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Competitive Binding (competitive + binding)
Selected AbstractsCooperative and Competitive Binding in Synergistic Mixtures of Thermobifida fuscaCellulases Cel5A, Cel6B, and Cel9ABIOTECHNOLOGY PROGRESS, Issue 4 2002Tina Jeoh Synergism between cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fuscaCel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled cellulase concentrations in binary component mixtures was in the range of 7,9%. Data at 5 °C show that binding levels of cellulases in mixture reactions are only 22,70% of the binding levels in single component reactions. At 50 °C, however, most of the cellulase components in the same mixtures bound to extents of 40,126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 °C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE < 1 was generally observed for the reactions at 5 °C indicating anti-synergistic behavior. Degrees of synergistic binding (DSB) were also calculated, where anti-synergistic mixtures had DSB < 1 and synergistic mixtures had DSB>1. We conclude that the lower extents of binding at 5 °C are due to competition for binding sites by the cellulase components in the mixtures and the enhanced binding extents at 50 °C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis. [source] Stability of Anion Binding with Monomers of a Cationic SurfactantCHEMPHYSCHEM, Issue 6 2008Anna Jakubowska Dr. Competitive binding: Electrospray ionisation mass spectrometry is used to probe the binding ability of different anions with a cationic surfactant. Bond strengths are estimated from plots of the intensity of the peak assigned to a given complex ion in the mass spectrum versus the cone voltage applied to induce the abstraction of the counterions from the monomers (see graph). [source] Determination of dissociation constants by competitive binding in partial filling capillary electrophoresisELECTROPHORESIS, Issue 7-8 2004Mikael Nilsson Abstract The determination of dissociation constands (Kd) by competitive ligand binding in partial filling capillary electrophoresis is demonstrated. Two different strategies were applied, one of which only uses a single reporter ligand and a more elaborated one which suppresses systemic disturbances by using a racemic mixture as reporter. The dissociation constants obtained by both alternatives were virtually identical and in good agreement with those previously reported. [source] Flow-through partial-filling affinity capillary electrophoresis can estimate binding constants of neutral ligands to receptors via a competitive assay techniqueELECTROPHORESIS, Issue 6 2003John Kaddis Abstract This work evaluates the use of a competitive binding assay using flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of neutral ligands to a receptor. We demonstrate this technique using, as a model system, carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides. In this technique, the capillary is first partially filled with a negatively charged ligand, a sample containing CAB and two noninteracting standards, and a neutral ligand, then electrophoresed. Upon application of a voltage the sample plug migrates into the plug of negatively charged ligand (L,) resulting in the formation of a CAB-L, complex. Continued electrophoresis results in mixing between the neutral ligand (L0) and the CAB-L, complex. L0 successfully competes out L, to form the new CAB-L0 complex. Analysis of the change in the relative migration time ratio (RMTR) of CAB relative to the noninteracting standards, as a function of neutral ligand concentration, yields a value for the binding constant. These values are in agreement with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented. [source] Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and humanENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2007Vickie S. Wilson Abstract Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor , (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols. [source] Characterization of putative ligands for a fish gonadal androgen receptor in a pulp mill effluentENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006D. G. Joakim Larsson Abstract Fish exposed to pulp and paper mill effluents often become masculinized. A plausible hypothesisis that this is caused by activation of androgen receptors. The present study aimed to investigate if ligands for the fish gonadal androgen receptor (AR2) are present in pulp mill effluent and to characterize/identify these compounds. Extracts of both primary and biologically treated effluents from a Swedish kraft pulp mill were fractionated chemically. Fractions were tested in competitive binding assays for AR2 from ovaries of the Atlantic croaker (Micropogonias undulatus). Primary effluent contained 96 ng dihydrotestosterone equivalents/L, whereas biologically treated effluent was 16 times less potent. Further fractionations and assays of binding activities were performed on the primary effluent. Eight final fractions displaced androgen in the binding assay, and gas chromatography/mass spectrometry (GC/MS) analyses revealed that these contained 37 detectable compounds that were not present in inactive fractions. The majority were moderately polar compounds between 200 and 400 g/mol with hydroxyl/carbonyl groups. Two compounds were ruled out because of their lack of binding to AR2. The mass spectra of a third compound matched that of 4-hydroxy-3 (2-(4-hydroxy-3methoxophenyl)ethyl)-5-metoxyacetophenon, but the remaining candidates could not be fully identified. A search for 21 known steroidal AR2 ligands showed that progesterone, a relatively strong AR2 ligand, was present in the primary effluent (1.6 ,g/L) but was removed during the biological treatment step. The detection of multiple fractions with significant binding activity indicates that a variety of compounds in effluents have the potential to masculinize fish near pulp mills via an androgen receptor-mediated mechanism. [source] Biotic ligand model of the acute toxicity of metals.ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2001Abstract The biotic ligand model (BLM) of acute metal toxicity to aquatic organisms is based on the idea that mortality occurs when the metal,biotic ligand complex reaches a critical concentration. For fish, the biotic ligand is either known or suspected to be the sodium or calcium channel proteins in the gill surface that regulate the ionic composition of the blood. For other organisms, it is hypothesized that a biotic ligand exists and that mortality can be modeled in a similar way. The biotic ligand interacts with the metal cations in solution. The amount of metal that binds is determined by a competition for metal ions between the biotic ligand and the other aqueous ligands, particularly dissolved organic matter (DOM), and the competition for the biotic ligand between the toxic metal ion and the other metal cations in solution, for example, calcium. The model is a generalization of the free ion activity model that relates toxicity to the concentration of the divalent metal cation. The difference is the presence of competitive binding at the biotic ligand, which models the protective effects of other metal cations, and the direct influence of pH. The model is implemented using the Windermere humic aqueous model (WHAM) model of metal,DOM complexation. It is applied to copper and silver using gill complexation constants reported by R. Playle and coworkers. Initial application is made to the fathead minnow data set reported by R. Erickson and a water effects ratio data set by J. Diamond. The use of the BLM for determining total maximum daily loadings (TMDLs) and for regional risk assessments is discussed within a probabilistic framework. At first glance, it appears that a large amount of data are required for a successful application. However, the use of lognormal probability distributions reduces the required data to a manageable amount. [source] Biotic ligand model of the acute toxicity of metals.ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2001Abstract The biotic ligand model (BLM) was developed to explain and predict the effects of water chemistry on the acute toxicity of metals to aquatic organisms. The biotic ligand is defined as a specific receptor within an organism where metal complexation leads to acute toxicity. The BLM is designed to predict metal interactions at the biotic ligand within the context of aqueous metal speciation and competitive binding of protective cations such as calcium. Toxicity is defined as accumulation of metal at the biotic ligand at or above a critical threshold concentration. This modeling framework provides mechanistic explanations for the observed effects of aqueous ligands, such as natural organic matter, and water hardness on metal toxicity. In this paper, the development of a copper version of the BLM is described. The calibrated model is then used to calculate LC50 (the lethal concentration for 50% of test organisms) and is evaluated by comparison with published toxicity data sets for freshwater fish (fathead minnow, Pimephales promelas) and Daphnia. [source] Functional approach to investigate Lp(a) in ischaemic heart and cerebral diseasesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2003A. De La Peña-Díaz Abstract Background Lp(a), a major cardiovascular risk factor, contains a specific apolipoprotein, apo(a), which by virtue of structural homology with plasminogen inhibits the formation of plasmin, the fibrinolytic enzyme. A number of clinical reports support the role of Lp(a) as a cardiovascular or cerebral risk factor, and experimental data suggest that it may contribute to atherothrombosis by inhibiting fibrinolysis. Design A well-characterized model of a fibrin surface and an apo(a)-specific monoclonal antibody were used to develop a functional approach to detect pathogenic Lp(a). The assay is based on the competitive binding of Lp(a) and plasminogen for fibrin, and quantifies fibrin-bound Lp(a). High Lp(a) binding to fibrin is correlated with decreased plasmin formation. In a transversal case,control study we studied 248 individuals: 105 had a history of ischaemic cardiopathy (IC), 52 had cerebro-vascular disease (CVD) of thrombotic origin, and 91 were controls. Results The remarkably high apo(a) fibrin-binding in CVD (0·268 ± 0·15 nmol L,1) compared with IC (0·155 ± 0·12 nmol L,1) suggests the existence of peculiar and poorly understood differences in pro- or anti-thrombotic mechanisms in either cerebral and/or coronary arteries. Conclusions Our results demonstrated that Lp(a) fibrin-binding and small Apo(a) isoforms are associated with athero-thrombotic disease. [source] A human-specific TNF-responsive promoter for Goodpasture antigen-binding proteinFEBS JOURNAL, Issue 20 2005Froilán Granero The Goodpasture antigen-binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head-to-head with POLK, the gene encoding for DNA polymerase kappa (pol ,), and shares with it a 140-bp promoter containing a Sp1 site, a TATA-like element, and a nuclear factor kappa B (NF,B)-like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NF,B that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NF,B recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NF,B to a DNA element encompassing a NF,B-like site that is pivotal for the 140-bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA-like element but is devoid of the NF,B-like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human-specific TNF-responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity. [source] Binding of the volatile general anesthetics halothane and isoflurane to a mammalian ,-barrel proteinFEBS JOURNAL, Issue 2 2005Jonas S. Johansson A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic,protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large ,-barrel internal cavity (515 ± 30 Å3) lined predominantly by aromatic and aliphatic residues. Halothane binding to the ,-barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the ,-barrel of porcine odorant binding protein with dissociation constants of 0.46 ± 0.10 mm and 0.43 ± 0.12 mm determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with Kd values of 80 ± 10 µm and 100 ± 10 µm, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by ,0.9 ± 0.1 kcal·mol,1. In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 Å resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules. [source] Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitroGENES TO CELLS, Issue 11 2000Aziz Taghbalout Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region. [source] Electrosynthesized Surface-Imprinted Conducting Polymer Microrods for Selective Protein RecognitionADVANCED MATERIALS, Issue 22 2009Anna Menaker Novel surface-imprinted conducting polymer microrods are shown to selectively recognize the template protein, as demonstrated by competitive binding assays using fluorescence detection. The electrochemical template synthesis provides means for controlled fabrication and spatial confinement of the PEDOT/PSS polymer proposed in this work, which exhibits extraordinary low nonspecific interactions and is effectively turned into a selective protein sorbent upon imprinting. [source] Hyaluronan synthase-3 is upregulated in metastatic colon carcinoma cells and manipulation of expression alters matrix retention and cellular growthINTERNATIONAL JOURNAL OF CANCER, Issue 5 2003Kelli M. Bullard Abstract HA is a glycosaminoglycan that is synthesized on the inner surface of the plasma membrane and secreted into the pericellular matrix. HA and its biosynthetic enzymes (HAS1, HAS2 and HAS3) are thought to participate in tumor growth and cancer progression. In our study, colon carcinoma cells isolated from a lymph node metastasis (SW620) produced more pericellular HA and expressed higher levels of HAS3 mRNA compared to cells isolated from a primary colon carcinoma (SW480). To assess functionality, HAS3 expression in SW620 cells was inhibited by transfection with an asHAS3 construct. Decreased HA secretion and cell-surface retention by asHAS3 transfectants were confirmed using competitive binding and particle exclusion assays. Anchorage-independent growth, a correlate of tumor growth in vivo, was assessed by colony formation in soft agar. SW620 cells stably transfected with asHAS3 demonstrated significant growth inhibition, as evidenced by fewer colonies and smaller colony area than either SW620 cells or cells transfected with vector alone. Addition of exogenous HA restored growth in asHAS3 transfectants. Thus, we demonstrate that pericellular HA secretion and retention and HAS3 expression are increased in metastatic colon carcinoma cells relative to cells derived from a primary tumor. Inhibition of HAS3 expression in these cells decreased the pericellular HA matrix and inhibited anchorage-independent growth. These data suggest that HA and HAS3 function in the growth and progression of colon carcinoma. © 2003 Wiley-Liss, Inc. [source] Deuterium isotope effects observed during competitive binding chiral recognition electrospray ionization,mass spectrometry of cinchona alkaloid-based systemsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2006Kevin A. Schug Abstract Deuterium isotope effects are reported for binding between tert -butylcarbamoyl-quinine/quinidine chiral selectors and isotopomeric quasienantiomers of N -(3,5-dinitrobenzoyl)leucine measured using electrospray ionization-mass spectrometry (ESI-MS) and competitive binding. Evaluation of mixtures of each selector with one labeled and one unlabeled enantiomeric selectand of identical configuration showed a significant difference in measured ion abundances of diastereomeric complexes between the selector and each selectand. It was found that in some cases, the complex containing the nondeuterated selectand was 15% more abundant than its deuterated counterpart. On the basis of an assessment of solution- and gas-phase isotope effects reported in the literature, a series of control experiments were performed to study the origin of the effects. On the basis of these measurements, our preliminary conclusion is that the differing gas-phase physicochemical nature of the deuterated versus nondeuterated selectand represents the strongest contribution to the observed effect in this chiral molecular recognition system. Copyright © 2006 John Wiley & Sons, Ltd. [source] Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite greenJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2009Xing Zhang Abstract In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13,50 nucleotide "guest" RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF "ribosomal RNA aptamer." Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo. Copyright © 2009 John Wiley & Sons, Ltd. [source] Amitriptyline has a dual effect on the conductive properties of the epithelial Na channelJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2002Florentina Pena This study was undertaken with the aim of testing the action of amitriptyline on the epithelial Na channel (ENaC), which belongs to the same family (Deg/ENaC) as ASICs (acid-sensing ion channels) and many other putative members in the brain. We assumed that, having a common protein structure, characterization of the amitriptyline-ENaC interaction could help to elucidate the analgesic mechanism of this tricyclic antidepressant. Na-channel characteristics were derived from the analysis of blocker-induced lorentzian noise produced by amiloride. The effect of amitriptyline, present in the mucosal bathing solution, on the transepithelial short-circuit current (1sc) and conductance (Gt), and on the blocker-induced noise of apical Na channels, was studied on isolated ventral skin of the frog Rana ridibunda. Amitriptyline exerted a dual effect on the macroscopic short-circuit current and conductance of the epithelia, increasing these two parameters in the concentration range 0.1,50 ,M, while at higher concentrations (100,1000 ,M) it showed an inhibitory action. The decrease in the association rate (k01) of amiloride to the apical Na channels from 15.6 ± 4.2 ,M,1 S,1 in control Cl-Ringer to 7.4 ± 1.7 ,M,1 S,1 at 200 ,M amitriptyline in a concentration-dependent manner suggests a competitive binding of amitriptyline to the pyrazine ring binding site for amiloride. [source] A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12 -dependent Escherichia coli 113/3 cellsMOLECULAR MICROBIOLOGY, Issue 3 2000Christopher N. Penfold The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343,418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1,497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12 -dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive. [source] Identification of the peptide motifs that interact with HLA-DR8 (DRB1*0802) in Streptococcus mutans proteinsMOLECULAR ORAL MICROBIOLOGY, Issue 4 2002Y. Nomura A glucosyltransferase (GTF) and a surface protein antigen (PAc) of Streptococcus mutans have been suggested as possible components of an effective dental caries vaccine. To identify antigenic peptides in GTF and PAc that bind to MHC class II (HLA-DR8, DRB1*0802) molecules, we investigated binding activities to DR8 molecules of overlapping synthetic peptides at several sites in GTF and in the alanine-rich repeating region of PAc using an ELISA-inhibition competitive binding assay for the interaction between the HLA-DR molecule and the PAc (316,334) peptide. Six GTF peptides and 10 PAc peptides strongly bound to the HLA-DR8 molecule. In a homology analysis of the amino acid sequences of the six GTF peptides, two binding motifs were found in L/Y, ,Y/L,A/N and Y/L, ,N/G/E, ,Y,V/L/P. Moreover, a new binding motif in PAc was found in L- ,Y-A. It is suggested that these binding motifs could be useful in designing a dental caries vaccine in humans. [source] GABAergic phthalide dimers from Angelica sinensis (Oliv.) DielsPHYTOCHEMICAL ANALYSIS, Issue 6 2006Shixin Deng Abstract The methanol extract of Angelica sinensis (Oliv.) Diels roots (Dang Gui) has been shown to exhibit competitive binding to the GABAa receptor, suggesting the presence of GABAergic ligands. Chromatographic fractionation of the methanol extract led to the isolation of two GABAergic dimeric phthalides 1 and 2. Gelispirolide (1) was elucidated as a new phthalide dimer composed of a Z -ligustilide and a Z -butylidenephthalide unit on the basis of spectroscopic approaches including one- and two-dimensional NMR, HRESIMS and HRESIMS-MS. Compound 2 was identified as the known dimeric phthalide, riligustilide, by comparison of its spectroscopic data with literature values. Its dimeric linkage and stereochemistry were ascertained by a single crystal X-ray diffraction experiment. Both dimers 1 and 2 were found to be active in an in vitro GABAa receptor-binding assay with IC50 values of 29 and 24 µm, respectively. Copyright © 2006 John Wiley & Sons, Ltd. [source] ,-Catenin dysregulation in cancer: interactions with E-cadherin and beyond,THE JOURNAL OF PATHOLOGY, Issue 2 2010Qun Lu Abstract Stable E-cadherin-based adherens junctions are pivotal in maintaining epithelial tissue integrity and are the major barrier for epithelial cancer metastasis. Proteins of the p120ctn subfamily have emerged recently as critical players for supporting this stability. The identification of the unique juxtamembrane domain (JMD) in E-cadherin that binds directly to ,-catenin/NPRAP/neurojungin (CTNND2) and p120ctn (CTNND1) provides a common motif for their interactions. Recently, crystallographic resolution of the JMD of p120ctn further highlighted possibilities of intervening between interactions of p120ctn subfamily proteins and E-cadherin for designing anti-cancer therapeutics. For most epithelial cancers, studies have demonstrated a reduction of p120ctn expression or alteration of its subcellular distribution. On the other hand, ,-catenin, a primarily neural-enriched protein in the brain of healthy individuals, is up-regulated in all cancer types that have been studied to date. Two research articles in the September 2010 issue of The Journal of Pathology increase our understanding of the involvement of these proteins in lung cancer. One reports the identification of rare p120ctn (CTNND1) gene amplification in lung cancer. One mechanism by which ,-catenin and p120ctn may play a role in carcinogenesis is their competitive binding to E-cadherin through the JMD. The other presents the first vigorous characterization of ,-catenin overexpression in lung cancer. Unexpectedly, the authors observed that ,-catenin promotes malignant phenotypes of non-small cell lung cancer by non-competitive binding to E-cadherin with p120ctn in the cytoplasm. Looking towards the future, the understanding of ,-catenin and p120ctn with and beyond their localization at the cell,cell junction should provide further insight into their roles in cancer pathogenesis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Invited Commentary for Castillo et al. Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer, Journal of Pathology, 2010; 222: 89,98. And for Zhang et al. ,-Catenin promotes malignant phenotype of non-small cell lung cancer by non-competative binding to E-cadherin with p120ctn in cytoplasm. Journal of Pathology, 2010; 222: 76,88. [source] Structure of human transthyretin complexed with bromophenols: a new mode of bindingACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2000Minakshi Ghosh The binding of two organohalogen substances, pentabromophenol (PBP) and 2,4,6-tribromophenol (TBP), to human transthyretin (TTR), a thyroid hormone transport protein, has been studied by in vitro competitive binding assays and by X-ray crystallography. Both compounds bind to TTR with high affinity, in competition with the natural ligand thyroxine (T4). The crystal structures of the TTR,PBP and TTR,TBP complexes show some unusual binding patterns for the ligands. They bind exclusively in the `reversed' mode, with their hydroxyl group pointing towards the mouth of the binding channel and in planes approximately perpendicular to that adopted by the T4 phenolic ring in a TTR,T4 complex, a feature not observed before. The hydroxyl group in the ligands, which was previously thought to be a key ingredient for a strong binding to TTR, does not seem to play an important role in the binding of these compounds to TTR. In the TTR,PBP complex, it is primarily the halogens which interact with the TTR molecule and therefore must account for the strong affinity of binding. The interactions with the halogens are smaller in number in TTR,TBP and there is a decrease in affinity, even though the interaction with the hydroxyl group is stronger than that in the TTR,PBP complex. [source] Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved-core alginate microspheresBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Ayesha Chaudhary Abstract The feasibility of dissolved-core alginate-templated fluorescent microspheres as "smart tattoo" glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye-labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution-phase counterparts. The glucose sensitivity of the encapsulated TRITC-Con A/FITC-dextran (500,kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC-apo-GOx/FITC-dextran (500,kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0,50,mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120,s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0,50,mM glucose, using near-infrared dyes (Alexa Fluor-647-labeled dextran as donor and QSY-21-conjugated apo-GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075,1085. © 2009 Wiley Periodicals, Inc. [source] | ||||||||||||||||