Accumulation of mu-type opiate receptor ligands during the culture of phytohemagglutinin-stimulated lymphocytes

Substances displacing labelled ligands from opiate receptors of the rat brain membrane fractions were found in the extracts of human blood lymphocytes. Pronase treatment has shown that at least part of the opiate receptor ligands present in lymphocyte extracts are of peptide nature. Opiate mu-receptor ligands content was identical in the extracts of the total population, T-lymphocyte and B-cell-enriched population. However, opiate delta-receptor ligands concentration in T-cell population is 15 times lower than in B-cell-enriched population. Opiate receptor ligands content is not changed during cultivation of nonstimulated lymphocytes. However, phytohemagglutinin activation results in a threefold increase of opiate mu-receptor ligands content in lymphocytes. The results obtained suggest that synthesis and processing of opiate mu-receptor ligands precursor occur in stimulated T-lymphocytes.     

Exploration of the structure-activity relationship of the diaryl anilide class of ligands for translocator protein-potential novel positron emitting tomography imaging agents

A series of novel ligands based on the diaryl anilide (DAA) class of translocator protein (TSPO) ligands was synthesised and evaluated as potential positron emitting tomography (PET) ligands for imaging TPSO in vivo. Fluorine-18 labelling of the molecules was achieved using direct radiolabelling or synthon based labelling approaches. Several of the ligands prepared have promising profiles as potential TSPO PET imaging ligands and will be evaluated further as potential clinical imaging agents.     

Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action

Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti-receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors.     

Kinetics of adsorption of extended ligands on DNA at small fillings

In the present work, the adsorption kinetics of extended ligands on DNA duplexes at small fillings when molecules of DNA duplexes are on the underlayer within diffusion layer has been investigated. Both diffusion of ligands in solution (diffusion stage) and adsorption of ligands (kinetic stage) are taken into consideration at adsorption of ligands on DNA duplexes. Nonlinear system of differential equations describing adsorption of ligands where not only diffusion stage but also kinetic stage is taken into account, is obtained, moreover the equations allow localizing duplexes in arbitrary place within diffusion layer. Numeric solution of the equations makes possible to investigate the filling kinetics of DNA duplexes by ligands depending on parameters controlling adsorption process. It has been shown that depending on relation between adsorption parameters different kinetic regimes of adsorption – kinetic, complex, and diffusion regimes may be realized.     

Studies of cation binding in ZnCl2-regenerated bacteriorhodopsin by x-ray absorption fine structures: effects of removing water molecules and adding Cl- ions.

The binding of Zn2+ in Zn2+-regenerated bacteriorhodopsin (bR) was studied under various conditions by x-ray absorption fine structures (XAFS). The 0.9:1 and 2:1 Zn2+:bR samples gave similar XAFS spectra, suggesting that Zn2+ might have only one strong binding site in bR. It was found that in aqueous bR solution, Zn2+ has an average of six oxygen or nitrogen ligands. Upon drying, two ligands are lost, suggesting the existence of two weakly bound water ligands near the cation-binding site in bacteriorhodopsin. When excess Cl- ions were present before drying in the Zn2+-regenerated bR samples, it was found that two of the ligands were replaced by Cl- ions in the dried film, whereas two remain unchanged. The above observations suggest that Zn2+ has three types of ligands in regenerated bR (referred to as types I, II, and III). Type I ligands are strongly bound. These ligands cannot be removed by drying or by exchanging with Cl- ions. Type II ligands cannot be removed by drying, but can be replaced by Cl- ligands. Type III ligands are weakly bound to the metal cation and are most likely water molecules that can be removed by evaporation under vacuum or by drying with anhydrous CaSO4. The results are discussed in terms of the possible structure of the strongly binding site of Zn2+ in bR.     

Thermodynamic models of binding ligands to nucleic acids

Models of adsorption were considered, which describe the binding of biologically active ligands on DNA templates. The binding is described most comprehensively and in greatest detail by the distribution function, which determines the probability of detecting the preset number of adsorbed ligands on the template. In the case of noncooperative binding, this function corresponds to the Gaussian distribution and is characterized by two quantities: the mean value of the occupation of the template by ligands and the dispersion of occupation. The accuracy of the occupation of the template by ligands is inversely proportional to dispersion. As the length of the template and the number of reaction sites covered by one ligand upon binding increase, the accuracy of the occupation of the template by ligands increases. An important characteristic of binding is the degree of coverage of the template by ligands. This characteristic represents the portion of template reaction sites covered by all ligands adsorbed on the template. If polycations are bound to nucleic acid molecules, the coverage of the template determines the transition of nucleic acids to a compact state. The degree of template coverage for extended ligands depends only slightly on the binding constant in a wide range of concentrations of a free ligand in solution. Different adsorption models are considered from the unified point of view. The classification of cooperative interactions for a wide class of systems is given, from situations when several ligands are bound on nucleic acid templates to a situation when templates change by the action of ligands and begin to interact with each other.     

Isotherm of ligand adsorption on DNA at multiplicative noise

Fluctuations of the number of ligands adsorbed on macromolecules are considered in the case when the number of ligands in the solution fluctuates under the action of fluctuations of the external medium (external noise). For the case of small filling, the multiplicative type of stochastic differential equation is obtained, describing the time variation of the number of ligands adsorbed on macromolecules. The isotherm of adsorption of ligands on DNA is obtained. It is shown that at small ligand concentrations, for some relations between adsorption parameters and the intensity of the external noise, no macromolecule adsorption of ligands takes place.     

Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands

BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.     

Affinity partitioning and extraction of proteins.

Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed. Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported. The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples. The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin. The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.     

Amphiregulin regulates the production of human chorionic gonadotropin in trophoblasts

AimsThe aim of this study was to investigate the significance of epidermal growth factor receptor (EGFR) ligands produced in syncytiotrophoblasts during normal pregnancy.Main methodsWe examined the expression of EGFR ligands in human pregnancy by real-time PCR, and analyzed the relationship between EGFR ligands and human chorionic gonadotropin (hCG) or human placental lactogen in amniotic fluid by ELISA. In addition, we also examined the EGFR ligands in syncytiotrophoblasts and the amount of hCG secretion in JAR, JEG3 and BeWo cells in the presence of each EGFR ligand.Key findingsIn order to identify possible candidates among the EGFR ligands, we examined the predominant expression of an EGFR ligand in the chorionic villi and amniotic fluid during normal pregnancy, and analyzed the relationship between EGFR ligands and hCG in trophoblastic model cells. Amphiregulin was primarily expressed throughout human pregnancy and stimulated the secretion of hCG, indicating that amphiregulin is a key molecule among EGFR ligands.SignificanceAmphiregulin may play a pivotal role in the development or maturation of placenta.     

Synthesis and pharmacological characterization of bivalent ligands of epibatidine at neuronal nicotinic acetylcholine receptors

A series of bivalent ligands 6a-d of epibatidine were synthesized. All four ligands showed nanomolar binding affinities at six neuronal nicotinic acetylcholine receptor (nAChR) subtypes in competition binding assays. In contrast to epibatidine, these bivalent ligands are weak partial agonists at the alpha3beta4 nAChR as shown by functional assays.     

Combinatorial selection of RNA ligands for complex cellular targets : the RNA liagands-based proteomics

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution. RNA ligands for the mixed nuclear targets were selected from around 6 x 10(14) RNA sequences through an iterated enrichment process. RNA ligands for various gene products of the extract were simultaneously selected and were shown to specifically interact with their target molecules. The target molecules were isolated from the nuclear extract by affinity chromatography using columns tagged with the RNA ligands, resolved on two-dimensional gels, and identified by mass spectrometry. These RNA ligands may be useful in characterizing novel functions of cellular proteins and modulating complex molecular events.     

Highly flexible ligand binding pocket of ecdysone receptor: a single amino acid change leads to discrimination between two groups of nonsteroidal ecdysone agonists

The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

Studies on endotoxin removal mechanism of adsorbents with amino acid ligands

In this paper, a series of adsorbents with different amino acid ligands for endotoxin removal were prepared and endotoxin adsorption capacities (EAC) in aqueous solution were studied using an affinity column. The results showed that the property and structure of amino acid ligands have great influence on EAC. As the increasing of isoelectric point and polarity of amino acids ligands, EACs of the adsorbents increased. In addition, computer simulation method was employed to a further investigation on the interaction between endotoxins and ligands. Based on the results, some adsorbents were applied to remove endotoxin from endotoxemia rabbit's serum. Similar adsorption results were observed and the removal efficiency of adsorbents with Arg, Ser ligands is up to 78%.     

Assessment of lead speciation by organic ligands using speciation models

Abstract

The biogeochemical behaviour of lead (Pb) in ecosystems greatly depends on its chemical species. Organic ligands strongly influence Pb species and mobility in soil solution. In the present study, two metal speciation models, Windermere Humic Aqueous Model (WHAM) and Visual Minteq are used to compute Pb speciation in nutrient solution in the presence and absence of organic ligands. The three organic ligands used include ethylenediamine tetraacetic acid (EDTA), citric acid (CA) and fulvic acid (FA). The results show that in the absence of organic ligands, Pb²⁺ is the dominant form under acidic conditions and Pb–OH under alkaline conditions. The presence of organic ligands strongly influences Pb speciation. EDTA is more effective than are CA and FA concerning its influence on Pb speciation due to high Pb binding capacity. The results also indicate that Pb binding capacity of organic ligands varies with solution pH.