peptide binding affinities

NASA Astrophysics Data System (ADS)

Zhou, Peng; Chen, Xiang; Shang, Zhicai

2009-03-01

In this article, the concept of multi conformation-based quantitative structure-activity relationship (MCB-QSAR) is proposed, and based upon that, we describe a new approach called the side-chain conformational space analysis (SCSA) to model and predict protein-peptide binding affinities. In SCSA, multi-conformations (rather than traditional single-conformation) have received much attention, and the statistical average information on multi-conformations of side chains is determined using self-consistent mean field theory based upon side chain rotamer library. Thereby, enthalpy contributions (including electrostatic, steric, hydrophobic interaction and hydrogen bond) and conformational entropy effects to the binding are investigated in terms of occurrence probability of residue rotamers. Then, SCSA was applied into the dataset of 419 HLA-A*0201 binding peptides, and nonbonding contributions of each position in peptide ligands are well determined. For the peptides, the hydrogen bond and electrostatic interactions of the two ends are essential to the binding specificity, van der Waals and hydrophobic interactions of all the positions ensure strong binding affinity, and the loss of conformational entropy at anchor positions partially counteracts other favorable nonbonding effects.

Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter

PubMed Central

Agarwal, Sorabh

2018-01-01

Abstract Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (−794 CATT5–8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders. PMID:29186613

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

PubMed

Patil, Hemangi; Cho, Kyoung-in; Lee, James; Yang, Yi; Orry, Andrew; Ferreira, Paulo A

2013-03-27

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

PubMed

Drané, Pascal; Brault, Marie-Eve; Cui, Gaofeng; Meghani, Khyati; Chaubey, Shweta; Detappe, Alexandre; Parnandi, Nishita; He, Yizhou; Zheng, Xiao-Feng; Botuyan, Maria Victoria; Kalousi, Alkmini; Yewdell, William T; Münch, Christian; Harper, J Wade; Chaudhuri, Jayanta; Soutoglou, Evi; Mer, Georges; Chowdhury, Dipanjan

2017-03-09

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

Media Competition Implementation for the Massachusetts Childhood Obesity Research Demonstration Study (MA-CORD): Adoption and Reach

PubMed Central

Criss, Shaniece; Cheung, Lilian; Giles, Catherine; Gortmaker, Steven; Viswanath, Kasisomayajula; Kwass, Jo-Ann; Davison, Kirsten

2016-01-01

The Massachusetts Childhood Obesity Research Demonstration Study (MA-CORD) was a multi-level, multi-sector community intervention with a media competition component to provide an overarching synergy and promote awareness of target behaviors to reduce childhood obesity. Students participating in the media competition were tasked with developing videos, song/rap lyrics, and artwork that reflected the goals. The aim of this study is to document the process used to develop and implement the media competition along with its reach and adoption. An adapted version of Neta and colleagues’ 2015 framework on dissemination and implementation was used to summarize the process by which the media competition was developed and implemented. Adoption was defined by whether eligible schools or afterschool programs decided to implement the media competition. Reach was defined by student participation rates within schools/programs and the number of votes cast for the finalists on the coalition website and students’ paper ballots. A total of 595 students participated in the media competition from 18 school and afterschool programs in two communities. Adoption of the media competitions ranged from 22% to 100% in programs and reach ranged from 3% to 33% of the student population. The documentation of the implementation should contribute to the replication of the media competition. PMID:27058549

Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

PubMed

Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

2015-07-08

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Complex Transcriptional Control of the Antibiotic Regulator afsS in Streptomyces: PhoP and AfsR Are Overlapping, Competitive Activators▿

PubMed Central

Santos-Beneit, Fernando; Rodríguez-García, Antonio; Martín, Juan F.

2011-01-01

The afsS gene of several Streptomyces species encodes a small sigma factor-like protein that acts as an activator of several pathway-specific regulatory genes (e.g., actII-ORF4 and redD in Streptomyces coelicolor). The two pleiotropic regulators AfsR and PhoP bind to overlapping sequences in the −35 region of the afsS promoter and control its expression. Using mutated afsS promoters containing specific point mutations in the AfsR and PhoP binding sequences, we proved that the overlapping recognition sequences for AfsR and PhoP are displaced by 1 nucleotide. Different nucleotide positions are important for binding of AfsR or PhoP, as shown by electrophoretic mobility shift assays and by reporter studies using the luxAB gene coupled to the different promoters. Mutant promoter M5 (with a nucleotide change at position 5 of the consensus box) binds AfsR but not PhoP with high affinity (named “superAfsR”). Expression of the afsS gene from this promoter led to overproduction of actinorhodin. Mutant promoter M16 binds PhoP with extremely high affinity (“superPhoP”). Studies with ΔafsR and ΔphoP mutants (lacking AfsR and PhoP, respectively) showed that both global regulators are competitive transcriptional activators of afsS. AfsR has greater influence on expression of afsS than PhoP, as shown by reverse transcriptase PCR (RT-PCR) and promoter reporter (luciferase) studies. These two high-level regulators appear to integrate different nutritional signals (particularly phosphate limitation sensed by PhoR), S-adenosylmethionine, and other still unknown environmental signals (leading to AfsR phosphorylation) for the AfsS-mediated control of biosynthesis of secondary metabolites. PMID:21378195

‘Partial’ competition of heterobivalent ligand binding may be mistaken for allosteric interactions: a comparison of different target interaction models

PubMed Central

Vauquelin, Georges; Hall, David; Charlton, Steven J

2015-01-01

Background and Purpose Non-competitive drugs that confer allosteric modulation of orthosteric ligand binding are of increasing interest as therapeutic agents. Sought-after advantages include a ceiling level to drug effect and greater receptor-subtype selectivity. It is thus important to determine the mode of interaction of newly identified receptor ligands early in the drug discovery process and binding studies with labelled orthosteric ligands constitute a traditional approach for this. According to the general allosteric ternary complex model, allosteric ligands that exhibit negative cooperativity may generate distinctive ‘competition’ curves: they will not reach baseline levels and their nadir will increase in par with the orthosteric ligand concentration. This behaviour is often considered a key hallmark of allosteric interactions. Experimental Approach The present study is based on differential equation-based simulations. Key Results The differential equation-based simulations revealed that the same ‘competition binding’ pattern was also obtained when a monovalent ligand binds to one of the target sites of a heterobivalent ligand, even if this process is exempt of allosteric interactions. This pattern was not strictly reciprocal when the binding of each of the ligands was recorded. The prominence of this phenomenon may vary from one heterobivalent ligand to another and we suggest that this phenomenon may take place with ligands that have been proposed to bind according to ‘two-domain’ and ‘charnière’ models. Conclusions and Implications The present findings indicate a familiar experimental situation where bivalency may give rise to observations that could inadvertently be interpreted as allosteric binding. Yet, both mechanisms could be differentiated based on alternative experiments and structural considerations. PMID:25537684

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

PubMed Central

Bosson, Andrew D.; Zamudio, Jesse R.; Sharp, Phillip A.

2016-01-01

SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. PMID:25449132

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PubMed Central

1988-01-01

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

Selective binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid to peroxisome proliferator-activated receptor gamma allows ligand identification and characterization.

PubMed

Zorrilla, Silvia; Garzón, Beatriz; Pérez-Sala, Dolores

2010-04-01

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARalpha and retinoid X receptor alpha (RXRalpha). ANS binding is competed by PPARgamma agonists such as rosiglitazone, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), and 9,10-dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) (CAY10410). Moreover, the affinity of PPARgamma for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARgamma agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ(2) that does not activate PPAR in cells. We found that although this compound bound to PPARgamma with low affinity, it failed to promote PPARgamma interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARgamma binding and interactions may provide valuable strategies to fully understand the role of PPARgamma ligands. Copyright 2009 Elsevier Inc. All rights reserved.

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function.

PubMed

Xie, Y; Cohen, J B

2001-01-26

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Using dental care resources optimally: quality-efficiency trade-offs in a competitive private market.

PubMed

Prasad, Banuru Muralidhara; Varatharajan, D

2011-01-01

Modern lifestyle changes led to increased dental care needs in India. Consequently, there has been a sharp rise in dentist numbers. Karnataka state alone produces 2,500 dentists annually, who are engaged in the non-government sector owing to inadequate public sector opportunities. This article aims to assess Karnataka private dental clinic quality and efficiency. Dentists were interviewed using a close-ended, structured interview schedule and their clinics were assessed using a checklist adopted from guidelines for providing machinery and equipment under the National Oral Health Care Programme (NOHCP). Dental "hotel" and clinical quality were scored based on this checklist. Clinical quality was "excellent" in 12 per cent of clinics and poor in 49 per cent. Clinics with better infrastructure charged higher price (p < 0.05). Multi-chair clinics charging fixed rates were high (81 per cent). According to 59.5 per cent of dentists, competition did not improve quality while 27 per cent felt that competition increased price, not quality. About 30.9 per cent of the poor quality clinics, 41 per cent average quality clinics and 26 per cent good quality clinics were technically efficient. The multi chair clinics offered better quality at higher prices and single chair clinics provided poorer quality at lower prices. In other words, they had a sub-optimal price-quality mix. Therefore, there is a need to regulate price and quality in all clinics to arrive at an optimal price-quality mix so that clients are not overburdened financially even while receiving good quality dental care. The article advocates that resources are used optimally as a way to achieve value for money and to achieve break-even points thereby providing quality care in a competitive market. Factors that influence dental practitioner behaviour are evaluated.

Characterization of bradykinin receptors in human lung fibroblasts using the binding of 3[H][Des-Arg10,Leu9]kallidin and [3H]NPC17731.

PubMed

Zhang, S P; Codd, E E

1998-01-01

Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD > [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD > DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 > DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.

Copper(II) binding by dissolved organic matter: Importance of the copper-to-dissolved organic matter ratio and implications for the Biotic Ligand Model

USGS Publications Warehouse

Craven, Alison M.; Aiken, George R.; Ryan, Joseph N.

2012-01-01

The ratio of copper to dissolved organic matter (DOM) is known to affect the strength of copper binding by DOM, but previous methods to determine the Cu2+–DOM binding strength have generally not measured binding constants over the same Cu:DOM ratios. In this study, we used a competitive ligand exchange–solid-phase extraction (CLE-SPE) method to determine conditional stability constants for Cu2+–DOM binding at pH 6.6 and 0.01 M ionic strength over a range of Cu:DOM ratios that bridge the detection windows of copper-ion-selective electrode and voltammetry measurements. As the Cu:DOM ratio increased from 0.0005 to 0.1 mg of Cu/mg of DOM, the measured conditional binding constant (cKCuDOM) decreased from 1011.5 to 105.6 M–1. A comparison of the binding constants measured by CLE-SPE with those measured by copper-ion-selective electrode and voltammetry demonstrates that the Cu:DOM ratio is an important factor controlling Cu2+–DOM binding strength even for DOM isolates of different types and different sources and for whole water samples. The results were modeled with Visual MINTEQ and compared to results from the biotic ligand model (BLM). The BLM was found to over-estimate Cu2+ at low total copper concentrations and under-estimate Cu2+ at high total copper concentrations.

Antagonistic Function of the RNA-binding Protein HuR and miR-200b in Post-transcriptional Regulation of Vascular Endothelial Growth Factor-A Expression and Angiogenesis*

PubMed Central

Chang, Sung-Hee; Lu, Yi-Chien; Li, Xi; Hsieh, Wan-Ying; Xiong, Yuquan; Ghosh, Mallika; Evans, Todd; Elemento, Olivier; Hla, Timothy

2013-01-01

HuR, also known as Elavl1, is an RNA-binding protein that regulates embryonic development, progenitor cell survival, and cell stress responses. The role of HuR in angiogenesis is not known. Using a myeloid-specific HuR knock-out mouse model (Elavl1Mø KO), we show that HuR expression in bone marrow-derived macrophages (BMDMs) is needed to maintain the expression of genes enriched in AU-rich elements and U-rich elements in the 3′-UTR. In addition, BMDMs from Elavl1Mø KO mice also showed alterations in expression of several miRNAs. Interestingly, computational analysis suggested that miR-200b, which is up-regulated in Elavl1Mø KO BMDMs, interacts with myeloid mRNAs very close to the HuR binding sites, suggesting competitive regulation of gene expression. One such mRNA encodes vascular endothelial growth factor (VEGF)-A, a major regulator of angiogenesis. Immunoprecipitation of RNA-protein complexes and luciferase reporter assays indicate that HuR antagonizes the suppressive activity of miR-200b, down-regulates miR-200b expression, and promotes VEGF-A expression. Indeed, Vegf-a and other angiogenic regulatory transcripts were down-regulated in Elavl1Mø KO BMDMs. Interestingly, tumor growth, angiogenesis, vascular sprouting, branching, and permeability were significantly attenuated in Elavl1Mø KO mice, suggesting that HuR-regulated myeloid-derived factors modulate tumor angiogenesis in trans. Zebrafish embryos injected with an elavl1 morpholino oligomer or miR-200b mimic showed angiogenesis defects in the subintestinal vein plexus, and elavl1 mRNA rescued the repressive effect of miR-200b. In addition, miR-200b and HuR morpholino oligomer suppressed the activity of a zVEGF 3′-UTR luciferase reporter construct. Together, these studies reveal an evolutionarily conserved post-transcriptional mechanism involving competitive interactions between HuR and miR-200b that controls angiogenesis. PMID:23223443

Apo states of calmodulin and CaBP1 control CaV1 voltage-gated calcium channel function through direct competition for the IQ domain.

PubMed

Findeisen, Felix; Rumpf, Christine H; Minor, Daniel L

2013-09-09

In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation and limits calcium entry, whereas CaBP1 blocks calcium-dependent inactivation (CDI) and allows sustained calcium influx. Here, we combine isothermal titration calorimetry with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca(2+)/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium-binding properties. The observation that the apo forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Simulation of a model nanopore sensor: Ion competition underlies device behavior.

PubMed

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-28

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Simulation of a model nanopore sensor: Ion competition underlies device behavior

NASA Astrophysics Data System (ADS)

Mádai, Eszter; Valiskó, Mónika; Dallos, András; Boda, Dezső

2017-12-01

We study a model nanopore sensor with which a very low concentration of analyte molecules can be detected on the basis of the selective binding of the analyte molecules to the binding sites on the pore wall. The bound analyte ions partially replace the current-carrier cations in a thermodynamic competition. This competition depends both on the properties of the nanopore and the concentrations of the competing ions (through their chemical potentials). The output signal given by the device is the current reduction caused by the presence of the analyte ions. The concentration of the analyte ions can be determined through calibration curves. We model the binding site with the square-well potential and the electrolyte as charged hard spheres in an implicit background solvent. We study the system with a hybrid method in which we compute the ion flux with the Nernst-Planck (NP) equation coupled with the Local Equilibrium Monte Carlo (LEMC) simulation technique. The resulting NP+LEMC method is able to handle both strong ionic correlations inside the pore (including finite size of ions) and bulk concentrations as low as micromolar. We analyze the effect of bulk ion concentrations, pore parameters, binding site parameters, electrolyte properties, and voltage on the behavior of the device.

Effects of competitive red blood cell binding and reduced hematocrit on the blood and plasma levels of (/sup 14/C)Indapamide in the rat

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

Lettieri, J.T.; Portelli, S.T.

1983-02-01

The effects of chlorthalidone and acetazolamide on the red blood cell binding of indapamide were investigated. Both drugs caused a substantial decrease in the amount of indapamide bound to the erythrocytes in vitro. This effect was demonstrated by a change in the indapamide blood/plasma ratio from approximately 6 in control samples, to a value of 1 when either of the displacing agents was added. Coadministration of acetazolamide with /sup 14/C-labeled indapamide to rats, resulted in a 5-fold drop in the blood levels of total radioactivity, relative to rats dosed with (/sup 14/C)indapamide alone. Concomitantly, there was a 2-fold increase inmore » the plasma levels of total radioactivity after acetazolamide coadministration. In rats whose hematocrits had been reduced by extensive bleeding, there were only minor alterations in the blood/plasma partitioning of (/sup 14/C)indapamide. Thus, chlorthalidone and acetazolamide were able to displace indapamide from erythrocytes in vitro and in vivo, possibly by competition at a carbonic anhydrase binding site. The pharmacokinetics of drugs which are extensively bound to erythrocytes may be significantly altered by the presence of other agents capable of competitive binding.« less

Ikaros controls isotype selection during immunoglobulin class switch recombination.

PubMed

Sellars, MacLean; Reina-San-Martin, Bernardo; Kastner, Philippe; Chan, Susan

2009-05-11

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG(2b) and IgG(2a), and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the gamma2b and gamma2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of gamma2b and gamma2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

Structure-based design of competitive ligands to target Spon2 in gastric cancer: An integration of molecular modeling and in vitro assay.

PubMed

Xu, Zhenglei; Yu, Zhichao; Nai, Shumei; Shi, Ruiyue; Tang, Qinhong; Zhang, Haiyang; Ye, Lijuan; Wang, Lisheng; Hong, Yincai

2017-10-01

Spon2 is a proto-oncogene matrix protein that plays an essential role in the tumorigenesis and metastasis of gastric cancer. The protein has recently been found to function as a guanine nucleotide exchange factor through the activation of RhoGTPase. Here, computational modeling and bioinformatics analysis were employed to investigate the molecular mechanism and biological implication underlying Spon2 autoinhibition. It is revealed that the binding of PxxP motif to SH domain can stabilize the intramolecular interaction between the N-terminal helix and DH domain of Spon2, thus shifting the protein into an autoinhibitory state. Here, we proposed releasing Spon2 autoinhibition by targeting SH domain with competitive peptide ligands. To verify this notion, the PxxP sequence was adopted as the start to derive an array of efficient SH binders by using a structure-based rational design strategy, which were then substantiated with fluorescence spectroscopy analysis and guanine nucleotide exchange test. Consequently, the obtained peptide ligands were determined to have a moderate or high affinity for SH domain; they can also enhance Spon2 exchange activity by 1.2-6.1 folds, exhibiting a significant correlation with their SH-binding affinity (Pearson's coefficient=0.92). In addition, neutral substitution of conserved residues in a high-affinity peptide ligand can largely reduce its Spon2-activating potency, confirming that the designed peptide activates Spon2 by competitively disrupting SH-PxxP interaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

PubMed

Peterson, R C; Reich, M F; Dunn, P E; Law, J H; Katzenellnbogen, J A

1977-05-17

A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.

PubMed

Henry, Brian L; Connell, Justin; Liang, Aiye; Krishnasamy, Chandravel; Desai, Umesh R

2009-07-31

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

Theoretical study on mode competition between fundamental and second harmonic modes in a 0.42 THz gyrotron with gradually tapered complex cavity

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

Zhao, Qixiang, E-mail: zxqi1105@gmail.com; Yu, Sheng; Zhang, Tianzhong

2015-10-15

In this paper, the nonlinear dynamics of mode competition in the complex cavity gyrotron are studied by using multi-frequency, time-dependent theory with the cold-cavity longitudinal profile approximation. Based on the theory, a code is written to simulate the mode competition in the gradually tapered complex cavity gyrotron operating at second harmonic oscillation. The simulations tracking seven competition modes show that single mode oscillation of the desired mode TE{sub 17.4} at 150 kW level can be expected with proper choice of operating parameters. Through studying on mode competition, it is proved that the complex cavity has a good capability for suppressing themore » mode competition. Meanwhile, it is found that TE{sub 17.3} could be excited in the first cavity as a competition mode when the gyrotron operating at large beam current, which leads to that TE{sub 17.3} and TE{sub 17.4} with different frequencies can coexist stably in the complex cavity gyrotron with very close amplitudes. Thus, the complex cavity might be used for multi-frequency output gyrotron.« less

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus

PubMed Central

Gao, Xiang; Esseili, Malak A.; Lu, Zhongyan; Saif, Linda J.

2016-01-01

ABSTRACT Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. PMID:26969699

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

PubMed

Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

2016-05-15

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Competitive fluorescent pseudo-immunoassay exploiting molecularly imprinted polymers for the detection of biogenic amines in fish matrix.

PubMed

Mattsson, Leena; Xu, Jingjing; Preininger, Claudia; Tse Sum Bui, Bernadette; Haupt, Karsten

2018-05-01

We developed a competitive fluorescent molecularly imprinted polymer (MIP) assay to detect biogenic amines in fish samples. MIPs synthesized by precipitation polymerization using histamine as template were used in a batch binding assay analogous to competitive fluoroimmunoassays. Introducing a complex sample matrix, such as fish extract, into the assay changes the environment and the binding conditions, therefore the importance of the sample preparation is extensively discussed. Several extraction and purification methods for fish were comprehensively studied, and an optimal clean-up procedure for fish samples using liquid-liquid extraction was developed. The feasibility of the competitive MIP assay was shown in the purified fish extract over a broad histamine range (1 - 430µM). The MIP had the highest affinity towards histamine, but recognized also the structurally similar biogenic amines tyramine and tryptamine, as well as spermine and spermidine, providing simultaneous analysis and assessment of the total amount of biogenic amines. Copyright © 2018 Elsevier B.V. All rights reserved.

Characteristics and Concepts of Dynamic Hub Proteins in DNA Processing Machinery from Studies of RPA

PubMed Central

Sugitani, Norie; Chazin, Walter J.

2015-01-01

DNA replication, damage response and repair require the coordinated action of multi-domain proteins operating within dynamic multi-protein machines that act upon the DNA substrate. These modular proteins contain flexible linkers of various lengths, which enable changes in the spatial distribution of the globular domains (architecture) that harbor their essential biochemical functions. This mobile architecture is uniquely suited to follow the evolving substrate landscape present over the course of the specific process performed by the multi-protein machinery. A fundamental advance in understanding of protein machinery is the realization of the pervasive role of dynamics. Not only is the machine undergoing dynamic transformations, but the proteins themselves are flexible and constantly adapting to the progression through the steps of the overall process. Within this dynamic context the activity of the constituent proteins must be coordinated, a role typically played by hub proteins. A number of important characteristics of modular proteins and concepts about the operation of dynamic machinery have been discerned. These provide the underlying basis for the action of the machinery that reads DNA, and responds to and repairs DNA damage. Here, we introduce a number of key characteristics and concepts, including the modularity of the proteins, linkage of weak binding sites, direct competition between sites, and allostery, using the well recognized hub protein replication protein A (RPA). PMID:25542993

Product reformulation in the food system to improve food safety. Evaluation of policy interventions.

PubMed

Marotta, Giuseppe; Simeone, Mariarosaria; Nazzaro, Concetta

2014-03-01

The objective of this study is to understand the level of attention that the consumer awards to a balanced diet and to product ingredients, with a twofold purpose: to understand whether food product reformulation can generate a competitive advantage for companies that practice it and to evaluate the most appropriate policy interventions to promote a healthy diet. Reformulation strategy, in the absence of binding rules, could be generated by consumers. Results from qualitative research and from empirical analysis have shown that the question of health is a latent demand influenced by two main factors: a general lack of information, and the marketing strategies adopted by companies which bring about an increase in the information asymmetry between producers and consumers. In the absence of binding rules, it is therefore necessary that the government implement information campaigns (food education) aimed at increasing knowledge regarding the effects of unhealthy ingredients, in order to inform and improve consumer choice. It is only by means of widespread information campaigns that food product reformulation can become a strategic variable and allow companies to gain a competitive advantage. This may lead to virtuous results in terms of reducing the social costs related to an unhealthy diet. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

PubMed Central

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki

2014-01-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. PMID:24958104

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

PubMed Central

Presley, Tennille; Vedam, Kaushik; Liu, Xiaoping; Zweier, Jay L.

2009-01-01

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV. PMID:19412660

Adsorption of heavy metal from aqueous solution by dehydrated root powder of long-root Eichhornia crassipes.

PubMed

Li, Qiang; Chen, Bo; Lin, Peng; Zhou, Jiali; Zhan, Juhong; Shen, Qiuying; Pan, Xuejun

2016-01-01

The root powder of long-root Eichhornia crassipes, as a new kind of biodegradable adsorbent, has been tested for aqueous adsorption of Pb, Zn, Cu, and Cd. From FT-IR, we found that the absorption peaks of phosphorous compounds, carbonyl, and nitrogenous compounds displayed obvious changes before and after adsorption which illustrated that plant characteristics may play a role in binding with metals. Surface properties and morphology of the root powders have been characterized by means of SEM and BET. Energy spectrum analysis showed that the metals were adsorbed on root powders after adsorption. Then, optimum quantity of powder, pH values, and metal ion concentrations in single-system and multi-system were detected to discuss the characteristics and mechanisms of metal adsorption. Freundlich model and the second-order kinetics equation could well describe the adsorption of heavy metals in single-metal system. The adsorption of Pb, Zn, and Cd in the multi-metal system decreased with the concentration increased. At last, competitive adsorption of every two metals on root powder proved that Cu and Pb had suppressed the adsorption performance of Cd and Zn.

Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

PubMed

Okada, N; Liszewski, M K; Atkinson, J P; Caparon, M

1995-03-28

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

Mechanistic insights into the link between visfatin gene C-1535T polymorphism and coronary artery disease: an in vitro study.

PubMed

Wang, Yong-Sheng; Gao, Wei; Li, Hong-Fen; Wang, Ze-Mu; Zhu, Jun; Zhao, Huan; Yan, Jian-Jun; Jia, En-Zhi; Yang, Zhi-Jian; Wang, Lian-Sheng

2012-04-01

Visfatin, a pro-inflammatory cytokine predominantly released from leucocytes, is correlated with coronary artery disease (CAD). We have previously reported that the -1535C>T polymorphism (rs1330082), which located on the promoter region of visfatin, was associated with decreased risk of CAD. Here, we investigated the underlying mechanism by which this polymorphism affects the genetic susceptibility to CAD. The difference of the promoter activities between -1535T variant and -1535C allele was tested by luciferase reporter gene assay. The difference of transcription factor binding activities between T and C allele was evaluated by electrophoretic mobility shift assay. In reporter gene assay, we showed that the T variant had a significantly reduced transcriptional activity compared with the C allele. The T-variant significantly attenuated the promoter binding affinity to nuclear transcription factors and this effect became much obvious after treatment with TNF-α. Moreover, competition experiment revealed that the retarded complex formed by T-1535- or C-1535-probe binding to nuclear extracts was nearly completely inhibited by unlabeled activator protein-1 (AP-1) specific probe, indicating that AP-1 might be the target nuclear effector. Taken together, our data provided potential mechanistic link between the visfatin -1535C>T polymorphism and reduced CAD risk.

From non-covalent binding to irreversible DNA lesions: nile blue and nile red as photosensitizing agents

PubMed Central

Gattuso, Hugo; Besancenot, Vanessa; Grandemange, Stéphanie; Marazzi, Marco; Monari, Antonio

2016-01-01

We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue. PMID:27329409

Inhibition of HMGA2 binding to DNA by netropsin

PubMed Central

Miao, Yi; Cui, Tengjiao; Leng, Fenfei; Wilson, W. David

2008-01-01

The design of small synthetic molecules that can be used to affect gene expression is an area of active interest for development of agents in therapeutic and biotechnology applications. Many compounds that target the minor groove in AT sequences in DNA are well characterized and are promising reagents for use as modulators of protein-DNA complexes. The mammalian high mobility group transcriptional factor, HMGA2, also targets the DNA minor groove and plays critical roles in disease processes from cancer to obesity. Biosensor-surface plasmon resonance methods were used to monitor HMGA2 binding to target sites on immobilized DNA and a competition assay for inhibition of the HMGA2-DNA complex was designed. HMGA2 binds strongly to the DNA through AT hook domains with KD values of 20 - 30 nM depending on the DNA sequence. The well-characterized minor groove binder, netropsin, was used to develop and test the assay. The compound has two binding sites in the protein-DNA interaction sequence and this provides an advantage for inhibition. An equation for analysis of results when the inhibitor has two binding sites in the biopolymer recognition surface is presented with the results. The assay provides a platform for discovery of HMGA2 inhibitors. PMID:18023407

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

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

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with boundmore » ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.« less

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

PubMed Central

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

2017-01-01

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. PMID:28760974

Structural Transformation Detection Contributes to Screening of Behaviorally Active Compounds: Dynamic Binding Process Analysis of DhelOBP21 from Dastarcus helophoroides.

PubMed

Yang, Rui-Nan; Li, Dong-Zhen; Yu, Guangqiang; Yi, Shan-Cheng; Zhang, Yinan; Kong, De-Xin; Wang, Man-Qun

2017-12-01

In light of reverse chemical ecology, the fluorescence competitive binding assays of functional odorant binding proteins (OBPs) is a recent advanced approach for screening behaviorally active compounds of insects. Previous research on Dastareus helophoroides identified a minus-C OBP, DhelOBP21, which preferably binds to several ligands. In this study, only (+)-β-pinene proved attractive to unmated adult beetles. To obtain a more in-depth explanation of the lack of behavioral activity of other ligands we selected compounds with high (camphor) and low (β-caryophyllene) binding affinities. The structural transformation of OBPs was investigated using well-established approaches for studying binding processes, such as fluorescent quenching assays, circular dichroism, and molecular dynamics. The dynamic binding process revealed that the flexibility of DhelOBP21 seems conducive to binding specific ligands, as opposed to broad substrate binding. The compound (+)-β-pinene and DhelOBP21 formed a stable complex through a secondary structural transformation of DhelOBP21, in which its amino-terminus transformed from random coil to an α-helix to cover the binding pocket. On the other hand, camphor could not efficiently induce a stable structural transformation, and its high binding affinities were due to strong hydrogen-bonding, compromising the structure of the protein. The other compound, β-caryophyllene, only collided with DhelOBP21 and could not be positioned in the binding pocket. Studying structural transformation of these proteins through examining the dynamic binding process rather than using approaches that just measure binding affinities such as fluorescence competitive binding assays can provide a more efficient and reliable approach for screening behaviorally active compounds.

The A2iA French handwriting recognition system at the Rimes-ICDAR2011 competition

NASA Astrophysics Data System (ADS)

Menasri, Farès; Louradour, Jérôme; Bianne-Bernard, Anne-Laure; Kermorvant, Christopher

2012-01-01

This paper describes the system for the recognition of French handwriting submitted by A2iA to the competition organized at ICDAR2011 using the Rimes database. This system is composed of several recognizers based on three different recognition technologies, combined using a novel combination method. A framework multi-word recognition based on weighted finite state transducers is presented, using an explicit word segmentation, a combination of isolated word recognizers and a language model. The system was tested both for isolated word recognition and for multi-word line recognition and submitted to the RIMES-ICDAR2011 competition. This system outperformed all previously proposed systems on these tasks.

Development, Validation, and Application of a Novel Ligand-Binding Assay to Selectively Measure PEGylated Recombinant Human Coagulation Factor VIII (BAX 855).

PubMed

Weber, Alfred; Engelmaier, Andrea; Hainzelmayer, Sandra; Minibeck, Eva; Anderle, Heinz; Schwarz, Hans Peter; Turecek, Peter L

2015-10-21

BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.

1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

PubMed

Hsu, Shang-Te Danny; Cabrita, Lisa D; Christodoulou, John; Dobson, Christopher M

2009-06-01

The gelation factor from Dictyostelium discoideum (ABP-120) is an actin binding protein consisting of six immunoglobulin (Ig) domains in the C-terminal rod domain. We have recently used the pair of domains 5 and 6 of ABP-120 as a model system for studying multi-domain nascent chain folding on the ribosome. Here we present the NMR assignments of domain 5 in its native and 8M urea-denatured states.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

PubMed

Meza-Romero, Roberto; Benedek, Gil; Leng, Lin; Bucala, Richard; Vandenbark, Arthur A

2016-04-01

Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal alpha(1,2)fucose residues in the cecal mucosa.

PubMed

Chessa, Daniela; Winter, Maria G; Jakomin, Marcello; Bäumler, Andreas J

2009-02-01

The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc) or by pretreatment of cells with alpha1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galbeta1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucalpha1-2Galbeta1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucalpha1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an alpha1-2 fucosylated receptor(s) present in the cecal mucosa.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal α (1,2)fucose residues in the cecal mucosa

PubMed Central

Chessa, Daniela; Winter, Maria G.; Jakomin, Marcello; Bäumler, Andreas J.

2013-01-01

SUMMARY The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucα1-2Galβ1-4GlcNAc) or by pretreatment of cells with α1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galβ1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucα1-2Galβ1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucα1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an α1-2 fucosylated receptor(s) present in the cecal mucosa. PMID:19183274

The Intrinsically Disordered Regions of the Drosophila melanogaster Hox Protein Ultrabithorax Select Interacting Proteins Based on Partner Topology

PubMed Central

Hsiao, Hao-Ching; Gonzalez, Kim L.; Catanese, Daniel J.; Jordy, Kristopher E.; Matthews, Kathleen S.; Bondos, Sarah E.

2014-01-01

Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues. PMID:25286318

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Protonation linked equilibria and apparent affinity constants: the thermodynamic profile of the alpha-chymotrypsin-proflavin interaction.

PubMed

Bruylants, Gilles; Wintjens, René; Looze, Yvan; Redfield, Christina; Bartik, Kristin

2007-12-01

Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K(obs), DeltaH(obs)(0) ). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural information. We present here a detailed study, using NMR and ITC, of the interaction between alpha-chymotrypsin and one of its competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process. The possible driving forces of the interaction between alpha-chymotrypsin and proflavin are discussed in the light of the experimental data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study of binding processes involving protonation/deprotonation equilibria.

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

PubMed

Bong, Ji-Hong; Song, Hyun-Woo; Kim, Tae-Hun; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2018-04-15

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Adsorption of Cd, Cu and Zn from aqueous solutions onto ferronickel slag under different potentially toxic metal combination.

PubMed

Park, Jong-Hwan; Kim, Seong-Heon; Kang, Se-Won; Kang, Byung-Hwa; Cho, Ju-Sik; Heo, Jong-Soo; Delaune, Ronald D; Ok, Yong Sik; Seo, Dong-Cheol

2016-01-01

Adsorption characteristics of potentially toxic metals in single- and multi-metal forms onto ferronickel slag were evaluated. Competitive sorption of metals by ferronickel slag has never been reported previously. The maximum adsorption capacities of toxic metals on ferronickel were in the order of Cd (10.2 mg g(-1)) > Cu (8.4 mg g(-1)) > Zn (4.4 mg g(-1)) in the single-metal adsorption isotherm and Cu (6.1 mg g(-1)) > Cd (2.3 mg g(-1)) > Zn (0.3 mg g(-1)) in the multi-metal adsorption isotherm. In comparison with single-metal adsorption isotherm, the reduction rates of maximum toxic metal adsorption capacity in the multi-metal adsorption isotherm were in the following order of Zn (93%) > Cd (78%) > Cu (27%). The Freundlich isotherm provides a slightly better fit than the Langmuir isotherm equation using ferronickel slag for potentially toxic metal adsorption. Multi-metal adsorption behaviors differed from single-metal adsorption due to competition, based on data obtained from Freundlich and Langmuir adsorption models and three-dimensional simulation. Especially, Cd and Zn were easily exchanged and substituted by Cu during multi-metal adsorption. Further competitive adsorption studies are necessary in order to accurately estimate adsorption capacity of ferronickel slag for potentially toxic metals in natural environments.

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces.

PubMed

Sanders, Michael R; Clifton, Luke A; Neylon, Cameron; Frazier, Richard A; Green, Rebecca J

2013-07-17

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. This study examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b, and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers using a combination of surface pressure measurements, external reflection FTIR spectroscopy, and neutron reflectometry. Results highlighted differences in the protein binding mechanisms and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

PubMed Central

Pfister, Klaus; Radosevich, Steven R.; Arntzen, Charles J.

1979-01-01

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H2O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (14C-radiolabel assays) with binding constants (K) of 1.4 × 10−8 molar and 4 × 10−8 molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10−8 molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts. We conclude that triazine resistance of both intact plants and isolated chloroplasts of Senecio vulgaris L. is based upon a minor modification of the protein in the photosystem II complex which is responsible for herbicide binding. This change results in a specific loss of atrazine (triazine)-binding capacity. PMID:16661120

International Validation of Two Human Recombinant Estrogen Receptor (ERa) Binding Assays

EPA Science Inventory

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay...

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

Application of multi-factorial design of experiments to successfully optimize immunoassays for robust measurements of therapeutic proteins.

PubMed

Ray, Chad A; Patel, Vimal; Shih, Judy; Macaraeg, Chris; Wu, Yuling; Thway, Theingi; Ma, Mark; Lee, Jean W; Desilva, Binodh

2009-02-20

Developing a process that generates robust immunoassays that can be used to support studies with tight timelines is a common challenge for bioanalytical laboratories. Design of experiments (DOEs) is a tool that has been used by many industries for the purpose of optimizing processes. The approach is capable of identifying critical factors and their interactions with a minimal number of experiments. The challenge for implementing this tool in the bioanalytical laboratory is to develop a user-friendly approach that scientists can understand and apply. We have successfully addressed these challenges by eliminating the screening design, introducing automation, and applying a simple mathematical approach for the output parameter. A modified central composite design (CCD) was applied to three ligand binding assays. The intra-plate factors selected were coating, detection antibody concentration, and streptavidin-HRP concentrations. The inter-plate factors included incubation times for each step. The objective was to maximize the logS/B (S/B) of the low standard to the blank. The maximum desirable conditions were determined using JMP 7.0. To verify the validity of the predictions, the logS/B prediction was compared against the observed logS/B during pre-study validation experiments. The three assays were optimized using the multi-factorial DOE. The total error for all three methods was less than 20% which indicated method robustness. DOE identified interactions in one of the methods. The model predictions for logS/B were within 25% of the observed pre-study validation values for all methods tested. The comparison between the CCD and hybrid screening design yielded comparable parameter estimates. The user-friendly design enables effective application of multi-factorial DOE to optimize ligand binding assays for therapeutic proteins. The approach allows for identification of interactions between factors, consistency in optimal parameter determination, and reduced method development time.

Comprehensive comparative analysis and identification of RNA-binding protein domains: multi-class classification and feature selection.

PubMed

Jahandideh, Samad; Srinivasasainagendra, Vinodh; Zhi, Degui

2012-11-07

RNA-protein interaction plays an important role in various cellular processes, such as protein synthesis, gene regulation, post-transcriptional gene regulation, alternative splicing, and infections by RNA viruses. In this study, using Gene Ontology Annotated (GOA) and Structural Classification of Proteins (SCOP) databases an automatic procedure was designed to capture structurally solved RNA-binding protein domains in different subclasses. Subsequently, we applied tuned multi-class SVM (TMCSVM), Random Forest (RF), and multi-class ℓ1/ℓq-regularized logistic regression (MCRLR) for analysis and classifying RNA-binding protein domains based on a comprehensive set of sequence and structural features. In this study, we compared prediction accuracy of three different state-of-the-art predictor methods. From our results, TMCSVM outperforms the other methods and suggests the potential of TMCSVM as a useful tool for facilitating the multi-class prediction of RNA-binding protein domains. On the other hand, MCRLR by elucidating importance of features for their contribution in predictive accuracy of RNA-binding protein domains subclasses, helps us to provide some biological insights into the roles of sequences and structures in protein-RNA interactions.

Periplasmic binding protein-based detection of maltose using liposomes: a new class of biorecognition elements in competitive assays.

PubMed

Edwards, Katie A; Baeumner, Antje J

2013-03-05

A periplasmic binding protein (PBP) was investigated as a novel binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10(-4) M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these proteins' two globular domains upon target binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose binding protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the protein. MBP conjugated fluorescent dye-encapsulating liposomes served to provide recognition and signal amplification in a competitive assay for maltose using amylose magnetic beads in a microtiter plate-based format. The development of appropriate binding buffers and competitive surfaces are described, with general observations expected to extend to PBPs for other analytes. The resulting assay was specific for d-(+)-maltose versus other sugar analogs including d-(+)-raffinose, sucrose, d-trehalose, d-(+)-xylose, d-fructose, 1-thio-β-d-glucose sodium salt, d-(+)-galactose, sorbitol, glycerol, and dextrose. Cross-reactivity with d-lactose and d-(+)-glucose occurred only at concentrations >10(4)-fold greater than d-(+)-maltose. The limit of detection was 78 nM with a dynamic range covering over 3 orders of magnitude. Accurate detection of maltose as an active ingredient in a pharmaceutical preparation was demonstrated. This method offers a significant improvement over existing enzymatic detection approaches that cannot discriminate between maltose and glucose and over existing fluorescence resonance energy transfer (FRET)-based detection methods that are sensitivity limited. In addition, it opens up a new strategy for the development of biosensors to difficult analytes refractory to immunological detection.

p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

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

Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

1990-08-01

The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

Changing Faces of Transcriptional Regulation Reflected by Zic3

PubMed Central

Winata, Cecilia Lanny; Kondrychyn, Igor; Deddens, J.C.; Korzh, Vladimir

2015-01-01

The advent of genomics in the study of developmental mechanisms has brought a trove of information on gene datasets and regulation during development, where the Zic family of zinc-finger proteins plays an important role. Genomic analysis of the modes of action of Zic3 in pluripotent cells demonstrated its requirement for maintenance of stem cells pluripotency upon binding to the proximal regulatory regions (promoters) of genes associated with cell pluripotency (Nanog, Sox2, Oct4, etc.) as well as cell cycle, proliferation, oncogenesis and early embryogenesis. In contrast, during gastrulation and neurulation Zic3 acts by binding the distal regulatory regions (enhancers, etc) associated with control of gene transcription in the Nodal and Wnt signaling pathways, including genes that act to break body symmetry. This illustrates a general role of Zic3 as a transcriptional regulator that acts not only alone, but in many instances in conjunction with other transcription factors. The latter is done by binding to adjacent sites in the context of multi-transcription factor complexes associated with regulatory elements. PMID:26085810

Multi-scale theoretical investigation of hydrogen storage in covalent organic frameworks.

PubMed

Tylianakis, Emmanuel; Klontzas, Emmanouel; Froudakis, George E

2011-03-01

The quest for efficient hydrogen storage materials has been the limiting step towards the commercialization of hydrogen as an energy carrier and has attracted a lot of attention from the scientific community. Sophisticated multi-scale theoretical techniques have been considered as a valuable tool for the prediction of materials storage properties. Such techniques have also been used for the investigation of hydrogen storage in a novel category of porous materials known as Covalent Organic Frameworks (COFs). These framework materials are consisted of light elements and are characterized by exceptional physicochemical properties such as large surface areas and pore volumes. Combinations of ab initio, Molecular Dynamics (MD) and Grand Canonical Monte-Carlo (GCMC) calculations have been performed to investigate the hydrogen adsorption in these ultra-light materials. The purpose of the present review is to summarize the theoretical hydrogen storage studies that have been published after the discovery of COFs. Experimental and theoretical studies have proven that COFs have comparable or better hydrogen storage abilities than other competitive materials such as MOF. The key factors that can lead to the improvement of the hydrogen storage properties of COFs are highlighted, accompanied with some recently presented theoretical multi-scale studies concerning these factors.

Community-level cohesion without cooperation.

PubMed

Tikhonov, Mikhail

2016-06-16

Recent work draws attention to community-community encounters ('coalescence') as likely an important factor shaping natural ecosystems. This work builds on MacArthur's classic model of competitive coexistence to investigate such community-level competition in a minimal theoretical setting. It is shown that the ability of a species to survive a coalescence event is best predicted by a community-level 'fitness' of its native community rather than the intrinsic performance of the species itself. The model presented here allows formalizing a macroscopic perspective whereby a community harboring organisms at varying abundances becomes equivalent to a single organism expressing genes at different levels. While most natural communities do not satisfy the strict criteria of multicellularity developed by multi-level selection theory, the effective cohesion described here is a generic consequence of resource partitioning, requires no cooperative interactions, and can be expected to be widespread in microbial ecosystems.

Competitive-Cooperative Automated Reasoning from Distributed and Multiple Source of Data

NASA Astrophysics Data System (ADS)

Fard, Amin Milani

Knowledge extraction from distributed database systems, have been investigated during past decade in order to analyze billions of information records. In this work a competitive deduction approach in a heterogeneous data grid environment is proposed using classic data mining and statistical methods. By applying a game theory concept in a multi-agent model, we tried to design a policy for hierarchical knowledge discovery and inference fusion. To show the system run, a sample multi-expert system has also been developed.

Addition of multiple limiting resources reduces grassland diversity.

PubMed

Harpole, W Stanley; Sullivan, Lauren L; Lind, Eric M; Firn, Jennifer; Adler, Peter B; Borer, Elizabeth T; Chase, Jonathan; Fay, Philip A; Hautier, Yann; Hillebrand, Helmut; MacDougall, Andrew S; Seabloom, Eric W; Williams, Ryan; Bakker, Jonathan D; Cadotte, Marc W; Chaneton, Enrique J; Chu, Chengjin; Cleland, Elsa E; D'Antonio, Carla; Davies, Kendi F; Gruner, Daniel S; Hagenah, Nicole; Kirkman, Kevin; Knops, Johannes M H; La Pierre, Kimberly J; McCulley, Rebecca L; Moore, Joslin L; Morgan, John W; Prober, Suzanne M; Risch, Anita C; Schuetz, Martin; Stevens, Carly J; Wragg, Peter D

2016-09-01

Niche dimensionality provides a general theoretical explanation for biodiversity-more niches, defined by more limiting factors, allow for more ways that species can coexist. Because plant species compete for the same set of limiting resources, theory predicts that addition of a limiting resource eliminates potential trade-offs, reducing the number of species that can coexist. Multiple nutrient limitation of plant production is common and therefore fertilization may reduce diversity by reducing the number or dimensionality of belowground limiting factors. At the same time, nutrient addition, by increasing biomass, should ultimately shift competition from belowground nutrients towards a one-dimensional competitive trade-off for light. Here we show that plant species diversity decreased when a greater number of limiting nutrients were added across 45 grassland sites from a multi-continent experimental network. The number of added nutrients predicted diversity loss, even after controlling for effects of plant biomass, and even where biomass production was not nutrient-limited. We found that elevated resource supply reduced niche dimensionality and diversity and increased both productivity and compositional turnover. Our results point to the importance of understanding dimensionality in ecological systems that are undergoing diversity loss in response to multiple global change factors.

Psychometric properties of the Spanish version of the Controlling Coach Behaviors Scale in the sport context.

PubMed

Castillo, Isabel; Tomás, Inés; Ntoumanis, Nikos; Bartholomew, Kimberley; Duda, Joan L; Balaguer, Isabel

2014-01-01

The purpose of this research was to translate into Spanish and examine the psychometric properties of the Spanish version of the Controlling Coach Behaviors Scale (CCBS) in male soccer players. The CCBS is a questionnaire designed to assess athletes' perceptions of sports coaches' controlling interpersonal style from the perspective of the self-determination theory. Study 1 tested the factorial structure of the translated scale using confirmatory factor analysis (CFA) and provided evidence of discriminant validity. Studies 2 and 3 examined the invariance across time and across competitive level via multi-sample CFA. Reliability analyses were also conducted. The CFA results revealed that a four-factor model was acceptable, indicating that a controlling interpersonal style is a multidimensional construct represented by four separate and related controlling coaching strategies. Further, results supported the invariance of the CCBS factor structure across time and competitive level and provided support for the internal consistency of the scale. Overall, the CCBS demonstrated adequate internal consistency, as well as good factorial validity. The Spanish version of the CCBS represents a valid and reliable adaptation of the instrument, which can be confidently used to measure soccer players' perceptions of their coaches' controlling interpersonal style.

Mechanical Unfolding Studies on Single-Domain SUMO and Multi-Domain Periplasmic Binding Proteins

NASA Astrophysics Data System (ADS)

Kotamarthi, Hema Chandra; Ainavarapu, Sri Rama Koti

Protein mechanics is a key component of many cellular and sub-cellular processes. The current review focuses on recent studies from our laboratory that probe the effect of sequence on the mechanical stability of structurally similar proteins and the unfolding mechanisms of multi-domain periplasmic binding proteins. Ubiquitin and small ubiquitin-related modifiers (SUMOs) are structurally similar and possess different mechanical stabilities, ubiquitin being stronger than SUMOs as revealed from their unfolding forces. These differences are plausibly due to the variation in number of inter-residue contacts. The unfolding potential widths determined from the pulling speed-dependent studies revealed that SUMOs are mechanically more flexible than ubiquitin. This flexibility of SUMOs plays a role in ligand binding and our single-molecule studies on SUMO interaction with SUMO binding motifs (SBMs) have shown that ligand binding decreases the SUMO flexibility and increases its mechanical stability. Studies on multi-domain periplasmic binding proteins have revealed that the unfolding energy landscape of these proteins is complex and they follow kinetic partitioning between two-state and multiple three-state pathways.

Epigenetic priors for identifying active transcription factor binding sites.

PubMed

Cuellar-Partida, Gabriel; Buske, Fabian A; McLeay, Robert C; Whitington, Tom; Noble, William Stafford; Bailey, Timothy L

2012-01-01

Accurate knowledge of the genome-wide binding of transcription factors in a particular cell type or under a particular condition is necessary for understanding transcriptional regulation. Using epigenetic data such as histone modification and DNase I, accessibility data has been shown to improve motif-based in silico methods for predicting such binding, but this approach has not yet been fully explored. We describe a probabilistic method for combining one or more tracks of epigenetic data with a standard DNA sequence motif model to improve our ability to identify active transcription factor binding sites (TFBSs). We convert each data type into a position-specific probabilistic prior and combine these priors with a traditional probabilistic motif model to compute a log-posterior odds score. Our experiments, using histone modifications H3K4me1, H3K4me3, H3K9ac and H3K27ac, as well as DNase I sensitivity, show conclusively that the log-posterior odds score consistently outperforms a simple binary filter based on the same data. We also show that our approach performs competitively with a more complex method, CENTIPEDE, and suggest that the relative simplicity of the log-posterior odds scoring method makes it an appealing and very general method for identifying functional TFBSs on the basis of DNA and epigenetic evidence. FIMO, part of the MEME Suite software toolkit, now supports log-posterior odds scoring using position-specific priors for motif search. A web server and source code are available at http://meme.nbcr.net. Utilities for creating priors are at http://research.imb.uq.edu.au/t.bailey/SD/Cuellar2011. t.bailey@uq.edu.au Supplementary data are available at Bioinformatics online.

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2010-01-01

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species

EPA Science Inventory

ABSTRACT In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human (hERα), quai...

Identification and characterization of cell-specific enhancer elements for the mouse ETF/Tead2 gene.

PubMed

Tanoue, Y; Yasunami, M; Suzuki, K; Ohkubo, H

2001-12-21

We have identified and characterized by transient transfection assays the cell-specific 117-bp enhancer sequence in the first intron of the mouse ETF (Embryonic TEA domain-containing factor)/Tead2 gene required for transcriptional activation in ETF/Tead2 gene-expressing cells, such as P19 cells. The 117-bp enhancer contains one GC-rich sequence (5'-GGGGCGGGG-3'), termed the GC box, and two tandemly repeated GA-rich sequences (5'-GGGGGAGGGG-3'), termed the proximal and distal GA elements. Further analyses, including transfection studies and electrophoretic mobility shift assays using a series of deletion and mutation constructs, indicated that Sp1, a putative activator, may be required to predominate over its competition with another unknown putative repressor, termed the GA element-binding factor, for binding to both the GC box, which overlapped with the proximal GA element, and the distal GA element in the 117-bp sequence in order to achieve a full enhancer activity. We also discuss a possible mechanism underlying the cell-specific enhancer activity of the 117-bp sequence.

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of σ-factor activity by proteolysis

PubMed Central

Jaiswal, Ravi K.; Prabha, Tangirala Surya; Manjeera, Gowravaram; Gopal, Balasubramanian

2013-01-01

The relative levels of different σ factors dictate the expression profile of a bacterium. Extracytoplasmic function σ factors synchronize the transcriptional profile with environmental conditions. The cellular concentration of free extracytoplasmic function σ factors is regulated by the localization of this protein in a σ/anti-σ complex. Anti-σ factors are multi-domain proteins with a receptor to sense environmental stimuli and a conserved anti-σ domain (ASD) that binds a σ factor. Here we describe the structure of Mycobacterium tuberculosis anti-σD (RsdA) in complex with the -35 promoter binding domain of σD (σD4). We note distinct conformational features that enable the release of σD by the selective proteolysis of the ASD in RsdA. The structural and biochemical features of the σD/RsdA complex provide a basis to reconcile diverse regulatory mechanisms that govern σ/anti-σ interactions despite high overall structural similarity. Multiple regulatory mechanisms embedded in an ASD scaffold thus provide an elegant route to rapidly re-engineer the expression profile of a bacterium in response to an environmental stimulus. PMID:23314154

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

PubMed Central

Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H.; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

2015-01-01

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

The effects of pargyline and 2-phenylethylamine on D1-like dopamine receptor binding.

PubMed

Berry, Mark D

2011-07-01

2-Phenylethylamine (PE) potentiates neuronal responses to dopamine by an unknown post-synaptic mechanism. Here, whether PE modifies D1-like receptor binding was examined. An unexpected effect of the monoamine oxidase inhibitor pargyline was observed, which did not involve competition for ligand binding. PE did not affect ligand binding in the presence or absence of pargyline. It is concluded that the effect of pargyline does not involve elevation of endogenous PE, and PE effects on dopaminergic neurotransmission are not due to altered D1-like receptor binding.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the β2-adrenoceptor

PubMed Central

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

2010-01-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')- , (R,S')-, and (S,R')-fenoterol, to the β2-adrenergic receptor (β2-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human β2-AR. Competitive displacement studies using [3H]CGP-12177 as the marker ligand were conducted at 4°, 15°, 25°, 30° and 37°C, the binding affinities calculated and the standard enthalpic (ΔH°) and standard entropic (ΔS°) contribution to the standard free energy change (ΔG°) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the β2-AR, and, therefore, the results of this study are inconsistent with the previously described “thermodynamic agonist-antagonist discrimination”, in which the binding of an agonist to the β-AR is entropy-driven and the binding of an antagonist is enthalpy driven. In addition, the data demonstrate that the chirality of the carbon atom containing the β-hydroxyl group of the fenoterol molecule (the β-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the β-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. PMID:20144591

The effect of stereochemistry on the thermodynamic characteristics of the binding of fenoterol stereoisomers to the beta(2)-adrenoceptor.

PubMed

Jozwiak, Krzysztof; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

2010-06-01

The binding thermodynamics of the stereoisomers of fenoterol, (R,R')-, (S,S')-, (R,S')-, and (S,R')-fenoterol, to the beta(2)-adrenergic receptor (beta(2)-AR) have been determined. The experiments utilized membranes obtained from HEK cells stably transfected with cDNA encoding human beta(2)-AR. Competitive displacement studies using [(3)H]CGP-12177 as the marker ligand were conducted at 4, 15, 25, 30 and 37 degrees C, the binding affinities calculated and the standard enthalpic (DeltaH degrees ) and standard entropic (DeltaS degrees ) contribution to the standard free energy change (DeltaG degrees ) associated with the binding process determined through the construction of van't Hoff plots. The results indicate that the binding of (S,S')- and (S,R')-fenoterol were predominately enthalpy-driven processes while the binding of (R,R')- and (R,S')-fenoterol were entropy-driven. All of the fenoterol stereoisomers are full agonists of the beta(2)-AR, and, therefore, the results of this study are inconsistent with the previously described "thermodynamic agonist-antagonist discrimination", in which the binding of an agonist to the beta-AR is entropy-driven and the binding of an antagonist is enthalpy-driven. In addition, the data demonstrate that the chirality of the carbon atom containing the beta-hydroxyl group of the fenoterol molecule (the beta-OH carbon) is a key factor in the determination of whether the binding process will be enthalpy-driven or entropy-driven. When the configuration at the beta-OH carbon is S the binding process is enthalpy-driven while the R configuration produces an entropy-driven process. Published by Elsevier Inc.

A hybrid multi-objective imperialist competitive algorithm and Monte Carlo method for robust safety design of a rail vehicle

NASA Astrophysics Data System (ADS)

Nejlaoui, Mohamed; Houidi, Ajmi; Affi, Zouhaier; Romdhane, Lotfi

2017-10-01

This paper deals with the robust safety design optimization of a rail vehicle system moving in short radius curved tracks. A combined multi-objective imperialist competitive algorithm and Monte Carlo method is developed and used for the robust multi-objective optimization of the rail vehicle system. This robust optimization of rail vehicle safety considers simultaneously the derailment angle and its standard deviation where the design parameters uncertainties are considered. The obtained results showed that the robust design reduces significantly the sensitivity of the rail vehicle safety to the design parameters uncertainties compared to the determinist one and to the literature results.

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

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

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

PubMed

Batsuli, Glaivy; Deng, Wei; Healey, John F; Parker, Ernest T; Baldwin, W Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete; Meeks, Shannon L

2016-10-20

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. © 2016 by The American Society of Hematology.

Competitive Adsorption and Oxidation Behavior of Heavy Metals on nZVI Coated with TEOS.

PubMed

Eglal, Mahmoud M; Ramamurthy, Amruthur S

2015-11-01

Zero valent iron nanoparticle (nanofer ZVI) is a powerful substance due to its coating with tetraethyl orthosilicate (TEOS). Tetraethyl orthosilicate imparts higher reactivity and decreases particle agglomeration. The competitive removal and displacement of multi-metals are influenced by time, pH, and initial concentration, the presence and properties of competing metals ion in the solution. For both the isotherm and kinetic studies performed for multi-metal removal experiments, compared to Pb II and Cd II, Cu II experienced a higher removal rate during the initial 5 minutes. After 120 minutes, all metals achieved removal efficiency in the range of 95 to 99%. The results of single and competitive kinetic tests for all three metals during the initial 5 minutes indicated that the presence of other metals generally reduce removal efficiency of metals. Both kinetic test and electron dispersive spectroscope (EDS) studies found that Cu II gets removed faster than the other metals. Pseudo-second order behavior was noted for the multi-metal removal systems.

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

PubMed

Frégeau, Chantal J; De Moors, Anick

2012-09-01

The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

PubMed

Swamy, N; Ghosh, S; Schneider, G B; Ray, R

2001-01-01

Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal conversion of DBP to DBP-maf requires the selective removal of galactose and sialic acid from the third domain of the protein. Hence, the differential effects of the two recombinant forms of DBP-maf is most likely related to glycosylation; E. coli expressed recombinant DBP is non-glycosylated, whereas the baculovirus expressed form is glycosylated. These data support the essential role of glycosylation for the osteoclast activating property of DBP-maf. Copyright 2001 Wiley-Liss, Inc.

Transcriptional analysis reveals the critical role of RNA polymerase-binding transcription factor, DksA, in regulating multi-drug resistance of Escherichia coli.

PubMed

Wang, Jiawei; Cao, Li; Yang, Xiaowen; Wu, Qingmin; Lu, Lin; Wang, Zhen

2018-05-07

The objective of this study was to comprehensively identify the target genes regulated by the RNA polymerase-binding transcription factor DksA in Escherichia coli, and to clarify the role of DksA in multi-drug resistance. A clinical E. coli strain, E8, was selected to construct the dksA gene deletion mutant by using the Red recombination system. The minimum inhibitory concentrations (MICs) of 12 antibiotics in the E8ΔdksA (mutant) were markedly lower than those in the wild-type strain, E8. Genes differentially expressed in the wild-type and dksA mutant were detected using RNA-Seq and were validated by performing quantitative real-time PCR (qRT-PCR). In total, 168 differentially expressed genes were identified in E8ΔdksA, including 81 up-regulated and 87 down-regulated genes. Many of the genes identified are involved in metabolism, two-component systems, transcriptional regulators, and transport/membrane proteins. Interestingly, genes encoding the transcriptional regulator, MarR, which is known to repress the multiple drug resistance operon, marRAB; MdfA, a transport protein that exhibits multidrug efflux activities; oligopeptide transport system proteins OppA and OppD were among those differentially expressed, and could potentially contribute to the increased drug susceptibility of E8ΔdksA. In conclusion, DksA plays an important role in the multi-drug resistance of this E. coli strain, and directly or indirectly regulates the expression of several genes related to antibiotic resistance. Copyright © 2018. Published by Elsevier B.V.

An affinity adsorption media that mimics heparan sulfate proteoglycans for the treatment of drug-resistant bacteremia

NASA Astrophysics Data System (ADS)

McCrea, Keith R.; Ward, Robert S.

2016-06-01

Removal of several drug-resistant bacteria from blood by affinity adsorption onto a heparin-functional media is reported. Heparin is a chemical analogue of heparan sulfate (HS) proteoglycans, found on transmembrane proteins of endothelial cells. Many blood-borne human pathogens, including bacteria, viruses, parasites, and fungi have been reported to target HS as an initial step in their pathogenesis. Here, we demonstrate the binding and removal of Methicillin-resistant Staphylococcus aureus (MRSA), Extended-Spectrum Betalactamase Klebsiella pneumoniae (ESBL), and two Carbapenem-resistant Enterobacteriaceae (both CRE Escherichia coli and CRE K. pneumoniae) using 300 μm polyethylene beads surface modified with end-point-attached heparin. Depending on the specific bacteria, the amount removed ranged between 39% (ESBL) and 99.9% (CRE). The total amount of bacteria adsorbed ranged between 2.8 × 105 and 8.6 × 105 colony forming units (CFU) per gram of adsorption media. Based on a polymicrobial challenge which showed no competitive binding, MRSA and CRE apparently utilize different binding sequences on the immobilized heparin ligand. Since the total circulating bacterial load during bacteremia seldom exceeds 5 × 105 CFUs, it appears possible to significantly reduce bacterial concentration in infected patients by multi-pass recirculation of their blood through a small extracorporeal affinity filter containing the heparin-functional adsorption media. This 'dialysis-like therapy' is expected to improve patient outcomes and reduce the cost of care, particularly when there are no anti-infective drugs available to treat the infection.

Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

PubMed Central

Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

1995-01-01

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered. PMID:7707507

Application of multi-tone mask technology in photolithographic fabrication of color filter components in LCD

NASA Astrophysics Data System (ADS)

Takada, Yoshihiro; Fukui, Matoko; Sai, Tsunehiro

2008-11-01

Recent progresses in the photoresists and photolithography for LCD industry applications have been primarily driven by the following two factors: advancement in the material performances (high resolution, high contrast ratio, low dielectric constant) for higher display quality, and cost reduction in the fabrication process. Along with crucial demand for cost competitiveness by improving production efficiency, environmental consciousness has been a major priority at fabrication process design to minimize the amount of waste produced. Having said the above, integration of two or more fabrication processes into a single process by using multi-tone mask technology has been the interest of research, due to its obvious advantage of reducing fabrication processes and cost. For example, multi-tone mask technology application has been widely employed on the TFT side to reduce the different types of photomasks being used. Similar trend has been employed on the CF side as well, where application of multi-tone mask technology is being investigated to integrate fabrication of multiple CF micro-components into a single process. In this presentation, we demonstrate a new approach of fabricating photospacer and peripheral CF components (MVA protrusion, sub-photospacers) in a single integrated process through multi-tone mask technology.

SAR by Oxime-Containing Peptide Libraries: Application to Tsg101 Ligand Optimization

PubMed Central

Liu, Fa; Stephen, Andrew G.; Waheed, Abdul A.; Aman, M. Javad; Freed, Eric O.; Fisher, Robert J.; Burke, Terrence R.

2008-01-01

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro (“PTAP”) motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15–20-fold enhancement in binding affinity was achieved by this approach. PMID:18655064

Characterization of a DNA damage-recognition protein mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

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

Donahue, B.A.; Augot, M.; Bellon, S.F.

1990-06-19

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, K{sub d}, of the protein from cisplatin-modified DNA was estimated to be (1-20) {times} 10{sup {minus}10} M. Protein binding is selective for DNAmore » modified with cisplatin, (Pt(en)Cl{sub 2}) (en, ethylenediamine), and (Pt(dach)Cl{sub 2}) (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating (Pt(dien)Cl)Cl (dien, diethylenetriamine) complexes. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl{sub 2}, CdCl{sub 2}, CoCl{sub 2}, or ZnCl{sub 2} and with 1 mM HgCl{sub 2}. This protein, alone or in conjunction with other cellular constituents, could be of general importance in the initial stages of processing of mammalian DNA damaged by cisplatin or other genotoxic agents and may belong to a wider class of such cellular damage-recognition proteins (DRPs).« less

Hepatitis B viral X protein interacts with tumor suppressor adenomatous polyposis coli to activate Wnt/β-catenin signaling.

PubMed

Hsieh, Antony; Kim, Hyeon-Seop; Lim, Seung-Oe; Yu, Dae-Yeul; Jung, Guhung

2011-01-28

HBV X protein is a transactivator of several cellular signaling pathways including Wnt which contributes to HBV associated neoplasia. The Wnt/β-catenin pathway is associated with HCC-initiating cells. Here we perform a functional screen for host factors involved in the transactivational properties of HBx. We identify adenomatous polyposis coli (APC) as a binding partner of HBx and further determine that HBx competitively binds APC to displace β-catenin from its degradation complex. This results in β-catenin upregulation in the nucleus and the activation of Wnt signaling. We show that Wnt inhibitors curcumin and quercetin target downstream β-catenin activity and effectively repress HBx-mediated regulation of c-MYC and E-cadherin. Our results provide a pathological mechanism of HBx induced malignant transformation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: A case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract.

PubMed

Wang, Zhiqiang; Kwon, Shin Hwa; Hwang, Seung Hwan; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

2017-03-24

The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD>30% and SBD>10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4',5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein. Copyright © 2017 Elsevier B.V. All rights reserved.

Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

PubMed

Du, W; Aloyo, V J; Harvey, J A

1997-10-03

Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

The E. coli Anti-Sigma Factor Rsd: Studies on the Specificity and Regulation of Its Expression

PubMed Central

Hofmann, Nina; Wurm, Reinhild; Wagner, Rolf

2011-01-01

Background Among the seven different sigma factors in E. coli σ70 has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting σ70-dependent transcription at the onset of stationary growth. Although binding of Rsd to σ70 has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. Methodology/Principal Findings We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with σ70, with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with σ38, the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and σ38- or σ70-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of σ70-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned out to be important for efficient rsd transcription. Conclusions/Significance The results contribute to a better understanding of the intricate mechanism of Rsd-mediated sigma factor specificity changes during stationary phase. PMID:21573101

The E. coli anti-sigma factor Rsd: studies on the specificity and regulation of its expression.

PubMed

Hofmann, Nina; Wurm, Reinhild; Wagner, Rolf

2011-05-06

Among the seven different sigma factors in E. coli σ(70) has the highest concentration and affinity for the core RNA polymerase. The E. coli protein Rsd is regarded as an anti-sigma factor, inhibiting σ(70)-dependent transcription at the onset of stationary growth. Although binding of Rsd to σ(70) has been shown and numerous structural studies on Rsd have been performed the detailed mechanism of action is still unknown. We have performed studies to unravel the function and regulation of Rsd expression in vitro and in vivo. Cross-linking and affinity binding revealed that Rsd is able to interact with σ(70), with the core enzyme of RNA polymerase and is able to form dimers in solution. Unexpectedly, we find that Rsd does also interact with σ(38), the stationary phase-specific sigma factor. This interaction was further corroborated by gel retardation and footprinting studies with different promoter fragments and σ(38)- or σ(70)-containing RNA polymerase in presence of Rsd. Under competitive in vitro transcription conditions, in presence of both sigma factors, a selective inhibition of σ(70)-dependent transcription was prevailing, however. Analysis of rsd expression revealed that the nucleoid-associated proteins H-NS and FIS, StpA and LRP bind to the regulatory region of the rsd promoters. Furthermore, the major promoter P2 was shown to be down-regulated in vivo by RpoS, the stationary phase-specific sigma factor and the transcription factor DksA, while induction of the stringent control enhanced rsd promoter activity. Most notably, the dam-dependent methylation of a cluster of GATC sites turned out to be important for efficient rsd transcription. The results contribute to a better understanding of the intricate mechanism of Rsd-mediated sigma factor specificity changes during stationary phase.

Effect of buffer at nanoscale molecular recognition interfaces - electrostatic binding of biological polyanions.

PubMed

Rodrigo, Ana C; Laurini, Erik; Vieira, Vânia M P; Pricl, Sabrina; Smith, David K

2017-10-19

We investigate the impact of an over-looked component on molecular recognition in water-buffer. The binding of a cationic dye to biological polyanion heparin is shown by isothermal calorimetry to depend on buffer (Tris-HCl > HEPES > PBS). The heparin binding of self-assembled multivalent (SAMul) cationic micelles is even more buffer dependent. Multivalent electrostatic molecular recognition is buffer dependent as a result of competitive interactions between the cationic binding interface and anions present in the buffer.

Cooperative action in eukaryotic gene regulation: Physical properties of a viral example

NASA Astrophysics Data System (ADS)

Werner, Maria; Zhu, Lizhe; Aurell, Erik

2007-12-01

The Epstein-Barr virus (EBV) infects more than 90% of the human population, and causes glandular fever as well as several more serious diseases. It is a tumor virus, and has been widely studied as a model system for cell transformation in humans. A central feature of the EBV life cycle is its ability to persist in human B cells in different latency states, denoted latency I, II, and III. In latency III the host cell is driven to cell proliferation and hence expansion of the viral population without entering the lytic pathway, while the latency I state is almost completely dormant. We here study the effective cooperativity of the viral C promoter, active in latency III EBV cell lines. We show that the unusually large number of binding sites of two competing transcription factors, one viral and one from the host, serves to make the switch sharper (higher Hill coefficient), either by cooperative binding between molecules of the same species when they bind, or by competition between the two species if there is sufficient steric hindrance.

Adsorption, Desorption, and Displacement Kinetics of H2O and CO2 on Forsterite, Mg2SiO4(011)

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

Smith, R. Scott; Li, Zhenjun; Dohnalek, Zdenek

We have examined the adsorbate-substrate interaction kinetics of CO2 and H2O on a natural forsterite crystal surface, Mg2SiO4(011), with 10-15% of substitutional Fe2+. We use temperature programmed desorption (TPD) and molecular beam techniques to determine the adsorption, desorption, and displacement kinetics for H2O and CO2. Neither CO2 nor H2O has distinct sub-monolayer desorption peaks but instead both have a broad continuous desorption feature that evolve smoothly into multilayer desorption. Inversion of the monolayer coverage spectra for both molecules reveals that the corresponding binding energies for H2O are greater than that for CO2 on all sites. The relative strength of thesemore » interactions is the dominant factor in the competitive adsorption/displacement kinetics. In experiments where the two adsorbates are co-dosed, H2O always binds to the highest energy binding sites available and displaces CO2. The onset of CO2 displacement by H2O occurs between 65 and 75 K.« less

The multi-zinc finger protein ZNF217 contacts DNA through a two-finger domain.

PubMed

Nunez, Noelia; Clifton, Molly M K; Funnell, Alister P W; Artuz, Crisbel; Hallal, Samantha; Quinlan, Kate G R; Font, Josep; Vandevenne, Marylène; Setiyaputra, Surya; Pearson, Richard C M; Mackay, Joel P; Crossley, Merlin

2011-11-04

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

The Multi-zinc Finger Protein ZNF217 Contacts DNA through a Two-finger Domain*

PubMed Central

Nunez, Noelia; Clifton, Molly M. K.; Funnell, Alister P. W.; Artuz, Crisbel; Hallal, Samantha; Quinlan, Kate G. R.; Font, Josep; Vandevenne, Marylène; Setiyaputra, Surya; Pearson, Richard C. M.; Mackay, Joel P.; Crossley, Merlin

2011-01-01

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif. PMID:21908891

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Long noncoding RNA PVT1 enhances the viability and invasion of papillary thyroid carcinoma cells by functioning as ceRNA of microRNA-30a through mediating expression of insulin like growth factor 1 receptor.

PubMed

Feng, Kun; Liu, Yu; Xu, Li-Juan; Zhao, Ling-Fei; Jia, Chao-Wen; Xu, Ming-Yan

2018-08-01

Invasion and metastasis of papillary thyroid carcinoma (PTC) significantly affects prognosis and quality of life of patients. Herein, we explored the binding relationship of long noncoding RNA PVT1 as ceRNA to microRNA-30a (miR-30a), and their effect on the development of PTC through regulating insulin like growth factor 1 receptor (IGF1R). PTC and adjacent normal tissues were collected, where the qRT-PCR and western blot assay were employed to evaluate the expression levels of PVT1, miR-30a and IGF1R. The correlation between PVT1 expression and clinicopathological characteristics of PTC patients was observed. PTC cell lines with the most/least significant difference from normal thyroid cells were selected and treated with siRNA PVT1 or overexpression PVT1 plasmids, miR-30a mimics or miR-30a inhibitors. Nucleus and cytoplasm segmentation was used to identify subcellular fractionation of PVT1. The binding relationship of PVT1 to miR-30a and the targeting relationship of miR-30a to IGF1R were confirmed by using bioinformatic prediction program, dual-luciferase reporter gene assay and RNA-pull down. Cell viability, cell cycle and apoptosis, invasion and migration capacities were assessed by MTT, flow cytometry, Transwell assay and scratch test, respectively. Western blot assay was employed to examine protein expression of IGF1R, apoptosis-related factors (caspase-3, cleaved capase-3) and epithelial-mesenchymal transition (EMT)-related factors (E-cadherin, Vimentin). In the PTC tissues and cells, PVT1 and IGF1R were highly expressed and miR-30a was poorly expressed. PVT1 exerted its effects on PTC mainly in the cytoplasm. The PVT1 expression was correlated with TNM staging, LNM and tumor infiltration of PTC. The competitive binding of PVT1 to miR-30a enhanced expression of IGF1R. In the in vitro experiments, BCPAP and TPC-1 cells were selected. When subjected to siRNA PVT1 or miR-30a mimics, BCPAP and TPC-1 cells exhibited inhibited proliferation, cell cycle progression, invasion, migration, EMT (increased E-cadherin and reduced Vimentin) and promoted apoptosis (reduced caspase-3 and increased cleaved capase-3), and moreover, the expression of IGF1R was reduced. This study provides evidence that long noncoding RNA PVT1 enhances the expression of IGF1R through competitive binding to miR-30a, whereby PVT1 facilitates the development of PTC. Copyright © 2018. Published by Elsevier Masson SAS.

Sodium and potassium competition in potassium-selective and non-selective channels

NASA Astrophysics Data System (ADS)

Sauer, David B.; Zeng, Weizhong; Canty, John; Lam, Yeeling; Jiang, Youxing

2013-11-01

Potassium channels selectively conduct K+, primarily to the exclusion of Na+, despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na+ and K+ binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K+ occupancy within the filter upon Na+ competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K+-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K+ sites. A double-barrier mechanism is proposed to explain K+ channel selectivity at low K+ concentrations.

Optimizing bi-objective, multi-echelon supply chain model using particle swarm intelligence algorithm

NASA Astrophysics Data System (ADS)

Sathish Kumar, V. R.; Anbuudayasankar, S. P.; Rameshkumar, K.

2018-02-01

In the current globalized scenario, business organizations are more dependent on cost effective supply chain to enhance profitability and better handle competition. Demand uncertainty is an important factor in success or failure of a supply chain. An efficient supply chain limits the stock held at all echelons to the extent of avoiding a stock-out situation. In this paper, a three echelon supply chain model consisting of supplier, manufacturing plant and market is developed and the same is optimized using particle swarm intelligence algorithm.

Verifying the competition between haloperidol and biperiden in serum albumin through a model based on spectrofluorimetry

NASA Astrophysics Data System (ADS)

Muniz da Silva Fragoso, Viviane; Patrícia de Morais e Coura, Carla; Paulino, Erica Tex; Valdez, Ethel Celene Narvaez; Silva, Dilson; Cortez, Celia Martins

2017-11-01

The aim of this work was to apply mathematical-computational modeling to study the interactions of haloperidol (HLP) and biperiden (BPD) with human (HSA) and bovine (BSA) serum albumin in order to verify the competition of these drugs for binding sites in HSA, using intrinsic tryptophan fluorescence quenching data. The association constants estimated for HPD-HSA was 2.17(±0.05) × 107 M-1, BPD-HSA was 2.01(±0.03) × 108 M-1 at 37 °C. Results have shown that drugs do not compete for the same binding sites in albumin.

A competitive binding model predicts the response of mammalian olfactory receptors to mixtures

NASA Astrophysics Data System (ADS)

Singh, Vijay; Murphy, Nicolle; Mainland, Joel; Balasubramanian, Vijay

Most natural odors are complex mixtures of many odorants, but due to the large number of possible mixtures only a small fraction can be studied experimentally. To get a realistic understanding of the olfactory system we need methods to predict responses to complex mixtures from single odorant responses. Focusing on mammalian olfactory receptors (ORs in mouse and human), we propose a simple biophysical model for odor-receptor interactions where only one odor molecule can bind to a receptor at a time. The resulting competition for occupancy of the receptor accounts for the experimentally observed nonlinear mixture responses. We first fit a dose-response relationship to individual odor responses and then use those parameters in a competitive binding model to predict mixture responses. With no additional parameters, the model predicts responses of 15 (of 18 tested) receptors to within 10 - 30 % of the observed values, for mixtures with 2, 3 and 12 odorants chosen from a panel of 30. Extensions of our basic model with odorant interactions lead to additional nonlinearities observed in mixture response like suppression, cooperativity, and overshadowing. Our model provides a systematic framework for characterizing and parameterizing such mixing nonlinearities from mixture response data.

Aminoglycoside antibiotics as a tool for the study of the biological role of calcium ions. Historical overview.

PubMed

Corrado, A P; de Morais, I P; Prado, W A

1989-01-01

Beginning with the pioneering work of Vital-Brazil and Corrado (1957), which suggested a possible interaction between aminoglycoside antibiotics (AGA) and calcium ions at the neuromuscular junction, the authors review the studies that demonstrated the existence of a competitive antagonism between AGA and calcium ions. In view of the low liposolubility of AGA and their inability to cross biological membranes, this antagonism seems to occur exclusively at calcium-binding sites at the level of the outer opening of calcium channels of the N-subtype, which are also the sites of interaction of omega-conotoxin. Being highly water soluble, AGA are easily removed from their binding sites with a consequent rapid reversal of their effects, a factor of primary importance to explain their wide use as tools in the pharmacological analysis of the study of the biological role of calcium ion on the membrane's outer surface. This use has advantages over the use of inorganic di- and trivalent cations such as Mg2+, Mn2+, Cd2+, Ni2+, La3+, etc., since the latter, though they are considered to be the most specific competitive antagonists of calcium ions, may induce biphasic effects due to their ability to cross the membranes and replace calcium and/or increase intracellular calcium concentration. The performance of AGA is also superior when compared with the so-called "specific" organic calcium antagonists--verapamil and nifedipine derivatives--since the latter, in addition to inducing possible biphasic effects, antagonize calcium in a non-competitive manner. Finally, the authors remark that AGA-Ca2+ antagonism relevance is not limited only to basic aspects and that it may have therapeutic implications since it provides alternatives for reducing the toxic adverse effects of this important group of antibiotics.

Hapten syntheses and antibody generation for the development of a polybrominated flame retardant ELISA.

PubMed

Shelver, Weilin L; Keum, Young-Soo; Kim, Hee-Joo; Rutherford, Drew; Hakk, Heldur H; Bergman, Ake; Li, Qing X

2005-05-18

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are increasingly an environmental concern. Several antibodies were developed for the polybrominated diphenyl ether flame retardant BDE-47 (1), often found in the highest concentration in human milk, plasma, and adipose tissue. Four haptens with different bromine and linker substitution patterns were synthesized and utilized to generate five polyclonal antibodies from goats and two polyclonal antibodies from rabbits. Competition was assessed using four different coating antigens for all seven antibodies. The coating antigen showed marked effects on competition. When the same hapten was used for antibody and the coating antigen less competition was observed. The effect of BDE structure on competition was evaluated by using BDE-47 (1), BDE-99 (2), BDE-100 (3), BDE-153 (4), and BDE-183 (5). None of the compounds showed high competition with antibody I-KLH, presumably because steric hindrance prevented formation of an efficient binding site. As predicted from structural considerations, BDE-47 (1) competed well with the remaining antibodies, whereas BDE-100 (3) competed well with only II-KLH. The remaining congeners (BDE-99 (2), BDE-153 (4), and BDE-183 (5)) contain bromines that cannot be positioned in binding sites and thus cross-react poorly. The competition study demonstrated that a bromine substitution on the congener could occupy a position analogous to the linker's position.

Towards understanding the epidemiology of Neisseria meningitidis in the African meningitis belt: a multi-disciplinary overview.

PubMed

Agier, Lydiane; Martiny, Nadège; Thiongane, Oumy; Mueller, Judith E; Paireau, Juliette; Watkins, Eleanor R; Irving, Tom J; Koutangni, Thibaut; Broutin, Hélène

2017-01-01

Neisseria meningitidis is the major cause of seasonal meningitis epidemics in the African meningitis belt. In the changing context of a reduction in incidence of serogroup A and an increase in incidence of serogroups W and C and of Streptococcus pneumoniae, a better understanding of the determinants driving the disease transmission dynamics remains crucial to improving bacterial meningitis control. The literature was searched to provide a multi-disciplinary overview of the determinants of meningitis transmission dynamics in the African meningitis belt. Seasonal hyperendemicity is likely predominantly caused by increased invasion rates, sporadic localized epidemics by increased transmission rates, and larger pluri-annual epidemic waves by changing population immunity. Carriage likely involves competition for colonization and cross-immunity. The duration of immunity likely depends on the acquisition type. Major risk factors include dust and low humidity, and presumably human contact rates and co-infections; social studies highlighted environmental and dietary factors, with supernatural explanations. Efforts should focus on implementing multi-country, longitudinal seroprevalence and epidemiological studies, validating immune markers of protection, and improving surveillance, including more systematic molecular characterizations of the bacteria. Integrating climate and social factors into disease control strategies represents a high priority for optimizing the public health response and anticipating the geographic evolution of the African meningitis belt. Copyright © 2016. Published by Elsevier Ltd.

Multi-age-grouping paradigm for young swimmers.

PubMed

Kojima, Kosuke; Jamison, Paul L; Stager, Joel M

2012-01-01

The purpose of this study was to examine the adequacy of "multi-age" classification systems in youth sports with a specific focus on the unisex multi-age-groupings used by USA Swimming. In addition, we offer an analytical rationale for the multi-age-groupings and potential alternatives. We examined the top 100 US swim performances for three years (2005, 2006, and 2007) for girls and boys in 15 age-groups (7 to 20 years and a singular group of 21 years and older). Data for each age and sex were pooled over the three years and means were calculated for each of seven competitive swim events. Swim times differed among each age up to the 14-year age-group in girls (F (14,30885) = 183.9, P < 0.01, Cohen's d = 1.19-3.72, large effect) and 16-year age-group in boys (F (14,30885) = 308.7, P < 0.01, Cohen's d = 0.81-3.64, large effect) for all events. Age-related differences in swim times continued later in boys than girls likely due to differences between the sexes in timing of growth and maturation. Because of the differences in swim performance in contemporary multi-age-groups, stratifying swimmers by a single age is the best means to ensure competitive fairness and equality, although there is no rationale for swimmers under the age of 8 years to compete in separate unisex competitive groups.

Relative Chemical Binding Affinities for Trout and Human Estrogen Receptor Using Different Competitive Binding Assays

EPA Science Inventory

Rainbow trout-based assays for estrogenicity are currently being used for development of predictive models based upon quantitative structure activity relationships. A predictive model based on a single species raises the question of whether this information is valid for other spe...

CONSTANTS FOR MERCURY BINDING BY DISSOLVED ORGANIC MATTER ISOLATES FROM THE FLORIDA EVERGLADES. (R827653)

EPA Science Inventory

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand appro...

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors.

PubMed

Nongonierma, Alice B; Mooney, Catherine; Shields, Denis C; FitzGerald, Richard J

2014-07-01

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure-activity relationship modeling, rather than on docking, in computationally selecting peptides for screening. Copyright © 2014 Elsevier Inc. All rights reserved.

The investigation of the binding of 6-mercaptopurine to site I on human serum albumin.

PubMed

Sochacka, Jolanta; Baran, Wojciech

2012-12-01

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.

A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

PubMed Central

Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

2013-01-01

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

Tomato ASR1 abrogates the response to abscisic acid and glucose in Arabidopsis by competing with ABI4 for DNA binding.

PubMed

Shkolnik, Doron; Bar-Zvi, Dudy

2008-05-01

The manipulation of transacting factors is commonly used to achieve a wide change in the expression of a large number of genes in transgenic plants as a result of a change in the expression of a single gene product. This is mostly achieved by the overexpression of transactivator or repressor proteins. In this study, it is demonstrated that the overexpression of an exogenous DNA-binding protein can be used to compete with the expression of an endogenous transcription factor sharing the same DNA-binding sequence. Arabidopsis was transformed with cDNA encoding tomato abscisic acid stress ripening 1 (ASR1), a sequence-specific DNA protein that has no orthologues in the Arabidopsis genome. ASR1-overexpressing (ASR1-OE) plants display an abscisic acid-insensitive 4 (abi4) phenotype: seed germination is not sensitive to inhibition by abscisic acid (ABA), glucose, NaCl and paclobutrazol. ASR1 binds coupling element 1 (CE1), a cis-acting element bound by the ABI4 transcription factor, located in the ABI4-regulated promoters, including that of the ABI4 gene. Chromatin immunoprecipitation demonstrates that ASR1 is bound in vivo to the promoter of the ABI4 gene in ASR1-OE plants, but not to promoters of genes known to be regulated by the transcription factors ABI3 or ABI5. Real-time polymerase chain reaction (PCR) analysis confirmed that the expression of ABI4 and ABI4-regulated genes is markedly reduced in ASR1-OE plants. Therefore, it is concluded that the abi4 phenotype of ASR1-OE plants is the result of competition between the foreign ASR1 and the endogenous ABI4 on specific promoter DNA sequences. The biotechnological advantage of using this approach in crop plants from the Brassicaceae family to reduce the transactivation activity of ABI4 is discussed.

In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125I Radiolabeling

PubMed Central

Chakroun, Maissa

2014-01-01

Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the 125I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops. PMID:25002420

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

NASA Technical Reports Server (NTRS)

Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

1996-01-01

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

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

Middleton, R.E.; Cohen, J.B.

1991-07-16

The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

PubMed

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

The action of stress hormones on the structure and function of erythrocyte membrane.

PubMed

Mokrushnikov, Pavel V; Panin, Lev E; Zaitsev, Boris N

2015-07-01

The action of a mixture of hormones (cortisol and adrenaline) on erythrocyte membrane during their binding was investigated. Changes in the membrane structure were elucidated by atomic force microscopy; microviscosity of the lipid bilayer and changes in the activity of Na(+),K(+)-ATPase at different concentrations of the hormones in erythrocyte suspension were estimated by the fluorescence method. Cortisol and adrenaline were shown to compete for the binding sites. A hormone that managed to bind nonspecifically to the membrane hindered the binding of another hormone. In a mixture of these hormones, cortisol won a competition for the binding sites; therewith, microviscosity of the membranes increased by 25%, which corresponds to a change in microviscosity produced by the action of cortisol alone. The competitive relationships affected also the Na(+),K(+)-ATPase activity, which was indicated by appearance of the second maximum of enzyme activity. It is assumed that an increase in microviscosity of erythrocyte membrane first raises the Na(+),K(+)-ATPase activity due to a growth of the maximum energy of membrane phonons, and then decreases the activity due to hindering of conformational transitions in the enzyme molecule.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

DOE PAGES

Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

2015-04-10

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

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

Ng, Simon; Lin, Edith; Kitov, Pavel I.

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Copper speciation and binding by organic matter in copper-contaminated streamwater

USGS Publications Warehouse

Breault, R.F.; Colman, J.A.; Aiken, G.R.; McKnight, D.

1996-01-01

Fulvic acid binding sites (1.3-70 ??M) and EDTA (0.0017-0.18 ??M) accounted for organically bound Cu in seven stream samples measured by potentiometric titration. Cu was 84-99% organically bound in filtrates with 200 nM total Cu. Binding of Cu by EDTA was limited by competition from other trace metals. Water hardness was inversely related to properties of dissolved organic carbon (DOC) that enhance fulvic acid binding: DOC concentration, percentage of DOC that is fulvic acid, and binding sites per fulvic acid carbon. Dissolved trace metals, stabilized by organic binding, occurred at increased concentration in soft water as compared to hard water.

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

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

NASA Astrophysics Data System (ADS)

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-01

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

PubMed

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

Gallium as a Therapeutic Agent: A Thermodynamic Evaluation of the Competition between Ga(3+) and Fe(3+) Ions in Metalloproteins.

PubMed

Nikolova, Valia; Angelova, Silvia; Markova, Nikoleta; Dudev, Todor

2016-03-10

Gallium has been employed (in the form of soluble salts) to fight various forms of cancer, infectious, and inflammatory diseases. The rationale behind this lies in the ability of Ga(3+) cation to mimic closely in appearance the native ferric ion, Fe(3+), thus interfering with the biological processes requiring ferric cofactors. However, Ga(3+) ion cannot participate in redox reactions and, when substituting for the "native" Fe(3+) ion in the enzyme active site, renders it inactive. Although a significant body of information on the Ga(3+)-Fe(3+) competition in biological systems has been accumulated, the intimate mechanism of the process is still not well understood and several questions remain: What are the basic physical principles governing the competition between the two trivalent cations in proteins? What type of metal centers are the most likely targets for gallium therapy? To what extent are the Fe(3+)-binding sites in the key enzyme ribonucleotide reductase vulnerable to Ga(3+) substitution? Here, we address these questions by studying the competition between Ga(3+) and Fe(3+) ions in model metal binding sites of various compositions and charge states. The results obtained are in line with available experimental data and shed light on the intimate mechanism of the Ga(3+)/Fe(3+) selectivity in various model metal binding sites and biological systems such as serum transferrin and ribonucleotide reductase.

Categorizing and Assessing Multi-Campus Universities in Contemporary Higher Education

ERIC Educational Resources Information Center

Pinheiro, Rómulo; Nordstrand Berg, Laila

2017-01-01

Multi-campus universities are not a new phenomenon per se, but they have become an increasing feature of contemporary higher education systems all over the world. In the case of Northern Europe, multi-campus universities are the consequence of contraction patterns resulting from overcapacity, fragmentation and rising competition. This paper has…

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

PubMed Central

Mendoza-Maldonado, Ramiro; Paolinelli, Roberta; Galbiati, Laura; Giadrossi, Sara; Giacca, Mauro

2010-01-01

Background The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication. PMID:21085491

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

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

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription of the tumor suppressor Rb gene via binding competition with Sp1 and recruitment of co-repressors.

PubMed

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-11-28

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

The fibrinogen-binding M1 protein reduces pharyngeal cell adherence and colonization phenotypes of M1T1 group A Streptococcus.

PubMed

Anderson, Ericka L; Cole, Jason N; Olson, Joshua; Ryba, Bryan; Ghosh, Partho; Nizet, Victor

2014-02-07

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

PubMed

Sadaie, Wakako; Harada, Yoshie; Matsuda, Michiyuki; Aoki, Kazuhiro

2014-09-01

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

PubMed

Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

2016-10-01

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

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

Zhao, Huaying, E-mail: zhaoh3@mail.nih.gov; Schuck, Peter, E-mail: zhaoh3@mail.nih.gov

2015-01-01

Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysicalmore » techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.« less

Long noncoding RNA FTX inhibits hepatocellular carcinoma proliferation and metastasis by binding MCM2 and miR-374a.

PubMed

Liu, F; Yuan, J-H; Huang, J-F; Yang, F; Wang, T-T; Ma, J-Z; Zhang, L; Zhou, C-C; Wang, F; Yu, J; Zhou, W-P; Sun, S-H

2016-10-13

It has long been known that males are more susceptible than females to hepatocellular carcinoma (HCC), but the reason remains elusive. In this study, we investigated the expression and function of the long noncoding RNA FTX (lnc-FTX), an X-inactive-specific transcript (XIST) regulator transcribed from the X chromosome inactivation center, in both HCC and HCC gender disparity. lnc-FTX is expressed at higher levels in female livers than in male livers and is significantly downregulated in HCC tissues compared with normal liver tissues. Patients with higher lnc-FTX expression exhibited longer survival, suggesting that lnc-FTX is a useful prognostic factor for HCC patients. lnc-FTX inhibits HCC cell growth and metastasis both in vitro and in vivo. Mechanistically, lnc-FTX represses Wnt/β-catenin signaling activity by competitively sponging miR-374a and inhibits HCC cell epithelial-mesenchymal transition and invasion. In addition, lnc-FTX binds to the DNA replication licensing factor MCM2, thereby impeding DNA replication and inhibiting proliferation in HCC cells. In conclusion, these findings suggest that lnc-FTX may act as a tumor suppressor in HCC through physically binding miR-374a and MCM2. It may also be one of the reasons for HCC gender disparity and may potentially contribute to HCC treatment.

Continuous Catalytic Production of Methyl Acrylates from Unsaturated Alcohols by Gold: The Strong Effect of C=C Unsaturation on Reaction Selectivity

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

Zugic, Branko; Karakalos, Stavros; Stowers, Kara J.

2016-03-04

Here we demonstrate the gas-phase catalytic production of methyl acrylates by oxygen-assisted coupling of methanol with the unsaturated alcohols allyl alcohol and methylallyl alcohol over nanoporous gold (npAu) at atmospheric pressure. Analogous investigations on O-activated Au(110) exhibit the same pattern of reactivity and are used to establish that the competition between methoxy and allyloxy (or methallyloxy) reaction intermediates for adsorption sites, mediated by the reactants themselves, determines the selectivity of reaction. Our results clearly show that the C=C bond substantially increases the binding efficacy of the allyloxy (or methallyloxy), thus requiring extremely high methanol mole fractions (>0.99) in order tomore » achieve comparable surface concentrations of methoxy and produce optimum yields of either methacrylate or methyl methacrylate. Allyloxy and methallyloxy were favored by factors of ~100 and ~450, respectively, vs methoxy. These values are more than 1 order of magnitude greater than those measured for competitive binding of ethoxy and 1-butoxy vs methoxy, demonstrating the strong effect of the carbon–carbon bond unsaturation. The 4.5-fold increase due to the addition of the methyl group in methylallyl alcohol vs allyl alcohol indicates the significant effect of the additional van der Waals interactions between the methyl group and the surface. Gas-phase acidity is also shown to be a good qualitative indicator for the relative binding strength of the alkoxides. This work provides insight into the control of reaction selectivity for coupling reactions and demonstrates the value of fundamental studies on single crystals for establishing key principles governing reaction selectivity. Notably, these oxygen-assisted coupling reactions occur without oxidation of the C=C bond.« less

Continuous Catalytic Production of Methyl Acrylates from Unsaturated Alcohols by Gold: The Strong Effect of C=C Unsaturation on Reaction Selectivity

DOE PAGES

Zugic, Branko; Karakalos, Stavros; Stowers, Kara J.; ...

2016-02-02

We demonstrate the gas-phase catalytic production of methyl acrylates by oxygen-assisted coupling of methanol with the unsaturated alcohols allyl alcohol and methylallyl alcohol over nanoporous gold (npAu) at atmospheric pressure. Analogous investigations on O-activated Au(110) exhibit the same pattern of reactivity and are used to establish that the competition between methoxy and allyloxy (or methallyloxy) reaction intermediates for adsorption sites, mediated by the reactants themselves, determines the selectivity of reaction. These results clearly show that the C=C bond substantially increases the binding efficacy of the allyloxy (or methallyloxy), thus requiring extremely high methanol mole fractions (>0.99) in order to achievemore » comparable surface concentrations of methoxy and produce optimum yields of either methacrylate or methyl methacrylate. Allyloxy and methallyloxy were favored by factors of ~100 and ~450, respectively, vs methoxy. These values are more than 1 order of magnitude greater than those measured for competitive binding of ethoxy and 1-butoxy vs methoxy, demonstrating the strong effect of the carbon–carbon bond unsaturation. The 4.5-fold increase due to the addition of the methyl group in methylallyl alcohol vs allyl alcohol indicates the significant effect of the additional van der Waals interactions between the methyl group and the surface. Gas-phase acidity is also shown to be a good qualitative indicator for the relative binding strength of the alkoxides. This work then provides insight into the control of reaction selectivity for coupling reactions and demonstrates the value of fundamental studies on single crystals for establishing key principles governing reaction selectivity. Notably, these oxygen-assisted coupling reactions occur without oxidation of the C=C bond.« less

Effects of Competition and Mode of Fire on Physiological Responses, Psychological Stress Reactions, and Shooting Performance

DTIC Science & Technology

1991-07-01

Alterations in serum cortisol and its binding characteristics in anorexia nervosa . J.urnal o•f Q n1raI £ndcrinaloag and M -bo.liam, Ui, 406-411. Clement...91 43. Comparison of Mean Pre-stress MAACL-R Depression Scores for SS Competition and SS Control Groups on Record-Fire Day With Those for Subjects in...independent non-stress contzol condition) .................. 92 5 44. Comparison of Mean Post-stress MAACL-R Depression Scores for SS Competition and SS

Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

PubMed Central

Lu, Xiaoming; Zoller, Erin E.; Weirauch, Matthew T.; Wu, Zhiguo; Namjou, Bahram; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan K.; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Sivils, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Joo, Young Bin; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Graham, Deborah Cunninghame; Vyse, Timothy J.; Guthridge, Joel M.; Gaffney, Patrick M.; Langefeld, Carl D.; Kelly, Jennifer A.; Greis, Kenneth D.; Kaufman, Kenneth M.; Harley, John B.; Kottyan, Leah C.

2015-01-01

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. PMID:25865496

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

PubMed

Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2007-11-01

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment

PubMed Central

Lee-Sayer, Sally S. M.; Dougan, Meghan N.; Cooper, Jesse; Sanderson, Leslie; Dosanjh, Manisha; Maxwell, Christopher A.

2018-01-01

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment. PMID:29684048

Structural basis for the binding of didemnins to human elongation factor eEF1A and rationale for the potent antitumor activity of these marine natural products.

PubMed

Marco, Esther; Martín-Santamaría, Sonsoles; Cuevas, Carmen; Gago, Federico

2004-08-26

Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability. On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP. We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3' terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu.GTP.tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1Balpha, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1Balpha complex. This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2. Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains. In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor. This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A. Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1Balpha or eEF1Bbeta, thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry. The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

Mechanism of reactant and product dissociation from the anthrax edema factor: a locally enhanced sampling and steered molecular dynamics study.

PubMed

Martínez, Leandro; Malliavin, Thérèse E; Blondel, Arnaud

2011-05-01

The anthrax edema factor is a toxin overproducing damaging levels of cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) from ATP. Here, mechanisms of dissociation of ATP and products (cAMP, PPi) from the active site are studied using locally enhanced sampling (LES) and steered molecular dynamics simulations. Various substrate conformations and ionic binding modes found in crystallographic structures are considered. LES simulations show that PPi and cAMP dissociate through different solvent accessible channels, while ATP dissociation requires significant active site exposure to solvent. The ionic content of the active site directly affects the dissociation of ATP and products. Only one ion dissociates along with ATP in the two-Mg(2+) binding site, suggesting that the other ion binds EF prior to ATP association. Dissociation of reaction products cAMP and PPi is impaired by direct electrostatic interactions between products and Mg(2+) ions. This provides an explanation for the inhibitory effect of high Mg(2+) concentrations on EF enzymatic activity. Breaking of electrostatic interactions is dependent on a competitive binding of water molecules to the ions, and thus on the solvent accessibility of the active site. Consequently, product dissociation seems to be a two-step process. First, ligands are progressively solvated while preserving the most important electrostatic interactions, in a process that is dependent on the flexibility of the active site. Second, breakage of the electrostatic bonds follows, and ligands diffuse into solvent. In agreement with this mechanism, product protonation facilitates dissociation.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

PubMed

Tugaeva, Kristina V; Faletrov, Yaroslav V; Allakhverdiev, Elvin S; Shkumatov, Vladimir M; Maksimov, Eugene G; Sluchanko, Nikolai N

2018-02-26

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

Activity Demands During Multi-Directional Team Sports: A Systematic Review.

PubMed

Taylor, Jeffrey B; Wright, Alexis A; Dischiavi, Steven L; Townsend, M Allison; Marmon, Adam R

2017-12-01

Late-stage rehabilitation programs often incorporate 'sport-specific' demands, but may not optimally simulate the in-game volume or intensity of such activities as sprinting, cutting, jumping, and lateral movement. The aim of this review was to characterize, quantify, and compare straight-line running and multi-directional demands during sport competition. A systematic review of PubMed, CINAHL, SPORTDiscus, and Cochrane Central Register of Controlled Trials databases was conducted. Studies that reported time-motion analysis data on straight-line running, accelerations/decelerations, activity changes, jumping, cutting, or lateral movement over the course of an entire competition in a multi-directional sport (soccer, basketball, lacrosse, handball, field hockey, futsal, volleyball) were included. Data was organized based on sport, age level, and sex and descriptive statistics of the frequency, intensity, time, and volume of the characteristics of running and multi-directional demands were extracted from each study. Eighty-one studies were included in the review (n = 47 soccer, n = 11 basketball, n = 9 handball, n = 7 field hockey, n = 3 futsal, n = 4 volleyball). Variability of sport demand data was found across sports, sexes, and age levels. Specifically, soccer and field hockey demanded the most volume of running, while basketball required the highest ratio of high-intensity running to sprinting. Athletes change activity between 500 and 3000 times over the course of a competition, or once every 2-4 s. Studies of soccer reported the most frequent cutting (up to 800 per game), while studies of basketball reported the highest frequency of lateral movement (up to 450 per game). Basketball (42-56 per game), handball (up to 90 per game), and volleyball (up to 35 per game) were found to require the most jumping. These data may provide an incomplete view of an athlete's straight-line running load, considering that only competition and not practice data was provided. Considerable variability exists in the demands of straight-line running and multi-directional demands across sports, competition levels, and sexes, indicating the need for sports medicine clinicians to design future rehabilitation programs with improved specificity (including the type of activity and dosage) to these demands.

Design, synthesis, manufacturing, and testing of a competitive FHSAE vehicle.

DOT National Transportation Integrated Search

2012-06-01

The goal of this multi-year project is to create a fully functional University of Idaho entry in the hybrid FSAE competition scheduled for : 2012. Vehicle integration has been completed as part of a variety of 2010-2011 senior design projects and 201...

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

PubMed

Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription.

PubMed

Bütepage, Mareike; Preisinger, Christian; von Kriegsheim, Alexander; Scheufen, Anja; Lausberg, Eva; Li, Jinyu; Kappes, Ferdinand; Feederle, Regina; Ernst, Sabrina; Eckei, Laura; Krieg, Sarah; Müller-Newen, Gerhard; Rossetti, Giulia; Feijs, Karla L H; Verheugd, Patricia; Lüscher, Bernhard

2018-04-30

Macrodomains are conserved protein folds associated with ADP-ribose binding and turnover. ADP-ribosylation is a posttranslational modification catalyzed primarily by ARTD (aka PARP) enzymes in cells. ARTDs transfer either single or multiple ADP-ribose units to substrates, resulting in mono- or poly-ADP-ribosylation. TARG1/C6orf130 is a macrodomain protein that hydrolyzes mono-ADP-ribosylation and interacts with poly-ADP-ribose chains. Interactome analyses revealed that TARG1 binds strongly to ribosomes and proteins associated with rRNA processing and ribosomal assembly factors. TARG1 localized to transcriptionally active nucleoli, which occurred independently of ADP-ribose binding. TARG1 shuttled continuously between nucleoli and nucleoplasm. In response to DNA damage, which activates ARTD1/2 (PARP1/2) and promotes synthesis of poly-ADP-ribose chains, TARG1 re-localized to the nucleoplasm. This was dependent on the ability of TARG1 to bind to poly-ADP-ribose. These findings are consistent with the observed ability of TARG1 to competitively interact with RNA and PAR chains. We propose a nucleolar role of TARG1 in ribosome assembly or quality control that is stalled when TARG1 is re-located to sites of DNA damage.

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters

PubMed Central

Hirayama, Bruce A; Díez-Sampedro, Ana; Wright, Ernest M

2001-01-01

Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na+/glucose (hSGLT1) and Na+/Cl−/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7×12Å. Important hydrogen bond interactions occur at five positions bordering this surface. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site ∼8Å from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors. PMID:11588102

An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates*

PubMed Central

Fredslund, Folmer; Vujičić Žagar, Andreja; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan

2016-01-01

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

A Computational Model for Docking of Noncompetitive Neuraminidase Inhibitors and Probing their Binding Interactions with Neuraminidase of Influenza Virus H5N1.

PubMed

Chintakrindi, Anand S; Martis, Elvis A F; Gohil, Devanshi J; Kothari, Sweta T; Chowdhary, Abhay S; Coutinho, Evans C; Kanyalkar, Meena A

2016-01-01

With cases of emergence of drug resistance to the current competitive inhibitors of neuraminidase (NA) such as oseltamivir and zanamavir, there is a present need for an alternative approach in the treatment of avian influenza. With this in view, some flavones and chalcones were designed based on quercetin, the most active naturally occurring noncompetitive inhibitor. We attempt to understand the binding of quercetin to H5N1-NA, and synthetic analogs of quercetin namely flavones and its precursors the chalcones using computational tools. Molecular docking was done using Libdock. Molecular dynamics (MD) simulations were performed using Amber14. We synthesized the two compounds; their structures were confirmed by infrared spectroscopy, 1H-NMR, and mass spectrometry. These molecules were then tested for H5N1-NA inhibition and kinetics of inhibition. Molecular docking studies yielded two compounds i.e., 4'-methoxyflavone and 2'-hydroxy-4-methoxychalcone, as promising leads which identified them as binders of the 150-cavity of NA. Furthermore, MD simulation studies revealed that quercetin and the two compounds bind and hold the 150 loop in its open conformation, which ultimately perturbs the binding of sialic acid in the catalytic site. Estimation of the free energy of binding by MM-PBSA portrays quercetin as more potent than chalcone and flavone. These molecules were then determined as non-competitive inhibitors from the Lineweaver-Burk plots rendered from the enzyme kinetic studies. We conclude that non-competitive type of inhibition, as shown in this study, can serve as an effective method to block NA and evade the currently seen drug resistance.

Dialysis, Albumin Binding, and Competitive Binding: A Laboratory Lesson Relating Three Chemical Concepts to Healthcare

ERIC Educational Resources Information Center

Domingo, Jennifer P.; Abualia, Mohammed; Barragan, Diana; Schroeder, Lianne; Wink, Donald J.; King, Maripat; Clark, Ginevra A.

2017-01-01

Introductory Chemistry laboratories must go beyond "cookbook" methods to illustrate how chemistry concepts apply to complex, real-world problems. In our case, we are preparing students to use their chemistry knowledge in the healthcare profession. The experiment described here explicitly models three important chemical concepts: dialysis…

Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening

EPA Science Inventory

The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across differ...

Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

EPA Science Inventory

The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

PubMed

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

PubMed

Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

2013-11-15

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

PubMed

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

PubMed Central

Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

2011-01-01

BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding. PMID:20958290

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

PubMed

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-09-19

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-07-11

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes.

PubMed

Lasarre, Breah; Aggarwal, Chaitanya; Federle, Michael J

2013-01-02

Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the -35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit. Rgg-family transcriptional regulators are widespread among low-G+C Gram-positive bacteria and in many cases contribute to bacterial physiology and virulence. Only recently was it discovered that several Rgg proteins function in cell-to-cell communication (quorum sensing [QS]) via direct interaction with signaling peptides. The mechanism(s) by which Rgg proteins mediate regulation is poorly understood, and further insight into Rgg function is anticipated to be of great importance for the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species. The results of this study on the Rgg2/3 QS circuit of S. pyogenes demonstrate that DNA binding of target promoters by the activator Rgg2 is directly inhibited by competitive binding by the repressor Rgg3, thereby preventing transcriptional activation of the target genes and premature induction of the QS circuit. This is a unique regulatory mechanism among Rgg proteins and other peptide-responsive QS regulators.

Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

PubMed Central

Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

2013-01-01

Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

Composite alginate gels for tunable cellular microenvironment mechanics

NASA Astrophysics Data System (ADS)

Khavari, Adele; Nydén, Magnus; Weitz, David A.; Ehrlicher, Allen J.

2016-08-01

The mechanics of the cellular microenvironment can be as critical as biochemistry in directing cell behavior. Many commonly utilized materials derived from extra-cellular-matrix create excellent scaffolds for cell growth, however, evaluating the relative mechanical and biochemical effects independently in 3D environments has been difficult in frequently used biopolymer matrices. Here we present 3D sodium alginate hydrogel microenvironments over a physiological range of stiffness (E = 1.85 to 5.29 kPa), with and without RGD binding sites or collagen fibers. We use confocal microscopy to measure the growth of multi-cellular aggregates (MCAs), of increasing metastatic potential in different elastic moduli of hydrogels, with and without binding factors. We find that the hydrogel stiffness regulates the growth and morphology of these cell clusters; MCAs grow larger and faster in the more rigid environments similar to cancerous breast tissue (E = 4-12 kPa) as compared to healthy tissue (E = 0.4-2 kpa). Adding binding factors from collagen and RGD peptides increases growth rates, and change maximum MCA sizes. These findings demonstrate the utility of these independently tunable mechanical/biochemistry gels, and that mechanical confinement in stiffer microenvironments may increase cell proliferation.

Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells.

PubMed

Sawada, Keigo; Takedachi, Masahide; Yamamoto, Satomi; Morimoto, Chiaki; Ozasa, Masao; Iwayama, Tomoaki; Lee, Chun Man; Okura, Hanayuki; Matsuyama, Akifumi; Kitamura, Masahiro; Murakami, Shinya

2015-08-14

Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Toward a modular multi-material nanoparticle synthesis and assembly strategy via bionanocombinatorics: bifunctional peptides for linking Au and Ag nanomaterials

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

Briggs, Beverly D.; Palafox-Hernandez, J. Pablo; Li, Yue

Materials-binding peptides represent a unique avenue towards controlling the shape and size of nanoparticles (NPs) grown under aqueous conditions. Here, employing a bionanocombinatorics approach, two such materials-binding peptides were linked at either end of a photoswitchable spacer, forming a multi-domain materials-binding molecule to control the in situ synthesis and organization of Ag and Au NPs under ambient conditions. These multi-domain molecules retained the peptides’ ability to nucleate, grow, and stabilize Ag and Au NPs in aqueous media. Disordered co-assemblies of the two nanomaterials were observed by TEM imaging of dried samples after sequential growth of the two metals, and showedmore » a clustering behavior that was not observed without both metals and the linker molecules. While TEM evidence indicated the formation of AuNP/AgNP assemblies upon drying, SAXS analysis indicated that no extended assemblies existed in solution, suggesting that sample drying plays an important role in facilitating NP clustering. Molecular simulations and experimental data revealed tunable materials-binding based upon the isomerization state of the photoswitchable unit and metal employed. This work is a first step in generating externally actuated biomolecules with specific material-binding properties that could be used as the building blocks to achieve multi-material switchable NP assemblies.« less

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

PubMed Central

Bertini, R; Barcelos, LS; Beccari, AR; Cavalieri, B; Moriconi, A; Bizzarri, C; Di Benedetto, P; Di Giacinto, C; Gloaguen, I; Galliera, E; Corsi, MM; Russo, RC; Andrade, SP; Cesta, MC; Nano, G; Aramini, A; Cutrin, JC; Locati, M; Allegretti, M; Teixeira, MM

2012-01-01

BACKGROUND AND PURPOSE DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [35S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys99 on CXCR1 and the non-conserved residue Asp293 on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases. PMID:21718305

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

PubMed Central

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Mannose-binding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications from infection, including disseminated intravascular coagulation

PubMed Central

Takahashi, Kazue; Chang, Wei-Chuan; Takahashi, Minoru; Pavlov, Vasile; Ishida, Yumi; La Bonte, Laura; Shi, Lei; Fujita, Teizo; Stahl, Gregory L.; Van Cott, Elizabeth M.

2010-01-01

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases. PMID:20399528

Motivation, study habits, and expectations of medical students in Singapore.

PubMed

Amin, Zubair; Tani, Massimiliano; Eng, Khoo Hoon; Samarasekara, Dujeepa D; Huak, Chan Yiong

2009-12-01

To determine the motivation and incentives in education, learning experience and teaching techniques, and expectations about future careers among medical students from a multi-ethnic Asian country. Pre-validated questionnaire-based survey with stratified random sampling among medical students. The questionnaire combined qualitative responses with semi-quantitative measures of available alternatives. The response rate was 83.1%. The most important factor for pursuing university study was 'prospect of finding an interesting challenging job' (rank 1-75%). Family made a significant contribution in decision making. Given the chance, a majority (67.2%) of respondents would prefer to study overseas. The main deterring factors were cost (67.7%), distance from home (28%), and local opportunity for post-graduation (23.4%). Despite their inclination of study overseas, the majority (73.9%) of the respondents indicated they were either very satisfied or satisfied with their current choice of university study. Only 20% of students were comfortable in asking questions in classroom as asking questions was deemed 'too risky' and 'unnecessary to get better grades'. Students adopted strategies related to assessment and competition to monitor their study. Senior students reported university education as less relevant to their future careers as compared to junior students (p = 0.002). Students' learning behaviour is determined by complex factors such as educational incentives, learning support, assessment and competition. Among several external factors, family, job prospects and expectations about the future play a critical role in education.

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E

2013-01-01

By coupling the protection and organization of ssDNA with the recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA manages to coordinate the biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA s DNA binding activity, combining small-angle x-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA s DNA-binding core. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than threemore » transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.« less

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants.

PubMed Central

Small, J M; Mitchell, T G

1986-01-01

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with 125I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal. PMID:3536747

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

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

Small, J.M.; Mitchell, T.G.

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with /sup 125/I, andmore » used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.« less

Product competitiveness analysis for e-commerce platform of special agricultural products

NASA Astrophysics Data System (ADS)

Wan, Fucheng; Ma, Ning; Yang, Dongwei; Xiong, Zhangyuan

2017-09-01

On the basis of analyzing the influence factors of the product competitiveness of the e-commerce platform of the special agricultural products and the characteristics of the analytical methods for the competitiveness of the special agricultural products, the price, the sales volume, the postage included service, the store reputation, the popularity, etc. were selected in this paper as the dimensionality for analyzing the competitiveness of the agricultural products, and the principal component factor analysis was taken as the competitiveness analysis method. Specifically, the web crawler was adopted to capture the information of various special agricultural products in the e-commerce platform ---- chi.taobao.com. Then, the original data captured thereby were preprocessed and MYSQL database was adopted to establish the information library for the special agricultural products. Then, the principal component factor analysis method was adopted to establish the analysis model for the competitiveness of the special agricultural products, and SPSS was adopted in the principal component factor analysis process to obtain the competitiveness evaluation factor system (support degree factor, price factor, service factor and evaluation factor) of the special agricultural products. Then, the linear regression method was adopted to establish the competitiveness index equation of the special agricultural products for estimating the competitiveness of the special agricultural products.

A stochastic multi-agent optimization model for energy infrastructure planning under uncertainty and competition.

DOT National Transportation Integrated Search

2017-07-04

This paper presents a stochastic multi-agent optimization model that supports energy infrastruc- : ture planning under uncertainty. The interdependence between dierent decision entities in the : system is captured in an energy supply chain network, w...

A discrete artificial bee colony algorithm for detecting transcription factor binding sites in DNA sequences.

PubMed

Karaboga, D; Aslan, S

2016-04-27

The great majority of biological sequences share significant similarity with other sequences as a result of evolutionary processes, and identifying these sequence similarities is one of the most challenging problems in bioinformatics. In this paper, we present a discrete artificial bee colony (ABC) algorithm, which is inspired by the intelligent foraging behavior of real honey bees, for the detection of highly conserved residue patterns or motifs within sequences. Experimental studies on three different data sets showed that the proposed discrete model, by adhering to the fundamental scheme of the ABC algorithm, produced competitive or better results than other metaheuristic motif discovery techniques.

Characterization of 12 GnRH peptide agonists - a kinetic perspective.

PubMed

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak-Reppel, Katrin; Fernández-Montalván, Amaury E; IJzerman, Adriaan P; Heitman, Laura H

2016-01-01

Drug-target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin-releasing hormone (GnRH) receptor for the treatment of hormone-dependent diseases. Surprisingly, the kinetic receptor-binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor-binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. A novel radioligand-binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [(125) I]-triptorelin. In addition to radioligand-binding studies, a homogeneous time-resolved FRET Tag-lite™ method was developed as an alternative assay for the same purpose. Two novel competition association assays were successfully developed and applied to determine the kinetic receptor-binding characteristics of 12 high-affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. © 2015 The British Pharmacological Society.

Characterization of 12 GnRH peptide agonists – a kinetic perspective

PubMed Central

Nederpelt, Indira; Georgi, Victoria; Schiele, Felix; Nowak‐Reppel, Katrin; Fernández‐Montalván, Amaury E.; IJzerman, Adriaan P.

2015-01-01

Background and Purpose Drug‐target residence time is an important, yet often overlooked, parameter in drug discovery. Multiple studies have proposed an increased residence time to be beneficial for improved drug efficacy and/or longer duration of action. Currently, there are many drugs on the market targeting the gonadotropin‐releasing hormone (GnRH) receptor for the treatment of hormone‐dependent diseases. Surprisingly, the kinetic receptor‐binding parameters of these analogues have not yet been reported. Therefore, this project focused on determining the receptor‐binding kinetics of 12 GnRH peptide agonists, including many marketed drugs. Experimental Approach A novel radioligand‐binding competition association assay was developed and optimized for the human GnRH receptor with the use of a radiolabelled peptide agonist, [125I]‐triptorelin. In addition to radioligand‐binding studies, a homogeneous time‐resolved FRET Tag‐lite™ method was developed as an alternative assay for the same purpose. Key Results Two novel competition association assays were successfully developed and applied to determine the kinetic receptor‐binding characteristics of 12 high‐affinity GnRH peptide agonists. Results obtained from both methods were highly correlated. Interestingly, the binding kinetics of the peptide agonists were more divergent than their affinities with residence times ranging from 5.6 min (goserelin) to 125 min (deslorelin). Conclusions and Implications Our research provides new insights by incorporating kinetic, next to equilibrium, binding parameters in current research and development that can potentially improve future drug discovery targeting the GnRH receptor. PMID:26398856

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

International Validation of Two Human Recombinant Estrogen ...

EPA Pesticide Factsheets

An international validation study has been successfully completed for 2 competitive binding assays using human recombinant ERa. Assays evaluated included the Freyberger-Wilson (FW) assay using a full length human ER, and the Chemical Evaluation and Research Institute (CERI) assay using a ligand-binding domain of the human ER. Twenty three compounds were tested in 6 laboratories for the FW assay and 5 for the CERJ assay, which included three controls (used with every run), 9 uncoded, and 14 coded chemicals across 3 subtasks. The overall goal of this validation study was to demonstrate the ability of each of the two assays to reliably classify the test chemicals as binders or non-binders. Laboratories had little trouble with the ER binders that produced a full binding curve when using either the CERI or FW assays. As is typical with all ER competitive binding assays, the weak binders proved to be more challenging. However, overall results from both the FW and CERI assays were consistent and in agreement with expected classifications regardless of the form of the hrER (i.e., full length ER versus an ER ligand binding domain) or the subtle differences in the protocols for conducting each assay. The reproducibility and accuracy for classification of chemicals as potential ER binders and non- binders using the FW and CERI hrER binding assays were comparable to that of the U.S.EPA’s existing ER binding test guideline OPPTS 890.1250, while providing an improved, highe

Towards Cooperative Predictive Data Mining in Competitive Environments

NASA Astrophysics Data System (ADS)

Lisý, Viliam; Jakob, Michal; Benda, Petr; Urban, Štěpán; Pěchouček, Michal

We study the problem of predictive data mining in a competitive multi-agent setting, in which each agent is assumed to have some partial knowledge required for correctly classifying a set of unlabelled examples. The agents are self-interested and therefore need to reason about the trade-offs between increasing their classification accuracy by collaborating with other agents and disclosing their private classification knowledge to other agents through such collaboration. We analyze the problem and propose a set of components which can enable cooperation in this otherwise competitive task. These components include measures for quantifying private knowledge disclosure, data-mining models suitable for multi-agent predictive data mining, and a set of strategies by which agents can improve their classification accuracy through collaboration. The overall framework and its individual components are validated on a synthetic experimental domain.

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques.

PubMed

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-03-30

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 10 5  M -1 ) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

PubMed Central

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-01-01

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M−1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA. PMID:28358124

Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

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

Kalow, W.; Tyndale, R.F.; Niznik, H.B.

1990-02-26

P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with ({sup 3}H)GBR-12935; 1-(2-(diphenylmethoxy) ethyl)-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity ({approximately}7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL)more » are both competitive inhibitors of P450IID6 activity and were found to inhibit ({sup 3}H)GBR-12935 binding. K{sub i} values of twelve compounds (known to interact with the DA transporter or P450IID6) for ({sup 3}H)GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with ({sup 3}H)GBR-12935.« less

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

NASA Astrophysics Data System (ADS)

Yang, Hongqin; Liu, Jiuyang; Huang, Yanmei; Gao, Rui; Tang, Bin; Li, Shanshan; He, Jiawei; Li, Hui

2017-03-01

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M-1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin.

PubMed

Gao, Shunxiang; Zheng, Xin; Hu, Bo; Sun, Mingjuan; Wu, Jihong; Jiao, Binghua; Wang, Lianghua

2017-03-15

In this study, we coupled biolayer interferometry (BLI) with competitive binding assay through an enzyme-linked aptamer and developed a real-time, ultra-sensitive, rapid quantitative method for detection of the marine biotoxin palytoxin. Horseradish peroxidase-labeled aptamers were used as biorecognition receptors to competitively bind with palytoxin, which was immobilized on the biosensor surface. The palytoxin: horseradish peroxidase-aptamer complex was then submerged in a 3,3'-diaminobenzidine solution, which resulted in formation of a precipitated polymeric product directly on the biosensor surface and a large change in the optical thickness of the biosensor layer. This change could obviously shift the interference pattern and generate a response profile on the BLI biosensor. The biosensor showed a broad linear range for palytoxin (200-700pg/mL) with a low detection limit (0.04pg/mL). Moreover, the biosensor was applied to the detection of palytoxin in spiked extracts and showed a high degree of selectivity for palytoxin, good reproducibility, and stability. This enzyme-linked, aptamer-based, competitive BLI biosensor offers a promising method for rapid and sensitive detection of palytoxin and other analytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding Mode Analyses and Pharmacophore Model Development for Stilbene Derivatives as a Novel and Competitive Class of α-Glucosidase Inhibitors

PubMed Central

Kim, Jun Young; Arooj, Mahreen; Kim, Siu; Hwang, Swan; Kim, Byeong-Woo; Park, Ki Hun; Lee, Keun Woo

2014-01-01

Stilbene urea derivatives as a novel and competitive class of non-glycosidic α-glucosidase inhibitors are effective for the treatment of type II diabetes and obesity. The main purposes of our molecular modeling study are to explore the most suitable binding poses of stilbene derivatives with analyzing the binding affinity differences and finally to develop a pharmacophore model which would represents critical features responsible for α-glucosidase inhibitory activity. Three-dimensional structure of S. cerevisiae α-glucosidase was built by homology modeling method and the structure was used for the molecular docking study to find out the initial binding mode of compound 12, which is the most highly active one. The initial structure was subjected to molecular dynamics (MD) simulations for protein structure adjustment at compound 12-bound state. Based on the adjusted conformation, the more reasonable binding modes of the stilbene urea derivatives were obtained from molecular docking and MD simulations. The binding mode of the derivatives was validated by correlation analysis between experimental Ki value and interaction energy. Our results revealed that the binding modes of the potent inhibitors were engaged with important hydrogen bond, hydrophobic, and π-interactions. With the validated compound 12-bound structure obtained from combining approach of docking and MD simulation, a proper four featured pharmacophore model was generated. It was also validated by comparison of fit values with the Ki values. Thus, these results will be helpful for understanding the relationship between binding mode and bioactivity and for designing better inhibitors from stilbene derivatives. PMID:24465730

Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors*

PubMed Central

Paczkowski, Jon E.; Mukherjee, Sampriti; McCready, Amelia R.; Cong, Jian-Ping; Aquino, Christopher J.; Kim, Hahn; Henke, Brad R.; Smith, Chari D.; Bassler, Bonnie L.

2017-01-01

Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms. PMID:28119451

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

PubMed

Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E

2016-11-11

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Next Generation Antibody Therapeutics Using Bispecific Antibody Technology.

PubMed

Igawa, Tomoyuki

2017-01-01

Nearly fifty monoclonal antibodies have been approved to date, and the market for monoclonal antibodies is expected to continue to grow. Since global competition in the field of antibody therapeutics is intense, we need to establish novel antibody engineering technologies to provide true benefit for patients, with differentiated product values. Bispecific antibodies are among the next generation of antibody therapeutics that can bind to two different target antigens by the two arms of immunoglobulin G (IgG) molecule, and are thus believed to be applicable to various therapeutic needs. Until recently, large scale manufacturing of human IgG bispecific antibody was impossible. We have established a technology, named asymmetric re-engineering technology (ART)-Ig, to enable large scale manufacturing of bispecific antibodies. Three examples of next generation antibody therapeutics using ART-Ig technology are described. Recent updates on bispecific antibodies against factor IXa and factor X for the treatment of hemophilia A, bispecific antibodies against a tumor specific antigen and T cell surface marker CD3 for cancer immunotherapy, and bispecific antibodies against two different epitopes of soluble antigen with pH-dependent binding property for the elimination of soluble antigen from plasma are also described.

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT3 receptors

PubMed Central

Thompson, A J; Lochner, M; Lummis, S C R

2007-01-01

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC 50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA 2 values of 4.92 (K B=12.0 μM) and 4.97 (K B=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with K i values of 15.0, 24.2 and 35.7 μ M. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists. PMID:17502851

Synergistic Blockade of Mitotic Exit by Two Chemical Inhibitors of the APC/C

PubMed Central

Sackton, Katharine L.; Dimova, Nevena; Zeng, Xing; Tian, Wei; Zhang, Mengmeng; Sackton, Timothy B.; Meaders, Johnathan; Pfaff, Kathleen L.; Sigoillot, Frederic; Yu, Hongtao; Luo, Xuelian; King, Randall W.

2014-01-01

Summary Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the Anaphase-Promoting Complex/Cyclosome (APC/C), a thirteen-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumor cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc205, which forms a co-receptor with the APC/C to recognize substrates containing a Destruction box (D-box)6-14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identified a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-L-arginine methyl ester (TAME), a small molecule that blocks the APC/C-Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine. PMID:25156254

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

PubMed

Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik; Jensen, Anders A; Gill, Avinash; Madden, Dean R; Kastrup, Jette S; Skottrup, Peter D

2016-11-01

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

Reconstruction of calmodulin single-molecule FRET states, dye interactions, and CaMKII peptide binding by MultiNest and classic maximum entropy

NASA Astrophysics Data System (ADS)

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-08-01

We analyzed single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy

PubMed Central

DeVore, Matthew S.; Gull, Stephen F.; Johnson, Carey K.

2013-01-01

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data. PMID:24223465

Reconstruction of Calmodulin Single-Molecule FRET States, Dye-Interactions, and CaMKII Peptide Binding by MultiNest and Classic Maximum Entropy.

PubMed

Devore, Matthew S; Gull, Stephen F; Johnson, Carey K

2013-08-30

We analyze single molecule FRET burst measurements using Bayesian nested sampling. The MultiNest algorithm produces accurate FRET efficiency distributions from single-molecule data. FRET efficiency distributions recovered by MultiNest and classic maximum entropy are compared for simulated data and for calmodulin labeled at residues 44 and 117. MultiNest compares favorably with maximum entropy analysis for simulated data, judged by the Bayesian evidence. FRET efficiency distributions recovered for calmodulin labeled with two different FRET dye pairs depended on the dye pair and changed upon Ca 2+ binding. We also looked at the FRET efficiency distributions of calmodulin bound to the calcium/calmodulin dependent protein kinase II (CaMKII) binding domain. For both dye pairs, the FRET efficiency distribution collapsed to a single peak in the case of calmodulin bound to the CaMKII peptide. These measurements strongly suggest that consideration of dye-protein interactions is crucial in forming an accurate picture of protein conformations from FRET data.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Competition between Hydrogen Bonding and Proton Transfer during Specific Anion Recognition by Dihomooxacalix[4]arene Bidentate Ureas.

PubMed

Martínez-González, Eduardo; González, Felipe J; Ascenso, José R; Marcos, Paula M; Frontana, Carlos

2016-08-05

Competition between hydrogen bonding and proton transfer reactions was studied for systems composed of electrogenerated dianionic species from dinitrobenzene isomers and substituted dihomooxacalix[4]arene bidentate urea derivatives. To analyze this competition, a second-order ErCrCi mechanism was considered where the binding process is succeeded by proton transfer and the voltammetric responses depend on two dimensionless parameters: the first related to hydrogen bonding reactions, and the second one to proton transfer processes. Experimental results indicated that, upon an increase in the concentration of phenyl-substituted dihomooxacalix[4]arene bidentate urea, voltammetric responses evolve from diffusion-controlled waves (where the binding process is at chemical equilibrium) into irreversible kinetic responses associated with proton transfer. In particular, the 1,3-dinitrobenzene isomer showed a higher proton transfer rate constant (∼25 M(-1) s(-1)) compared to that of the 1,2-dinitrobenzene (∼5 M(-1) s(-1)), whereas the 1,4-dinitrobenzene did not show any proton transfer effect in the experimental conditions employed.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

NASA Astrophysics Data System (ADS)

Knott, Michael; Best, Robert B.

2014-05-01

Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an "induced fit" or "conformational selection" mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

Development of the Muscle Dysmorphia Inventory (MDI).

PubMed

Rhea, D J; Lantz, C D; Cornelius, A E

2004-12-01

The development of the 6-factor, 27-item Muscle Dysmorphia Inventory (MDI) was based on Lantz et al. proposed model of characteristics associated with Muscle Dysmorphia. quantitative procedures including item-to-total correlations, exploratory and confirmatory factor analyses, and structure equation modeling confirmed the construct validity of the scale. Convergent validity was also tested. bodybuilding and powerlifting competition venues, weight training facilities, and university athletic venues. the 1(st) study consisted of 77 experienced male free weight lifters. The 2(nd) study consisted of 156 male non-competitive bodybuilders and weight lifters and 168 elite level powerlifters and bodybuilders. The 3(rd) study consisted of 151 male and female bodybuilders and weight lifters. each participant completed demographic information, the MDI, Drive for Thinness subscale of the Eating Disorder Inventory, and the Training Dependency subscale of the Bodybuilding Dependence Scale. Reliability estimates (Cronbach's a) ranged from 0.72 to 0.94. Factor loadings in all 3 studies supported the 6-factor structure (size/symmetry, supplement use, exercise dependence, pharmacological use, dietary behavior, and physique protection). Much of the scale validation was focused on construct validity, however, correlations with the MDI's subscales and the Training Dependency subscale of the Bodybuilding Dependence Scale and the Drive for Thinness subscale of the Eating Disorder Inventory provided evidence of convergent validity also. From these preliminary results, the MDI appears to contribute to the identification of a newly formed disorder by offering a multi-dimensional measure of factors related to Muscle Dysmorphia.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

PubMed

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-02-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Convection, diffusion and reaction in a surface-based biosensor: modeling of cooperativity and binding site competition on the surface and in the hydrogel.

PubMed

Lebedev, Konstantin; Mafé, Salvador; Stroeve, Pieter

2006-04-15

We study theoretically the transport and kinetic processes underlying the operation of a biosensor (particularly the surface plasmon sensor "Biacore") used to study the surface binding kinetics of biomolecules in solution to immobilized receptors. Unlike previous studies, we concentrate mainly on the modeling of system-specific phenomena rather than on the influence of mass transport limitations on the intrinsic kinetic rate constants determined from binding data. In the first problem, the case of two-site binding where each receptor unit on the surface can accommodate two analyte molecules on two different sites is considered. One analyte molecule always binds first to a specific site. Subsequently, the second analyte molecule can bind to the adjacent unoccupied site. In the second problem, two different analytes compete for one binding site on the same surface receptor. Finally, the third problem considers the case of positive cooperativity among bound molecules in the hydrogel using a simple mean-field approach. The transport in both the flow channel and the hydrogel phases of the biosensor is taken into account in this case (with few exceptions, most previous studies assume a simpler model in which the hydrogel is treated as a planar surface with the receptors). We consider simultaneously diffusion and convection through the flow channel together with diffusion and cooperativity binding on the surface and in the hydrogel. In each case, typical results for the concentration contours of the free and bound molecules in the flow channel and hydrogel regions are presented together with the time-dependent association/dissociation curves and reaction rates. For binding site competition, the analysis predicts overshoot phenomena.

Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W.

PubMed

Pearson, David S; Swartz, Darl R; Geeves, Michael A

2008-11-18

We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.

Fast Pressure Jumps Can Perturb Calcium and Magnesium Binding to Troponin C F29W

PubMed Central

Pearson, David S.; Swartz, Darl R.; Geeves, Michael A.

2009-01-01

We have used rapid pressure jump and stopped-flow fluorimetry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL.mol-1). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000 s-1 and 100 s-1. Between pCa 8-5.4 and at troponin C concentrations of 8-28 μM, the slow relaxation times were invariant indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200 - 300 μM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo. PMID:18942859

Keeping up with the Globe.

ERIC Educational Resources Information Center

Woods, Jacqueline

2000-01-01

States that new populations with diverse learning needs, fierce competition in curriculum development and delivery, new trends and uses of technology, and a growing international marketplace requiring multi-cultural, multi-skilled workforces are driving major structural and philosophical changes in America's educational systems. Asserts that focus…

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

PubMed

Ojelabi, Ogooluwa A; Lloyd, Kenneth P; Simon, Andrew H; De Zutter, Julie K; Carruthers, Anthony

2016-12-23

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with K i (app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with K i (app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site*

PubMed Central

Ojelabi, Ogooluwa A.; Lloyd, Kenneth P.; Simon, Andrew H.; De Zutter, Julie K.; Carruthers, Anthony

2016-01-01

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 μm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 μm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites. PMID:27836974

A new structural framework for integrating replication protein A into DNA processing machinery

PubMed Central

Brosey, Chris A.; Yan, Chunli; Tsutakawa, Susan E.; Heller, William T.; Rambo, Robert P.; Tainer, John A.; Ivanov, Ivaylo; Chazin, Walter J.

2013-01-01

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways. PMID:23303776

Understanding the mechanisms of protein-DNA interactions

NASA Astrophysics Data System (ADS)

Lavery, Richard

2004-03-01

Structural, biochemical and thermodynamic data on protein-DNA interactions show that specific recognition cannot be reduced to a simple set of binary interactions between the partners (such as hydrogen bonds, ion pairs or steric contacts). The mechanical properties of the partners also play a role and, in the case of DNA, variations in both conformation and flexibility as a function of base sequence can be a significant factor in guiding a protein to the correct binding site. All-atom molecular modeling offers a means of analyzing the role of different binding mechanisms within protein-DNA complexes of known structure. This however requires estimating the binding strengths for the full range of sequences with which a given protein can interact. Since this number grows exponentially with the length of the binding site it is necessary to find a method to accelerate the calculations. We have achieved this by using a multi-copy approach (ADAPT) which allows us to build a DNA fragment with a variable base sequence. The results obtained with this method correlate well with experimental consensus binding sequences. They enable us to show that indirect recognition mechanisms involving the sequence dependent properties of DNA play a significant role in many complexes. This approach also offers a means of predicting protein binding sites on the basis of binding energies, which is complementary to conventional lexical techniques.

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris; Yan, Chunli; Tsutakawa, Susan

2013-01-17

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamicmore » on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.« less

Localised and delocalised optically induced conversion of composite glow peak 5 in LiF:Mg,Ti (TLD-100) to glow peak 4 as a function of postirradiation annealing temperature.

PubMed

Horowitz, Y S; Einav, Y; Biderman, S; Oster, L

2002-01-01

The composite structure of glow peak 5 in LiF:Mg,Ti (TLD-100) has been investigated using optical bleaching by 310 nm (4 eV) light. The glow peak conversion efficiency of peak 5a (Tm = 187 degrees C) to peak 4 traps is very high at a value of 3+/-0.5 (1 SD) whereas the glow peak conversion efficiency of peak 5 (Tm = 205 degrees C) to peak 4 traps is 0.0026+/-0.0012 (1 SD). The high conversion efficiency of peak 5a to peak 4 arises from direct optical ionisation of the electron in the electron-hole pair. leaving behind a singly-trapped hole (peak 4), a direct mechanism, relatively free of competitive mechanisms. Optical ionisation of the 'singly-trapped' electron (peak 5), however, can lead to peak 4 only via multi-stage mechanisms involving charge carrier transport in the valence and conduction bands, a mechanism subject to competitive processes. The conduction/valence band competitive processes lead to the factor of one thousand decrease in the conversion efficiency of peak 5 compared to peak 5a.

Biological evaluation and molecular docking of baicalin and scutellarin as Helicobacter pylori urease inhibitors.

PubMed

Yu, Xiao-Dan; Zheng, Rong-Bo; Xie, Jian-Hui; Su, Ji-Yan; Huang, Xiao-Qi; Wang, Yong-Hong; Zheng, Yi-Feng; Mo, Zhi-Zhun; Wu, Xiao-Li; Wu, Dian-Wei; Liang, Ye-er; Zeng, Hui-Fang; Su, Zi-Ren; Huang, Ping

2015-03-13

Baicalin and scutellarin are the principal bioactive components of Scutellaria baicalensis Georgi which has extensively been incorporated into heat-clearing and detoxification formulas for the treatment of Helicobacter pylori-related gastrointestinal disorders in traditional Chinese medicine. However, the mechanism of action remained to be defined. To explore the inhibitory effect, kinetics and mechanism of Helicobacter pylori urease (the vital pathogenetic factor for Helicobacter pylori infection) inhibition by baicalin and scutellarin, for their therapeutic potential. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of baicalin and scutellarin was characterized with IC50 values, compared to acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor. Lineweaver-Burk and Dixon plots for the Helicobacter pylori urease inhibition of baicalin and scutellarin was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni(2+) binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. Moreover, cytotoxicity experiment using Gastric Epithelial Cells (GES-1) was evaluated. Baicalin and scutellarin effectively suppressed Helicobacter pylori urease in dose-dependent and time-independent manner with IC50 of 0.82±0.07 mM and 0.47±0.04 mM, respectively, compared to AHA (IC50=0.14±0.05 mM). Structure-activity relationship disclosed 4'-hydroxyl gave flavones an advantage to binding with Helicobacter pylori urease. Kinetic analysis revealed that the types of inhibition were non-competitive and reversible with inhibition constant Ki of 0.14±0.01 mM and 0.18±0.02 mM for baicalin and scutellarin, respectively. The mechanism of urease inhibition was considered to be blockage of the SH groups of Helicobacter pylori urease, since thiol reagents (L,D-dithiothreitol, L-cysteine and glutathione) abolished the inhibitory action and competitive active site Ni(2+) binding inhibitors (boric acid and sodium fluoride) carried invalid effect. Molecular docking study further supported the structure-activity analysis and indicated that baicalin and scutellarin interacted with the key residues Cys321 located on the mobile flap through S-H·π interaction, but did not interact with active site Ni(2+). Moreover, Baicalin (at 0.59-1.05 mM concentrations) and scutellarin (at 0.23-0.71 mM concentrations) did not exhibit significant cytotoxicity to GES-1. Baicalin and scutellarin were non-competitive inhibitors targeting sulfhydryl groups especially Cys321 around the active site of Helicobacter pylori urease, representing potential to be good candidate for future research as urease inhibitor for treatment of Helicobacter pylori infection. Furthermore, our work gave additional scientific support to the use of Scutellaria baicalensis in traditional Chinese medicine (TCM) to treat gastrointestinal disorders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Intra- versus Intermolecular Hydrogen Bonding: Solvent-Dependent Conformational Preferences of a Common Supramolecular Binding Motif from 1 H NMR and Vibrational Circular Dichroism Spectra.

PubMed

Demarque, Daniel P; Merten, Christian

2017-12-19

When predicting binding properties of small molecules or larger supramolecular aggregates, intra- and intermolecular hydrogen bonds are often considered the most important factor. Spectroscopic techniques such as 1 H NMR spectroscopy are typically utilized to characterize such binding events, but interpretation is often qualitative and follows chemical intuition. In this study, we compare the effects of intramolecular hydrogen bonding and solvation on two chiral 2,6-pyridinediyl-dialkylamides. In comparison with 1 H NMR spectroscopy, vibrational circular dichroism (VCD) spectroscopy proved to be more sensitive to conformational changes. In fact, the change of the solvent from CDCl 3 to [D 6 ]DMSO generates mirror-image VCD spectra for the same enantiomer. Here, the common sense that the sterically less hindered group is more prone to solvation proved to be wrong according predicted VCD spectra, which clearly show that both asymmetric amide hydrogens are equally likely to be solvated, but never simultaneously. The competition between intra- and intermolecular hydrogen bonding and their importance for a correct prediction of spectral properties are discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S⃞

PubMed Central

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-01-01

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. PMID:18801742

Comparison of potassium and sodium binding in vivo and in agarose samples using TQTPPI pulse sequence

NASA Astrophysics Data System (ADS)

Schepkin, Victor D.; Neubauer, Andreas; Nagel, Armin M.; Budinger, Thomas F.

2017-04-01

Potassium and sodium specific binding in vivo were explored at 21.1 T by triple quantum (TQ) magnetic resonance (MR) signals without filtration to achieve high sensitivities and precise quantifications. The pulse sequence used time proportional phase increments (TPPI). During simultaneous phase-time increments, it provided total single quantum (SQ) and TQ MR signals in the second dimension at single and triple quantum frequencies, respectively. The detection of both TQ and SQ signals was performed at identical experimental conditions and the resulting TQ signal equals 60 ± 3% of the SQ signal when all ions experience sufficient time for binding. In a rat head in vivo the TQ percentage relative to SQ for potassium is 41.5 ± 3% and for sodium is 16.1 ± 1%. These percentages were compared to the matching values in an agarose tissue model with MR relaxation times similar to those of mammalian brain tissue. The sodium TQ signal in agarose samples decreased in the presence of potassium, suggesting a competitive binding of potassium relative to sodium ions for the same binding sites. The TQTPPI signals correspond to almost two times more effective binding of potassium than sodium. In vivo, up to ∼69% of total potassium and ∼27% of total sodium can be regarded as bound or experiencing an association time in the range of several milliseconds. Experimental data analyses show that more than half of the in vivo total sodium TQ signal could be from extracellular space, which is an important factor for quantification of intracellular MR signals.

Determining antibody-binding site of streptococcal pyrogenic exotoxin B to protect mice from group a streptococcus infection.

PubMed

Tsao, Nina; Cheng, Miao-Hui; Yang, Hsiu-Chen; Wang, Yu-Chieh; Liu, Yi-Ling; Kuo, Chih-Feng

2013-01-01

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. SPE B binds and cleaves antibody isotypes and further impairs the immune system by inhibiting complement activation. In this study, we examined the antibody-binding site of SPE B and used it to block SPE B actions during GAS infection. We constructed different segments of the spe B gene and induced them to express different recombinant fragments of SPE B. Using an enzyme-linked immunosorbent assay (ELISA), we found that residues 345-398 of the C-terminal domain of SPE B (rSPE B(345-398)), but not the N-terminal domain, was the major binding site for antibody isotypes. Using a competitive ELISA, we also found that rSPE B(345-398) bound to the Fc portion of IgG. The in vitro functional assays indicate that rSPE B(345-398) not only interfered with cleavage of antibody isotypes but also interfered with SPE B-induced inhibition of complement activation. Immunization of BALB/c mice using rSPE B(345-398) was able to induce production of a high titer of anti-rSPE B(345-398) antibodies and efficiently protected mice from GAS-induced death. These findings suggest that SPE B uses its C-terminal domain to bind the Fc portion of IgG and that immunization of mice with this binding domain (rSPE B(345-398)) could protect mice from GAS infection.

EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

PubMed

Zhou, Juan; Joshi, Bishnu P; Duan, Xiyu; Pant, Asha; Qiu, Zhen; Kuick, Rork; Owens, Scott R; Wang, Thomas D

2015-07-16

Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy. We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging. We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence. After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference. We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

Biochemical Study of Anti-Inflammatory Proteins vCCI and vMIP-II

DTIC Science & Technology

2014-07-17

protein ), where we showed that vCCI is able to bind so many different chemokines due to its general negatively charged surface , allowing it to bind...sample of these competition curves. Our conclusion from the data in Table 1 and Figure 1 is that the negatively charged surface of vCCI allows it to...Similar to our mutagenesis results, the overall data indicate that vCCI uses a negatively charged surface to bind positive charges on the chemokine

Changes in circulating cytokines and markers of muscle damage in elite cyclists during a multi-stage competition.

PubMed

Córdova Martínez, Alfredo; Martorell Pons, Miquel; Sureda Gomila, Antoni; Tur Marí, Josep A; Pons Biescas, Antoni

2015-09-01

The purpose of this study was to determine the changes in the basal and post-exercise plasma markers of muscular damage, lipid peroxidation and cytokines in eight male well-trained semiprofessional cyclists, in response to a three consecutive-day cycling competition. Serum markers of oxidative and muscular damage - creatine kinase activity, lactate dehydrogenase activity, myoglobin and malondialdehyde (MDA), creatinine and nitrite levels - followed a sawtooth-type representation throughout the competition. MDA showed an accumulative pattern, evidenced in the post-race values of the third stage which were significantly higher with respect to the values of the first stage. Cortisol levels were significantly influenced by an interaction between the exercise and the stage factors, with higher values on the 4th day. Plasma cytokine levels were only determined before the first stage and post-race, after the third stage. The exercise increased TNFα, IL6, IL2 and IFNγ levels, whereas IL1β was unchanged. In conclusion, cyclist stages induced oxidative and cellular muscle damage which is partially recovered to basal values by the next morning. Repetitive stages during the cycling competition accumulated plasma muscular damage and lipid peroxidation markers and pro-inflammatory cytokines, probably as a result of local inflammatory responses. © 2014 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd.

X-ray absorption spectroscopic evidence for binding of the competitive inhibitor 2-mercaptoethanol to the nickel sites of Jack bean urease. A new Ni-Ni interaction in the inhibited enzyme

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

Clark, P.A.; Wilcox, D.E.; Scott, R.A.

The enzyme Jack bean urease has been identified as the first nickel-containing metalloenzyme to catalyze the hydrolysis of urea to carbon dioxide and ammonia. Competitive inhibitors such as 2-mercaptoethanol (2-ME) have been shown to dramatically affect the ground-state electronic properties of the urease Ni(II) ions. Results of preliminary structural investigations using x-ray absorption spectroscopy of the nickel salts of urease in its native and 2-ME bound forms are presented. The binding of 2-ME to Ni(II) through the thiolate sulfur is confirmed by the results of this study. 17 refs., 2 figs., 2 tabs.

Competition between crystallization and vitrification of the rigid amorphous fraction in poly(3-hydroxybutyrate)

NASA Astrophysics Data System (ADS)

Di Lorenzo, Maria Laura; Righetti, Maria Cristina; Gazzano, Massimo

2012-07-01

Semicrystalline polymers have a metastable nanophase structure, where the various nanophases can be crystal, liquid, glass, or mesophase. This multi-level structure is determined by a competition among self-organization, crystallization, and vitrification of the amorphous segments and is established during material processing. The kinetics of such competition is here determined for poly(3-hydroxybutyrate) (PHB), as vitrification/devitrification of the rigid amorphous fraction strongly affects crystallization kinetics of PHB.

65 nm LP/GP mix low cost platform for multi-media wireless and consumer applications

NASA Astrophysics Data System (ADS)

Tavel, B.; Duriez, B.; Gwoziecki, R.; Basso, M. T.; Julien, C.; Ortolland, C.; Laplanche, Y.; Fox, R.; Sabouret, E.; Detcheverry, C.; Boeuf, F.; Morin, P.; Barge, D.; Bidaud, M.; Biénacel, J.; Garnier, P.; Cooper, K.; Chapon, J. D.; Trouiller, Y.; Belledent, J.; Broekaart, M.; Gouraud, P.; Denais, M.; Huard, V.; Rochereau, K.; Difrenza, R.; Planes, N.; Marin, M.; Boret, S.; Gloria, D.; Vanbergue, S.; Abramowitz, P.; Vishnubhotla, L.; Reber, D.; Stolk, P.; Woo, M.; Arnaud, F.

2006-04-01

A complete 65 nm CMOS platform, called LP/GP Mix, has been developed employing thick oxide transistor (IO), Low Power (LP) and General Purpose (GP) devices on the same chip. Dedicated to wireless multi-media and consumer applications, this new triple gate oxide platform is low cost (+1mask only) and saves over 35% of dynamic power with the use of the low operating voltage GP. The LP/GP mix shows competitive digital performance with a ring oscillator (FO = 1) speed equal to 7 ps per stage (GP) and 6T-SRAM static power lower than 10 pA/cell (LP). Compatible with mixed-signal design requirements, transistors show high voltage gain, low mismatch factor and low flicker noise. Moreover, to address mobile phone demands, excellent RF performance has been achieved with FT = 160 GHz for LP and 280 GHz for GP nMOS transistors.

Aerospace Workforce Development: The Nebraska Proposal; and Native Connections: A Multi-Consortium Workforce Development Proposal

NASA Technical Reports Server (NTRS)

Bowen, Brent; Vlasek, Karisa; Russell, Valerie; Teasdale, Jean; Downing, David R.; deSilva, Shan; Higginbotham, Jack; Duke, Edward; Westenkow, Dwayne; Johnson, Paul

2004-01-01

This report contains two sections, each of which describes a proposal for a program at the University of Nebraska. The sections are entitled: 1) Aerospace Workforce Development Augmentation Competition; 2) Native Connections: A Multi-Consortium Workforce Development Proposal.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*

PubMed Central

Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.

2016-01-01

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

PubMed

Mutti, Elena; Hunger, Miriam; Fedosov, Sergey; Nexo, Ebba; Kräutler, Bernhard

2017-11-16

The synthesis and structural characterization of Co-(dN) 25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) 39 -Cbl, which are organometallic DNA-B 12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) 25 -Cbl, or radiolabeled vitamin B 12 ( 57 Co-CNCbl) and Co-(dN) 25 -Cbl or Co-(dN) 39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) 18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development

PubMed Central

Li, Shutian

2015-01-01

The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate internal developmental cues with external environmental stimuli to orchestrate plant development. PMID:26039357

The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development.

PubMed

Li, Shutian

2015-01-01

The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate internal developmental cues with external environmental stimuli to orchestrate plant development.

Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

PubMed

Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

2001-02-28

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

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

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release

DOE PAGES

Warren, Christopher; Matsui, Tsutomu; Karp, Jerome M.; ...

2017-12-20

Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowedmore » us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.« less

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

PubMed

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Development of machine learning models to predict inhibition of 3-dehydroquinate dehydratase.

PubMed

de Ávila, Maurício Boff; de Azevedo, Walter Filgueira

2018-04-20

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD-targeted model to predict binding affinity. Combination of high-resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure. © 2018 John Wiley & Sons A/S.

Linear scaffolds for multivalent targeting of melanocortin receptors.

PubMed

Dehigaspitiya, Dilani Chathurika; Anglin, Bobbi L; Smith, Kara R; Weber, Craig S; Lynch, Ronald M; Mash, Eugene A

2015-12-21

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Obama Pushes Pre-K, Competitive Grants in Budget

ERIC Educational Resources Information Center

Klein, Alyson

2013-01-01

President Barack Obama's latest budget proposal envisions a sweeping, multi-billion-dollar expansion of prekindergarten programs and doubles down on the administration's strategy of using competitive grants to drive big change in states and districts--all as school districts try to cope with the largest cuts to federal education spending in recent…

The interaction between predator strategy and prey competition in a pair of multi-predator multi-prey lattices

NASA Astrophysics Data System (ADS)

Abernethy, Gavin M.; McCartney, Mark; Glass, David H.

2018-03-01

A computational study of a system of ten prey phenotypes and either one or ten predator phenotypes with a range of foraging behaviours, arranged on two separate one-dimensional lattices, is presented. Mutation between nearest neighbours along the prey lattice occurs at a constant rate, and mutation may or may not be enabled for the predators. The significance of competition amongst the prey is investigated by testing a variety of distributions of the relative intraspecific and interspecific competition. We also study the influence this has on the survival and population size of predator phenotypes with a variety of foraging strategies. Our results indicate that the distribution of competition amongst prey is of little significance, provided that intraspecific is stronger than the interspecific, and that it is typically preferable for a predator to adopt a foraging strategy that scales linearly with prey population sizes if it is alone. In an environment of multiple predator phenotypes, the least or most-focused predators are most likely to persist, dependent on the feeding parameter.

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

PubMed Central

Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

2015-01-01

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

Development of a Luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT).

PubMed

Anderson, George P; Lamar, Jacqueline D; Charles, Paul T

2007-04-15

Previously, a displacement immunoassay for 2,4,6-trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples; then biotinylated anti-TNT antibody and the reporter molecule, Streptavidin-R-Phycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (<1.0 ng/mL) and a dynamic range up to 1 microg/mL. Other factors such as reaction time, cross reactivity to other nitro-compounds, evaluation of acetone extracts of TNT contaminated soils, testing in environmental matrices such as fresh water and seawater were all completed. Finally, a multiplex assay for TNT and three protein toxins was successfully conducted using the competitive format.