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Articles de revues sur le sujet "NMR ligand-receptor interaction studies"
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Giragossian, Craig, Maria Pellegrini, and Dale F. Mierke. "NMR studies of CCK-8/CCK1 complex support membrane-associated pathway for ligand-receptor interaction." Canadian Journal of Physiology and Pharmacology 80, no. 5 (2002): 383–87. http://dx.doi.org/10.1139/y02-031.
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The interaction of peptide ligands with their associated G-protein-coupled receptors has been examined by a number of different experimental approaches over the years. We have been developing an approach utilizing high-resolution NMR to determine the structural features of the peptide ligand, well-designed fragments of the receptor, and the ligandreceptor complexes formed upon titration of the peptide hormone. The results from these investigations provide evidence for a membrane-associated pathway for the initial interaction of peptide ligands with the receptor. Here, our results from the investigation of the interaction of CCK-8 with the CCK1 receptor are described. Our spectroscopic results clearly show that both CCK-8 and the regions of CCK1 with which it interacts are closely associated with the zwitterionic interface of the lipids utilized in our solution spectroscopic studies.Key words: G-protein-coupled receptors, NMR structural characterization, cholecystokinin, CCK-8, cholecystokinin receptor, subtype 1, CCK1, peptide hormones.
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Ruan, Ke-He. "High resolution nuclear magnetic resonance spectroscopy-guided mutagenesis for characterization of membrane-bound proteins: Experimental designs and applications." Spectroscopy 18, no. 1 (2004): 13–29. http://dx.doi.org/10.1155/2004/802728.
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High resolution Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for determining the solution structures of peptides and small proteins, and their ligand binding functions. Molecular biology mutagenesis is a widely used and powerful approach for identification of the protein functions. We have developed a strategy integrating NMR experiments with mutagenesis studies to advance and extend the approaches used for structure/function relationship studies of proteins, especially for membrane-bound proteins, which play important roles in physiopathological processes. The procedures include the design of the functional protein domain, identification of the solution structure and intermolecular contacts between the protein segment and its ligand. These determinations are resolved by high-resolution 2D NMR spectroscopy, and followed by site-directed mutagenesis of the residues suggested from the NMR experiment for the membrane-bound proteins. The residues important to the protein functions, identified by the mutagenesis, were further used to re-assign the NMR spectra and finalize the docking of the protein with its ligand. A structural model of the protein/ligand interaction can be constructed at an atomic level based on the NMR spectroscopy and mutagenesis results. As an application, the strategy has enhanced our knowledge in the understanding of the structure/function relationship for a membrane-bound G protein coupling receptor, the thromboxane A2receptor (TP receptor), interacting with its ligand, and a microsomal P450, prostacyclin synthase (PGIS), docking with its substrate in the endoplasmic reticulum (ER) membrane. In this review, we have summarized the principles and applications for this newly developed technique.
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Braitsch, Michaela, Hanspeter Kählig, Georg Kontaxis, et al. "Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR." Beilstein Journal of Organic Chemistry 8 (March 27, 2012): 448–55. http://dx.doi.org/10.3762/bjoc.8.51.
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A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces.
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Assadi-Porter, Fariba, James Radek, Hongyu Rao, and Marco Tonelli. "Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties." Molecules 23, no. 10 (2018): 2531. http://dx.doi.org/10.3390/molecules23102531.
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Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPCR) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to the difficulty in producing large homogeneous quantities of the taste-receptor protein and the lack of reliable in vitro assays to precisely measure productive ligand binding modes that lead to activation of the receptor protein. We report here a multimodal high throughput assay to monitor ligand binding, receptor stability and conformational changes to model the molecular ligand-receptor interactions. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.
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Morales, Paula, Marta Bruix та M. Angeles Jiménez. "Structural Insights into β-arrestin/CB1 Receptor Interaction: NMR and CD Studies on Model Peptides". International Journal of Molecular Sciences 21, № 21 (2020): 8111. http://dx.doi.org/10.3390/ijms21218111.
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Activation of the cannabinoid CB1 receptor induces different cellular signaling cascades through coupling to different effector proteins (G-proteins and β-arrestins), triggering numerous therapeutic effects. Conformational changes and rearrangements at the intracellular domain of this GPCR receptor that accompany ligand binding dictate the signaling pathways. The GPCR-binding interface for G proteins has been extensively studied, whereas β-arrestin/GPCR complexes are still poorly understood. To gain knowledge in this direction, we designed peptides that mimic the motifs involved in the putative interacting region: β-arrestin1 finger loop and the transmembrane helix 7-helix 8 (TMH7-H8) elbow located at the intracellular side of the CB1 receptor. According to circular dichroism and NMR data, these peptides form a native-like, helical conformation and interact with each other in aqueous solution, in the presence of trifluoroethanol, and using zwitterionic detergent micelles as membrane mimics. These results increase our understanding of the binding mode of β-arrestin and CB1 receptor and validate minimalist approaches to structurally comprehend complex protein systems.
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Pintilie, Lucia, Amalia Stefaniu, Alina Ioana Nicu, Maria Maganu, and Miron Teodor Caproiu. "Design, Synthesis and Docking Studies of Some Novel Fluoroquinolone Compounds with Antibacterial Activity." Revista de Chimie 69, no. 4 (2018): 815–22. http://dx.doi.org/10.37358/rc.18.4.6207.
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A new series of fluoroquinolone compounds have been obtained by Gould-Jacobs method. The compounds have been characterized by physic-chemical methods (elemental analysis, FTIR, NMR, UV-Vis) and by antimicrobial activity against Gram-positive and Gram-negative microorganisms. For the synthesized compounds have been performed calculations of characteristics and molecular properties, using Spartan�14 Software from Wavefunction, Inc. Irvine, CA. and molecular docking studies using CLC Drug Discovery Workbench 2.4 software, to identify and visualize the most likely interaction ligand (fluoroquinolone) with the receptor protein.
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Nguyen, Hoa, Tianwei Jing та Xu Wang. "The Q163C/Q309C mutant of αMI-domain is an active variant suitable for NMR characterization". PLOS ONE 18, № 1 (2023): e0280778. http://dx.doi.org/10.1371/journal.pone.0280778.
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Integrin αMβ2 (Mac-1, CD11b/CD18, CR3) is an important adhesion receptor expressed on monocytes. Mac-1 is responsible for mediating cell migration, phagocytosis, degranulation as well as cell-cell fusion. It is also the most promiscuous integrin in terms of ligand specificity with over 100 ligands, most of which use the αMI-domain as their binding site. Despite the importance of αMI-domain in defining ligand interactions of Mac-1, structural studies of αMI-domain’s interactions with ligands are lacking. In particular, solution NMR studies of αMI-domain’s interaction with ligands have not been possible because the most commonly used active αMI-domain mutants (I316G and ΔK315) are not sufficiently stable and soluble to be used in solution NMR. The goal of this study is to identify an αMI-domain active mutant that’s amenable to NMR characterization. By screening known activating mutations of αMI-domain, we determined that the Q163C/Q309C mutant, which converts the αMI-domain into its active form through the formation of an intramolecular disulfide bond, can be produced with a high yield and is more stable than other active mutants. In addition, the Q163C/Q309C mutant has better NMR spectral quality than other active mutants and its affinity for ligands is comparable to other active mutants. Analysis of the Co2+-induced pseudocontact shifts in the Q163C/Q309C mutant showed the structure of the mutant is consistent with the active conformation. Finally, we show that the minor fraction of the Q163C/Q309C mutant without the disulfide bond can be removed through the use of carboxymethyl sepharose chromatography. We think the availability of this mutant for NMR study will significantly enhance structural characterizations of αMI-domain-ligand interactions.
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Bhat, Ishwar K. "SYNTHESIS, DOCKING STUDY AND ANTI-INFLAMMATORY STUDIES OF SOME FLAVANONES." INDIAN DRUGS 58, no. 02 (2021): 54–60. http://dx.doi.org/10.53879/id.58.02.12228.
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In this work, a series of flavanones (P1-P9) was synthesized by cyclization of substituted (hydroxyphenyl)- 3-(phenyl) prop-2-en-1-ones (S1-S9). The structures of the synthesized compounds were characterized by IR, 1 H NMR and mass spectral data. These derivatives were evaluated for anti-inflammatory activity. Compounds P1, P3, P6 and P7 showed excellent anti-inflammatory activity as compared to standard drug diclofenac sodium. Molecular docking of these flavanones (P1-P9) was also performed with receptor phosphoinositide-3-kinase PI3Kδ (PDB code: 5ITD). All the flavanones (P1-P9) were docked into same groove of the binding site of native co-crystallized (5-{4-[3-(4-acetylpiperazine-1-carbonyl) phenyl] quinazolin-6-yl}-2-methoxypyridine carbonitrile) ligand for activity explanation and exhibited good ligand interaction and binding affinity were of range -4.57 to -8.79kcal/mol.
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Pistolesi, Sara, Nico Tjandra, and Guillermo A. Bermejo. "Solution NMR studies of periplasmic binding proteins and their interaction partners." BioMolecular Concepts 2, no. 1-2 (2011): 53–64. http://dx.doi.org/10.1515/bmc.2011.005.
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AbstractPeriplasmic binding proteins (PBPs) are a crucial part of ATP-binding cassette import systems in Gram-negative bacteria. Central to their function is the ability to undergo a large-scale conformational rearrangement from open-unliganded to closed-liganded, which signals the presence of substrate and starts its translocation. Over the years, PBPs have been extensively studied not only owing to their essential role in nutrient uptake but also because they serve as excellent models for both practical applications (e.g., biosensor technology) and basic research (e.g., allosteric mechanisms). Although much of our knowledge at atomic level has been inferred from the detailed, static pictures afforded by crystallographic studies, nuclear magnetic resonance (NMR) has been able to fill certain gaps in such body of work, particularly with regard to dynamic processes. Here, we review NMR studies on PBPs, and their unique insights on conformation, dynamics, energetics, substrate binding, and interactions with related transport proteins. Based on the analysis of recent paramagnetic NMR results, as well as crystallographic and functional observations, we propose a mechanism that could explain the ability of certain PBPs to achieve a closed conformation in absence of ligand while others seem to remain open until ligand-mediated closure.
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Kharche, Shalmali, Manali Joshi, Amitabha Chattopadhyay, and Durba Sengupta. "Conformational plasticity and dynamic interactions of the N-terminal domain of the chemokine receptor CXCR1." PLOS Computational Biology 17, no. 5 (2021): e1008593. http://dx.doi.org/10.1371/journal.pcbi.1008593.
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The dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of chemokine receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces.
Thèses sur le sujet "NMR ligand-receptor interaction studies"
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Guzzetti, I. "INTEGRIN AND CADHERIN LIGANDS: INTERACTION STUDIES BY COMPUTATIONAL METHODS AND BIOAFFINITY NMR ON INTACT CELLS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/243616.
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On a molecular level protein – ligand interactions are central to a number of biological processes, but their investigation is inherently difficult due to several problems, especially for membrane proteins. The study of this type of interactions poses a whole set of challenges, including the characterization of the dynamic behaviour and of the conformational properties of the ligands in complex with the target macromolecules. A variety of biophysical methods have been developed to study protein – ligand interactions and several NMR spectroscopic techniques have emerged as powerful methods to identify and characterize the binding of ligands with receptor proteins. Ligand-based methods do not require labeled protein, since only the ligand NMR signals are detected and only a small amount of protein is required. These techniques are particularly useful in the medium–low affinity range and, therefore they have been adopted to detect ligand interactions in various systems. Among the ligand-based NMR techniques, Saturation Transfer Difference (STD) and transferred-NOE focus on the NMR signals of the ligand and utilize NOE effects between protein and ligand. They are used for: i) the definition of the bioactive conformation of the ligand in the bound state (tr-NOESY), ii) the identification and characterization of the binding mode of the ligand to the receptor with epitope mapping of the ligand itself (STD). The use of the technique is limited to molecules that exhibit a dissociation constant Kd between 10-3 M and 10-7 M.
During my PhD, I had the highly qualifying opportunity to grasp these new potent NMR methods, and to apply them for assessing the interactions of cell surface proteins with peptidomimetics. Since membrane proteins, such as integrins, change their conformation if extracted from their environment, it is clear the importance of working in the biophysical neighbourhood of the membrane itself and not in an isotropic extracellular medium. For this reason, when appropriate to the project, I have carried out NMR experiments using intact cells overexpressing the proteins of interest.
Specifically, two main topics have been addressed:
1. The first and second year of my PhD have been mainly focused on the conformational study of peptidomimetic integrin ligands and on the investigation of their interaction with platelets and cancer cell overexpressing integrins on their membrane. This study has been developed within the framework of a PRIN project (MIUR-PRIN project 2010NRREPL “Synthesis and Biomedical Applications of Tumor-Targeting Peptidomimetics”) in collaboration with the research groups of Proff. Gennari and Piarulli (University of Insubria) as regards the synthetic activities and with the group of Dr. Belvisi as regards the computational and design studies.
2. The second part of my PhD was mainly focused on cadherins, a class of cell adhesion proteins that promote homophilic interactions. This work is at an early stage and has been developed within the framework of a FIRB project coordinated by Dr. Civera (MIUR-FIRB “Futuro in Ricerca” RBFR088ITV “Computer-aided design, synthesis and biological evaluation of peptidomimetics targeting N-cadherin as anticancer agents”). The NMR study has been aimed at obtaining a thorough understanding of the interaction of peptidomimetic molecules with isolated cadherin constructs containing relevant extracellular domains.
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Tengel, Tobias. "Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1472.
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AIROLDI, CRISTINA. "Development of new potential antitumor drugs based on Ras protein inhibition." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2007. http://hdl.handle.net/10281/116562.
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Because of their role in oncogenesis, inhibition of Ras proteins, particularly of their tumorigenic variants, represents today one of the principal strategies finalized to the obtainment of new antitumoral therapies. Among the different possible approaches, one of the most innovative and less explored is represented by the inhibition of this protein activation, key event for the explication of their biological activity, but also for the Ras-induced tumoral cell transformation.
Objective of this thesis has been the development of new small molecules able to inhibit, at least partially (total inhibition in fact would result lethal for cell), Ras protein activation, in particular the GEFs-promoted GDP/GTP
nucleotide exchange.
Inhibitors able of inactivating Ras have been previously described by Schering-Plough. All these molecules contain a phenylhydroxylamino group that binds Ras in a region close to the nucleotide binding site and one aromatic group. Nevertheless, they present some negative characteristics that prevent their employment as potential drugs: (1) they are chemically unstable and (2) they are insoluble in water and in the most commonly used organic solvents.
In order to obtain new more efficient inhibitors, we adopted the rational drug design strategy.
Firstly, we studied the structure-activity relationship (SAR) of Schering-Plough compounds and of new molecules containing variants of their functional groups that we designed and synthesized. The data collected
demonstrated that the phenylhydroxylamino group is an essential pharmacophore, while other positions are not so critical for the biological activity.Keeping in mind this, we prepared new compounds in which the phenylhydroxylamino moiety is supported on glycidic templates, in an attempt to try to take advantage of carbohydrate capability of orienting substituents in space, in this case in a suitable manner for the interaction with Ras proteins. In addition, the sugar portion can improve compound pharmacokinetic properties and decrease their toxicity.
In this way, a new class of Ras inhibitors was obtained, their biological activity and the nature of their interaction with the molecular target were characterized.
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MERLO, SILVIA. "Characterization of biomedical relevant ligand-protein interactions using Nuclear Magnetic Resonance (NMR) Spectroscopy." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/40953.
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NMR techniques allow to obtain structural information useful for the comprehension of biological processes. The aim of this work is to investigate interactions between biological macromolecules and binding partners, including other macromolecules, small ligands and therapeutically relevant compounds. These results will be exploited for the design and development of new potential drugs and medical devices.
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Edwards, Rachel. "Ligand interactions of glutamate receptor 2 studied by NMR." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495042.
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Neurotransmission is the action of communication between neurons; the most common neurotransmitter in the mammalian CNS is glutamate. Excessive activation of glutamate receptors have been implicated in a number of neurodegenerative diseases; they are therefore of great interest for investigation as drug targets. The ionotropic glutamate receptor GluR2 has previously been expressed as a soluble construct representing the ligand binding domain known as S1S2J. GluR2 S1S2J contains two separate binding sites for different types of ligands; the glutamate binding site and the allosteric modulator binding site which is located the dimer interface formed between two GluR2 S1S2J monomers. Binding of ligands to the different sites has been implicated in two different mechanisms for receptor inactivation; both sites are targets for lead drug design. The GluR2 S1S2J construct has been studied in complex with its agonists and antagonists by X-ray crystallography however some questions remain about the exact structure of the ligand within the site and the allosteric modulator binding site has yet to be studied extensively.
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Curtis, Nicola. "Tritium NMR studies of protein-ligand interactions." Thesis, University of Surrey, 1994. http://epubs.surrey.ac.uk/842983/.
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Tritium NMR studies provide a convenient way of obtaining detailed information about conformational equilibria, dynamic processes and specific interactions in protein-ligand complexes provided that suitably 3H-labelled molecules are available. In this study [7,9-3H]- and [3',5',7-3H]folic acid, and [3',5',7-3H]methotrexate were synthesised and the NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analysed as a function of pH (DHFR-folate complexes) and temperature (DHFR-methotrexate complexes). From these data it was possible to obtain further evidence about the orientation of the pteridine ring in the complexes, and to monitor the dynamic processes in the bound ligands. In the 3H NMR spectra of the ternary complexes of the 3H-labelled folic acids with DHFR and NADP+, each labelled tritium gave rise to multiple signals, confirming previous findings that there are three interconverting, pH dependent, conformational forms of bound folate (forms I, IIa and IIb) in the ternary complex. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the methotrexate binary complex and also the ternary complex with NADPH were shown to exist as a single conformational state with the benzoyl ring flipping rate being too slow to give a single averaged signal for the 3',5'-tritium nuclei over the temperature range 283 - 313 K. 3h{1h} Nuclear Overhauser enhancement experiments have been conducted on the small molecules, [3H]dimethyl sulphoxide, [3',5',7-3H]folic acid and [3',5',7-3H]methotrexate as a prelude to 3H-1H heteronuclear NOE experiments on binary and ternary complexes formed using Lactobacillus casei DHFR and the ligands [3',5',7-3H]methotrexate and [3',5',7-3H]folic acid.
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Miyoshi, Emi. "Platinum(II) complexes : studied by diffusion NMR." Thesis, View thesis, 2008. http://handle.uws.edu.au:8081/1959.7/33587.
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Six novel platinum(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = ethylenediamine (en), 1R,2R-diaminocyclohexane (R,R-dach), or 1S,2S-diaminocyclohexane (S,S-dach) and IL = 4,7-dihydroxy-1,10-phenanthroline (4,7-dhp) or 4,7-dicarboxy-1,10-phenanthroline (4,7-dcp), were synthesised. All complexes were prepared by the addition of the intercalating ligand followed by the addition of the diamine ancillary ligand. The complexes with 4,7-dhp were soluble in DMSO and were characterised by 1H, 13C, and 195Pt NMR, elemental analysis, UV-vis, ESI-MS, and CD. The complexes with 4,7-dcp were only soluble in a highly acidic solution and, therefore, were characterised only by 1H NMR and elemental analysis. The cytotoxicity of the 4,7-dhp complexes was tested in the L1210 murine leukaemia cell line. [Pt(S,S-dach)(4,7-dhp)]Cl2 showed an IC50 value of > 80 μM. The antitumour and antibacterial activities of all six complexes were tested in vitro using the Kirby-Bauer disc diffusion method with Staphylococcus aureus and Agrobacterium tumefaciens. The 4,7-dhp complexes showed no activity to these bacteria strains. The activities of the 4,7-dcp complexes were not able to be tested due to their solubility only in acidic solutions, which itself inhibits cell growth. The diffusion coefficients of the Pt(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = en, R,R-dach, or S,S-dach and IL = phen, 4-mp, 4,7-dmp, 4,7-dhp, 4,7-dcp or 3,4,7,8-tmp and various starting materials used during the synthesis of these complexes were measured using pulsed gradient spin-echo (PGSE) NMR. The diffusion coefficients of both 4,7-dcp and [Pt(4,7-dcp)Cl2] were observed to be lower than other compounds with similar molecular weights indicating dimerisation of the compounds. The binding studies of the systems, [Pt(en)(phen)]Cl2 to (i) BSA, (ii) delipidated BSA, and (iii) d(GTCGAC)2 were studied using a simple two-site binding model with diffusion NMR. The binding of [Pt(en)(phen)]Cl2 – BSA was well described by the model giving the values Kd = 0.0021 ± 0.0002 M and n = 5.85 ± 0.31. On the contrary, the binding of [Pt(en)(phen)]Cl2 – delipidated BSA showed a poor fit to the model. From the poor fit of the data, it was speculated that the transverse relaxation of BSA largely affected the system. The binding of [Pt(en)(phen)]Cl2 – d(GTCGAC)2 showed results where the diffusion coefficient decreases as the concentration of the drug increases but an opposite effect was observed from the point where the drug reached equimolar concentrations to d(GTCGAC)2. It was speculated that the drug undergoes allosteric binding to the biomolecule or that a conformational change occurred as the drug concentration increases in the system. A further study of [Pt(en)(phen)]Cl2 and K2PtCl4 using 195Pt diffusion NMR was conducted giving a diffusion coefficient of 3.08 ± 0.04 × 10-10 m2 s-1 for K2PtCl4. The diffusion coefficient of [Pt(en)(phen)]Cl2, however, were unobtainable due to the short transverse relaxation of the Pt complex.
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Miyoshi, Emi. "Platinum(II) complexes studied by diffusion NMR /." View thesis, 2008. http://handle.uws.edu.au:8081/1959.7/33587.
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Thesis (M.Sc.) (Hons)--University of Western Sydney, 2008.<br>A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Master of Science (Honours). Includes bibliographies.
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Åkerberg, Helena. "Functional Studies of the Neuropeptide Y System : Receptor-Ligand Interaction and Regulation of Food Intake." Doctoral thesis, Uppsala universitet, Institutionen för neurovetenskap, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9533.
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The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.
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Björnerås, Johannes. "The opioid peptide dynorphin A : Biophysical studies of peptide–receptor and peptide–membrane interactions." Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-107766.
Texte intégralRésumé :
The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.<br><p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>
Chapitres de livres sur le sujet "NMR ligand-receptor interaction studies"
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Ciaramelli, C., A. Palmioli, and C. Airoldi. "Chapter 6. NMR-based Ligand–Receptor Interaction Studies under Conventional and Unconventional Conditions." In New Developments in NMR. Royal Society of Chemistry, 2022. http://dx.doi.org/10.1039/9781839165702-00142.
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Spiga, Ottavia, Maria Scarselli, Arianna Ciutti, et al. "NMR Studies of the Interaction of α-Bungarotoxin with a Mimotope of the Nicotinic Acetylcholine Receptor." In Peptides: The Wave of the Future. Springer Netherlands, 2001. http://dx.doi.org/10.1007/978-94-010-0464-0_432.
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Kearns, David R. "One- and Two-Dimensional NMR Studies of the Structures of Simple Sequence DNAs." In DNA—Ligand Interactions. Springer US, 1987. http://dx.doi.org/10.1007/978-1-4684-5383-6_2.
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Feeney, J. "NMR Studies of Protein Ligand Interactions." In NMR of Biological Macromolecules. Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-642-79158-1_6.
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Goldflam, Michael, Teresa Tarragó, Margarida Gairí, and Ernest Giralt. "NMR Studies of Protein–Ligand Interactions." In Methods in Molecular Biology. Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-480-3_14.
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Hiroaki, Hidekazu, and Daisuke Kohda. "Protein–Ligand Interactions Studied by NMR." In Experimental Approaches of NMR Spectroscopy. Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-5966-7_21.
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Roberts, G. C. K. "NMR Studies of Protein-Ligand Interactions: Dihydrofolate Reductase." In NMR in the Life Sciences. Springer US, 1986. http://dx.doi.org/10.1007/978-1-4684-8178-5_6.
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Gerothanassis, Ioannis P. "NMR Studies of Ligand-Protein Interactions Involving Quadrupolar Nuclei." In NMR of Biological Macromolecules. Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-642-79158-1_7.
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Feeney, J. "NMR Studies of Protein-Ligand and Protein-Protein Interactions Involving Proteins of Therapeutic Interest." In NMR in Supramolecular Chemistry. Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4615-9_18.
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Nakamura, Akira, Norikazu Ueyama, and Kizashi Yamaguchi. "Dynamic, Electronic and Magnetic Properties of Metal-ligand and Metal-metal Interaction Systems Studied by Solid-state High-resolution Multinuclear NMR." In Springer Series in Chemical Physics. Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-662-05339-3_7.
Texte intégralActes de conférences sur le sujet "NMR ligand-receptor interaction studies"
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Dzichenka, Yaraslau, Michail Shapira, Sergei Usanov, et al. "NOVEL LIGANDS OF HUMAN CYP7 ENZYMES – POSSIBLE MODULATORS OF CHOLESTEROL BLOOD LEVEL: COMPUTER SIMULATION STUDIES." In 1st INTERNATIONAL Conference on Chemo and BioInformatics. Institute for Information Technologies, University of Kragujevac, 2021. http://dx.doi.org/10.46793/iccbi21.435d.
Texte intégralRésumé :
Our in vitro studies showed that a couple of perspective steroidal derivatives showed previously biomedical potential via enzyme inhibition, receptor binding or antiproliferative effect against the cancer cells of reproductive tissues are able to bind to human CYP7 enzymes – key enzymes taking part in hydroxylation of cholesterol, 25-, 27-hydroxycholesterol and a number of steroidal hormones. In silico screening of binding affinity of the modified steroids toward CYP7 enzymes showed that interaction energy for the new ligands is comparable with consequent values, calculated for the ‘essential’ substrates of the enzymes – cholestenone (CYP7A1) and DHEA (CYP7B1). However, no correlation between binding energy and the affinity of the ligand was found. Novel ligands interact with conserved amino acids taking part in stabilization of natural substrates of CYP7 enzymes. A couple of structural features, governing ligand binding, were identified. Among which are planar structure of A-ring for CYP7A1 ligands, absence of many polar fragments in side-chain and presence of polar group at C3 position. Analysis of the docking results showed that CYP7B1 higher selectivity in comparison with CYP7A1 is connected by the structure of the cavity formed by α-helices I and B`. The data obtained will be used for the explanation of ligand specificity of human sterol- hydroxylases.
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Gustafson, E. J., H. Lukasiewicz, A. H. Schmaier, S. Niewiarowski, and R. W. Colman. "FIBRINOGEN BINDS TO HUMAN NEUTROPHILS AT A SITE DISTINCT FROM GPIIb/IIIa." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643850.
Texte intégralRésumé :
Many observations suggest a potential role for neutrophils in the modulation of hemostasis and thrombosis. Arterial thrombi are characterized by the presence of large numbers of neutrophils lining the perimeter of platelet aggregates. While investigating binding of high molecular weight kininogen (HMWK) to neutrophils, we found that fibrinogen (Fb) could inhibit binding of 125I-HMWK as well as displace HMWK already bound to neutrophils. We therefore initiated studies to determine whether Fb could bind to human neutrophils. Both Zn++ and Ca++ were required for maximal binding of 125I-Fb to neutrophils. Binding did not occur with Ca++ (ZmM) alone and was only 1/3 the maximal amount with Zn++ (50 μM) alone. At 4° the amount of 125I-Fb bound to neutrophils reached a plateau by 15 minutes and remained at this level over the next 30 minutes. At 23° and 37° the amount of 125I-Fb bound peaked by 4 minutes and then decreased over the next 30 minutes indicating receptor-mediated internalization. Excess Fb inhibited binding of 125I-Fb to neutrophils while prekal1ikrein, factor XII, and fibronectin did not. Binding of 125I-Fb was 99% reversible at 4° within 10 minutes with a 50-fold molar excess of Fb and 90% displaceable by excess HMWK. The apparent Kd was approximately 0.45 μM. Arg-Gly-Asp-Ser (RGDS) is a tetrapeptide common to Fb, fibronectin, vitronectin and other cel 1-attachment proteins. Fb has been demonstrated to bind to the glycoprotein IIb/111 a (GPIIb/IIIa) complex which is the platelet membrane receptor for RGDS. Although this RGDS-GPIIb/IIIa interaction occurs with Fb binding to platelets, it is apparently not involved with Fb binding to monocytes. To investigate if Fb binding to neutrophils involved this interaction of GPIIb/II la -RGDS we performed further studies. Binding of 125I-Fb to neutrophils was not inhibited by RGDS nor was it inhibited by a monoclonal antibody (10E5) to the platelet GPI I b/IIIa complex. In addition, the amount of 125I-Fb that hound to neutrophils from a patient with Glanzman's thrombosthenia was the same as that bound to normal neutrophils. These studies indicate that human neutrophils specifically bind Fb at a site similar to HMWK and distinct from GPIIb/IIIa.
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Belin, D., D. Baccino, A. Wohlwend, A. Estreicher, J. Hurate, and J.-D. Vassalli. "A CELLULAR RECEPTOR FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642957.
Texte intégralRésumé :
Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.The available information on the cellular binding of u-PA can be summarized as follows:1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional state of the cells. In some cases all sites are occupied by “endogenous” u-PA.2. Binding does not require u-PA activity, and prou-PA binds with the same affinity as does the active enzyme.3. The Kd for u-PA binding is between 1 and 10×10-10 M. The binding site appears to be specific for u-PA.4. Binding requires the presence of the A chain of u-PA; the growth factor module of the A chain is involved in this interaction.5. Bound enzyme does not dissociate readily, nor is it rapidly endocytosed; most importantly, it retains catalytic activity.Studies in progress are aimed at further defining the u-PA determinants responsible for binding. In this context it is noteworthy that there is a tight species specificity of binding: human and murine u-PA, for instance, bind only to cells of the homologous species. Characterization of the u-PA binding site suggests that it is an integral membrane protein that includes at least one Mf 50.000 polypeptide chain.In addition to allowing for the peri-cellular focalisation of u-PA catalysed proteolysis, expression of the u-PA binding site provides a mecanism whereby one cell type can acquire membrane-bound u-PA activity following secretion of the (pro)enzyme by another cell population. A striking example of this is the binding of u-PA, synthesized by the epithelial layer of the male genital tract, to the head region of murine spermatozoa.
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Hawiger, J. "PLATELET RECEPTOR RECOGNITION DOMAINS AND THEIR SYNTHETIC PEPTIDE ANALOGS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643726.
Texte intégralRésumé :
Adhesive molecules and their receptorsplay an essential role in hemostasis and thrombosis. Platelet thrombi are formed through the interaction of cell adhesion molecules (CAMs) with intercellular adhesion molecules (IAMs)and substrate adhesion molecules (SAMs). Platelet CAMs encompass membrane glycoproteins lb, lib, Ilia,and possibly la and IV, which constitutemembrane receptors for IAMs(e.g., fibrinogen) and for SAMs encompassingvon Willebrand Factor (vWF), fibronectin, vitronectin, collagen, and thrcmbospondin. Receptorfunction of platelet CAMs can be specific,i.e., only one adhesive protein among IAMs and SAMs is selected forbinding as exemplified by GPIb and vWF. Alternatively,more than one adhesive protein can interact with platelet CAMs comprising the GPIIb/IIIa complex.This common adhesive receptor mechanism switched on by thrombin, ADP, phorbol ester or ionophore A23187 is turned off by a rise in intraplatelet cyclic AMP which provides a negative control.Fibrinogen, the most abundant adhesiveprotein in plasma, interacts with platelet CAMs via receptor recognition domains on gamma and alpha chains. Pinpointing platelet receptor recognition domain to a carboxy-terminal segment of the gamma chain encompassing residues 400-411gave rise to a series of synthetic peptide analogs which do not interfere with themetabolic pathways of platelets but blockbinding of I fibrinogen to its receptors on stimulated platelets, inhibit their aggregation in vitro, and formation of a platelet thrombus in vivo. The alpha chain of human fibrinogen contains the sequenceRGD (residues 95-97 and 572-574). Synthetpeptide analogs of the RGD sequence, which constitute the "cell adhesion site" of fibronectin, also inhibit binding of 125I-fibrinogen to stimulated platelets. However, these synthetic peptides are not "specific" for fibrinogen chains because thealpha chain of human fibrinogen which hasnosequence homology with gamma 400-411 is prevented by a peptide gamma 400-411 from interaction with platelet receptors. Viceversa, the human gamma chain is blocked by tetrapeptide RGDS not expressed in the human gamma chain. Interaction of human vWF with human platelets is blocked by synthetic peptide analogs of gamma 400-411 (not present in vWF)and of RGD sequence (present in vWF).These synthetic peptides inhibite "common" receptor pathwaystimulated with ADP, thrombin, or phorbolester, but they do not interfere with binding of 125I-vWF via a "specific" pathvoy induced with ristocetin and involving GPIb.The design of synthetic peptide analogs which inhibit platelet receptors for adhesive molecules includes the following considerations: ligand specificity (is thepeptide inhibitory toward binding of one or more adhesive molecules?),cell speciicity (is the peptide specific for platelets or does it perturb the adhesive properties of other cells, e.g.,endothelium?);the hydrophilic character; protection against degradation by peptidases; and a sufficiently long half-life to achieve platelet inhibitory potency in vivo without overloading the blood with excessive amounts of peptide.This is accomplished by constructing a peptide-albumin conjugate with ahalf-life extended at least 30 times.Whenpeptides are modeled with predominantly hydrophilic or hydrophobic residues, only the hydrophilic peptide remained active to block the platelet receptor. This agreed with the general observation that sequences on adhesive molecules that are knownto interact with cellular receptors have a hydrophilic rather than a hydrophobic character. Furthermore, changing the charge of synthetic peptides toward the negative reduced the reactivity, whereas introducing additional arginine residues enhanced the reactivity toward platelet receptors. Localization of the functionally important binding domain in the flexible segment of an adhesive protein increases the likelihood that the synthetic peptide will assume the conformation mimicking such a domain in the native adhesive protein. Structure-function studies of the receptor recognition domains on adhesive molecules led to development of a new class of platelet inhibitors acting at the membranereceptors responsible for anchoring of platelets to the vessel wall and linking them to each other.
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Subramaniam, Dhananjay Radhakrishnan, and David J. Gee. "Hydrodynamic Recruitment of Leukocytes Is Influenced by Adherent Cell Morphology." In ASME 2018 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2018. http://dx.doi.org/10.1115/imece2018-86502.
Texte intégralRésumé :
Innate immunity depends on the coordinated activity of multiple leukocytes at or near the site of tissue injury. Previous numerical studies have shown that an adherent leukocyte can hydrodynamically recruit a free-stream leukocyte towards the endothelial surface. Using a computer model we created, we numerically investigated the hydrodynamic recruitment of circulating cells due to the presence of a nearby adherent deformed cell. For circulating cells positioned one diameter or more above the reactive surface and subsequently involved in a glancing (out-of-plane) collision with an adherent cell, the simulation indicated that the free-stream cell could be driven closer to the surface. This behavior was seen to depend, in part, on the offset glancing distance. Furthermore, for a deformed adherent cell a similar effect was observed, but beginning at smaller offset glancing distances. We also examined the binary interaction for a free-stream cell initially less than one diameter above the surface. For fixed offset glancing distances, the binary interaction with a more significantly deformed adherent cell resulted in enhanced recruiting effectiveness, as quantified by the time needed for the cell to descend to a height where receptor-ligand interactions were possible.
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Goldsmith, Harry L. "Observing Human Blood Cells in Flow Through Microchannels." In ASME 2010 8th International Conference on Nanochannels, Microchannels, and Minichannels collocated with 3rd Joint US-European Fluids Engineering Summer Meeting. ASMEDC, 2010. http://dx.doi.org/10.1115/fedsm-icnmm2010-31330.
Texte intégralRésumé :
For the rheologist, blood is essentially a concentrated suspension of biconcave 8-μm diameter red cells (40–45% by volume) that circulates within the body in vessels from 25 mm down to 5 μm diameter. Here, we describe in vitro tracking of blood cells in a traveling microtube apparatus and in a counter-rotating micro cone-plate device at low Reynolds numbers, Re. Observations of the flow behavior of individual red cells reveal a marked and continuously changing deformation and interaction of the cells in shear, and this, together with their migration away from the vessel wall accounts for the low whole blood overall viscosity compared to other concentrated suspensions and emulsions. Red cells also strongly affect the flow behavior and interactions of platelets and of white cells, which although present at much lower concentrations (0.3% by volume), play key roles in thrombosis, hemostasis, and inflammation. Studies of the kinetics of the formation and break-up of receptor-ligand bonds between membranes of platelets and of white cells in shear flow revealed single bond strengths of 50 −200 nN. Such micro particle image velocimetry (μPIV) studies have recently been considerably refined and extended to in vivo vessels such as postcapillary venules. Using submicron fluorescent latex spheres, the existence of an impermeable and hydrodynamically effective surface layer (< 0.5 μm thick) extending out from the vessel endothelium has been confirmed. The lecture is illustrated by movies of blood flow in vitro and in vivo.
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Apitz Castro, R., E. Ledezma, A. Jorquera, and M. K. Jain. "REVERSIBLE BLOCKADE OF PLATELET ACTIVATION DURING CARDIO-PUIMONAR BYPASS IN DOGS AFTER IV ADMINISTRATION OF AJOENE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644820.
Texte intégralRésumé :
Surgery with extracorporeal circulation (ECC) is associated with platelet activation, which greatly contribute to prolonged postoperative bleeding and increased blood loss after surgery. Antiplatelet ccnpounds which induce rapid and reversible inhibition of platelet function, without affecting platelet adhesiveness would be potentially useful in the management of the platelet-dependent hemostatic disorder observed in ECC. Ajoene, an organosulfur originally obtained from garlic, inhibits platelet release and aggregation induced ex vivo by all know agonists. It does not affect shape change or adhesion to collagen nor interfere with metabolic pathways relevant to the platelet reaction. Ajoene action is related to its direct interaction with the fibrinogen receptor on the platelet surface which irrpairs fibrinogen binding to stimulated or chymotrypsin treated platelets. IV administration of ajoene (15 mg/Kg) to mongrel dogs, inhibits platelet aggregation induced ex vivo by collagen (2-5 g/ml) or ADP (10 M). Ccnplete inhibition is attained after 20-35min and recuperation of platelet reactivity is obtained after 2.5-3.5 hours. To study the potential benefit of ajoene for the prevention of platelet activation during cardio-pulnonary bypass, ajoene was administered to anesthesized (heparin-anticoagulated) dogs, as described above, 40min before establishing the ECC. Circulation was mantained at 1.5L/min for a period of lOOmin Platelet count, and aggregation induced esc vivo by ADP or collagen were meassured immediately before ajoene administration, lOmin after the end of ECC and thereafter, hourly. Platelet count lOmin after end of ECC, in ncn-treated dogs fell to about 57% of prepump values, while in ajoene-treated animals circulating platelets represented 80% of pre-ECC values. Recovery of platelet function in ajoene-treated dogs started 2 hr after end of ECC (about 4 hr. after ajoene administration) reaching 70% 4 hr after end of ECC. Surgical bleeding in treated-dogs was not different frrm controls. Moderate bradicardia and hypotension, which in atrqpi-nized dogs returned to normal values within 3 min was observed. Although detailed pharmacological studies are still needed, our results suggest that ajoene is a potentially useful drug for the prevention of platelet activation induced by ECC.
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Authi, K. S., B. J. Evenden та N. Crawford. "ACTION OF GTPγS [GUANOSINE 5∲-0-(3-THIOPHOSPHATE)] ON SAPONIN-PERMEABILISED PLATELETS: INVOLVEMENT OF 'G' PROTEINS IN PLATELET ACTIVATION". У XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644514.
Texte intégralRésumé :
Certain ligand-receptor interactions at cell surfaces lead to the phospholipase-C (PLC) hydrolysis of phosphatidyl inositol (4.5) bisphosphate (PIP2). The products serve as intracellular second messengers, e.g. inositol (1.4.5) trisphosphate (IP3) releases Ca2+ from intracellular stores and diacylglycerol activates protein kinase-C. From studies using GTP and analogues (e.g. GTPγS) there is evidence of a key role for a guanine nucleotide binding protein(s) as a link between receptors and PIP2 hydrolysis. We report the actions of GTPγS on washed human platelets permeabilised with saponin (12-14 μg/ml) to allow penetration of low MWt polar substances. The responses to GTPγS are dose dependent (range 9-60 μM) and at 60 μM the agent induces shape change, aggregation and the secretion of 50% of previously incorporated [14C]-5HT. No effect of GTPγS is seen with intact cells. Shape change occurs 25-30 sec after GTPγS; aggregation and secretion is complete after 3 min. When GTP was used (up to 135 μM) with similarly permeabilised platelets no responses were initiated. Phosphatidylinositol turnover was monitored using 32P-labelling before permeabilisation. The addition of 90 μM GTPγS resulted in a 143 ± 23% (n=4) increase in 32P-phosphatidic acid (PA) with respect to the basal levels of “saponised control” cells. These findings suggest that GTPγS stimulates PLC activity through a ‘G’ protein interaction. The GDP analogue (GDPβS) produced no activation responses in saponised platelets but inhibited responses induced by GTPγS in a dose dependent manner (0-480 μM, max inhibition 480 μM). At 960 μM, GDPβS totally inhibited aggregation and secretion initiated by low doses of thrombin (0.1 U/ml) and collagen (1 μg/ml). Identical inhibition by GDPβS of thrombin and collagen-induced activation of intact platelets was observed indicating membrane penetration of this analogue. Shape change effects were not inhibited by GDPSS. The inhibitory effects of GDPSS towards thrombin and collagen induced secretion could be progressively overcome at higher doses of thrombin (0.2 U/ml - 2 U/ml) and collagen (5 μg/ml - 60 μg/ml) suggesting that at higher concentrations these agonists may exert effects through 'G' protein-independent mechanisms.
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Bussel, J. "FOR MODULATION AS A MEANS OF ELEVATING THE PLATELET COUNT IN ITP." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644761.
Texte intégralRésumé :
ITP is an autoantibody-mediated disease which would logically be treated by decreasing the level of autoantibody. However, the most exciting developments in understanding the pathophysiology of the thrombocytopenia and its treatment involve a better understanding of the MPS FcR system and ways in which it can be modulated. This work has focussed on phagocytic paralysis or FcR blockade (FcRBl): the slowing of destruction of antibody-coated platelets despite the persistent presence of antibody on the surface of the platelet.Several areas have been explored in learning about the MPS system. Investigation by Kurlander among others have revealed that at least 2 FcR's exist on mononuclear phagocytes: one with high and one with low affinity for monomeric IgG. Study of the high affinity FcR expressed by circulating monocytes, by Schreiber among others, has explored the effect of Danazol to decrease the expression of this FcR. The clinical relevance of this receptor is uncertain however because it is saturated in vitro by physiologic concentrations of IgG. Unkeless defined the properties of the low affinity "immune complex" FcR, expressed on macrophages and neutrophils, via monoclonal antibody 3G8 (see below) which blocks ligand binding to this FcR. The exact roles of these two, and possibly more, FcR's are being explored. Another still unsolved controversy involves whether the interaction Fc portions of antibodies coating particles with FcR's is mediated by a conformational change of the Fc portion or by a multipoint attachment of several Fc parts.Studies by Mollison in the 60's demonstrated that the MPS had a limited capacity for removal of antibody-coated (red) cells. Shulman pursued MPS modulation by exploring the inhibition of thrombocytopenia caused by infusion of ITP plasma into normals. Kelton demonstrated that "compensated" ITP may be caused by a decreased clearance of antibody-coated cells and that the rate of clearance of antibody-coated cells may be correlated with rate of clearance of antibody-coated cells may be correlated with the intrinsic levels of IgG. Stossel investigated FcRBl as a mechanism of effect of corticosteroids and related it clinically. Subsequently intravenous gammaglobulin (IVGG) was introducedas a treatment of ITP and Fehr et al first demonstrated FcRBl as the mechanism of effect of IVGG. Exploration of the mechanism of the FcRBl caused by IVGGled Salama and Mueller-Eckhardt to demonstrate the therapeutic effect of I anti-D, which apparentlycoats RBC with antibody and causes their destruction atthe coats RBC with antibody and causes their destruction at the expense of antibody-coated platelets. A similar degree of FcRBl has been shown for aldometrelated to the development of antibody on RBC.Our studies, including Drs. Clarkson, Kimberly, Nachman, and Unkeless, have focussed on the role of the low affinity or "Immune complex" FcR by using monoclonal antibody 3G8 in vivo. An infusion of 1 mg/kg of 3G8 in chimpanzees caused a reproducible FcRBl demonstrable by a slowing of the destruction of antibody-coated RBC for > 10 days (JEM, 1986). Less effect of 3G8 to inhibit CIC removal was seen using DNA-anti-DNA as the immune complex. In view of the wel1-documented effects of IVGG infusion to create FcRBl, we infused 3G8 into 6 adults with refractory ITP (NEJM, 1986). Specifically these patients were refractory to all forms of conventional therapy including splenectomy, steroids, vinca alkaloid infusion, immunosuppressives and danazol . 3 of the 6 patients had peak platelet responses to >80,000/ul. The other 3 had short-lived platelet increases from 10 to 30,000/ul. These responses confirmed the effect of FcRBl, specifically of the low affinity FcR, to underlie a dramatic platelet increase in therapy of ITP. Surprisingly 3 of the patients had apparent longterm effects of this therapy demonstrable in 2 cases as a maintenance of the platelet count >20,0C0/ul without any further therapy and in 1 case as a clearly enhanced responsiveness to other therapies following 3G8 infusion. Since Natural Killer activity was (transiently) ablated by 3G8 infusion, we speculate that an alternation of regulation of (auto) antibody production by NK cells may be responsible for this effect and that FcR interactions include regulatory roles in addition to their primary function of removal of CIC.
Rapports d'organisations sur le sujet "NMR ligand-receptor interaction studies"
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Altstein, Miriam, and Ronald J. Nachman. Rational Design of Insect Control Agent Prototypes Based on Pyrokinin/PBAN Neuropeptide Antagonists. United States Department of Agriculture, 2013. http://dx.doi.org/10.32747/2013.7593398.bard.
Texte intégralRésumé :
The general objective of this study was to develop rationally designed mimetic antagonists (and agonists) of the PK/PBAN Np class with enhanced bio-stability and bioavailability as prototypes for effective and environmentally friendly pest insect management agents. The PK/PBAN family is a multifunctional group of Nps that mediates key functions in insects (sex pheromone biosynthesis, cuticular melanization, myotropic activity, diapause and pupal development) and is, therefore, of high scientific and applied interest. The objectives of the current study were: (i) to identify an antagonist biophores (ii) to develop an arsenal of amphiphilic topically active PK/PBAN antagonists with an array of different time-release profiles based on the previously developed prototype analog; (iii) to develop rationally designed non-peptide SMLs based on the antagonist biophore determined in (i) and evaluate them in cloned receptor microplate binding assays and by pheromonotropic, melanotropic and pupariation in vivo assays. (iv) to clone PK/PBAN receptors (PK/PBAN-Rs) for further understanding of receptor-ligand interactions; (v) to develop microplate binding assays for screening the above SMLs. In the course of the granting period A series of amphiphilic PK/PBAN analogs based on a linear lead antagonist from the previous BARD grant was synthesized that incorporated a diverse array of hydrophobic groups (HR-Suc-A[dF]PRLa). Others were synthesized via the attachment of polyethylene glycol (PEG) polymers. A hydrophobic, biostablePK/PBAN/DH analog DH-2Abf-K prevented the onset of the protective state of diapause in H. zea pupae [EC50=7 pmol/larva] following injection into the preceding larval stage. It effectively induces the crop pest to commit a form of ‘ecological suicide’. Evaluation of a set of amphiphilic PK analogs with a diverse array of hydrophobic groups of the formula HR-Suc-FTPRLa led to the identification of analog T-63 (HR=Decyl) that increased the extent of diapause termination by a factor of 70% when applied topically to newly emerged pupae. Another biostablePK analog PK-Oic-1 featured anti-feedant and aphicidal properties that matched the potency of some commercial aphicides. Native PK showed no significant activity. The aphicidal effects were blocked by a new PEGylated PK antagonist analog PK-dF-PEG4, suggesting that the activity is mediated by a PK/PBAN receptor and therefore indicative of a novel and selective mode-of-action. Using a novel transPro mimetic motif (dihydroimidazole; ‘Jones’) developed in previous BARD-sponsored work, the first antagonist for the diapause hormone (DH), DH-Jo, was developed and shown to block over 50% of H. zea pupal diapause termination activity of native DH. This novel antagonist development strategy may be applicable to other invertebrate and vertebrate hormones that feature a transPro in the active core. The research identifies a critical component of the antagonist biophore for this PK/PBAN receptor subtype, i.e. a trans-oriented Pro. Additional work led to the molecular cloning and functional characterization of the DH receptor from H. zea, allowing for the discovery of three other DH antagonist analogs: Drosophila ETH, a β-AA analog, and a dF analog. The receptor experiments identified an agonist (DH-2Abf-dA) with a maximal response greater than native DH. ‘Deconvolution’ of a rationally-designed nonpeptide heterocyclic combinatorial library with a cyclic bis-guanidino (BG) scaffold led to discovery of several members that elicited activity in a pupariation acceleration assay, and one that also showed activity in an H. zea diapause termination assay, eliciting a maximal response of 90%. Molecular cloning and functional characterization of a CAP2b antidiuretic receptor from the kissing bug (R. prolixus) as well as the first CAP2b and PK receptors from a tick was also achieved. Notably, the PK/PBAN-like receptor from the cattle fever tick is unique among known PK/PBAN and CAP2b receptors in that it can interact with both ligand types, providing further evidence for an evolutionary relationship between these two NP families. In the course of the granting period we also managed to clone the PK/PBAN-R of H. peltigera, to express it and the S. littoralis-R Sf-9 cells and to evaluate their interaction with a variety of PK/PBAN ligands. In addition, three functional microplate assays in a HTS format have been developed: a cell-membrane competitive ligand binding assay; a Ca flux assay and a whole cell cAMP ELISA. The Ca flux assay has been used for receptor characterization due to its extremely high sensitivity. Computer homology studies were carried out to predict both receptor’s SAR and based on this analysis 8 mutants have been generated. The bioavailability of small linear antagonistic peptides has been evaluated and was found to be highly effective as sex pheromone biosynthesis inhibitors. The activity of 11 new amphiphilic analogs has also been evaluated. Unfortunately, due to a problem with the Heliothis moth colony we were unable to select those with pheromonotropic antagonistic activity and further check their bioavailability. Six peptides exhibited some melanotropic antagonistic activity but due to the low inhibitory effect the peptides were not further tested for bioavailability in S. littoralis larvae. Despite the fact that no new antagonistic peptides were discovered in the course of this granting period the results contribute to a better understanding of the interaction of the PK/PBAN family of Nps with their receptors, provided several HT assays for screening of libraries of various origin for presence of PK/PBAN-Ragonists and antagonists and provided important practical information for the further design of new, peptide-based insecticide prototypes aimed at the disruption of key neuroendocrine physiological functions in pest insects.
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Eyal, Yoram, and Sheila McCormick. Molecular Mechanisms of Pollen-Pistil Interactions in Interspecific Crossing Barriers in the Tomato Family. United States Department of Agriculture, 2000. http://dx.doi.org/10.32747/2000.7573076.bard.
Texte intégralRésumé :
During the evolutionary process of speciation in plants, naturally occurring barriers to reproduction have developed that affect the transfer of genes within and between related species. These barriers can occur at several different levels beginning with pollination-barriers and ending with hybrid-breakdown. The interaction between pollen and pistils presents one of the major barriers to intra- and inter-specific crosses and is the focus of this research project. Our long-term goal in this research proposal was defined to resolve questions on recognition and communication during pollen-pistil interactions in the extended tomato family. In this context, this work was initiated and planned to study the potential involvement of tomato pollen-specific receptor-like kinases (RLK's) in the interaction between pollen and pistils. By special permission from BARD the objectives of this research were extended to include studies on pollen-pistil interactions and pollination barriers in horticultural crops with an emphasis on citrus. Functional characterization of 2 pollen-specific RLK's from tomato was carried out. The data shows that both encode functional kinases that were active as recombinant proteins. One of the kinases was shown to accumulate mainly after pollen germination and to be phosphorylated in-vitro in pollen membranes as well as in-vivo. The presence of style extract resulted in dephosphorylation of the RLK, although no species specificity was observed. This data implies a role for at least one RLK in pollination events following pollen germination. However, a transgenic plant analysis of the RLK's comprising overexpression, dominant-negative and anti-sense constructs failed to provide answers on their role in pollination. While genetic effects on some of the plants were observed in both the Israeli and American labs, no clear functional answers were obtained. An alternative approach to addressing function was pursued by screening for an artificial ligand for the receptor domain using a peptide phage display library. An enriched peptide sequence was obtained and will be used to design a peptide-ligand to be tested for its effect o pollen germination and tube growth. Self-incompatibility (SI) in citrus was studied on 3 varieties of pummelo. SI was observed using fluorescence microscopy in each of the 3 varieties and compatibility relations between varieties was determined. An initial screen for an S-RNase SI mechanism yielded only a cDNA homologous to the group of S-like RNases, suggesting that SI results from an as yet unknown mechanism. 2D gel electrophoresis was applied to compare pollen and style profiles of different compatibility groups. A "polymorphic" protein band from style extracts was observed, isolated and micro-sequenced. Degenerate primers designed based on the peptide sequence date will be used to isolate the relevant genes i order to study their potential involvement in SI. A study on SI in the apple cultivar Top red was initiated. SI was found, as previously shown, to be complete thus requiring a compatible pollinator variety. A new S-RNase allele was discovered fro Top red styles and was found to be highly homologous to pear S-RNases, suggesting that evolution of these genes pre-dated speciation into apples and pears but not to other Rosaceae species. The new allele provides molecular-genetic tools to determine potential pollinators for the variety Top red as well as a tool to break-down SI in this important variety.