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Saturation Binding (saturation + binding)
Selected AbstractsCompetitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and humanENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2007Vickie S. Wilson Abstract Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor , (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols. [source] Adenosine A2A Receptors are Up-regulated in Pick's Disease Frontal CortexBRAIN PATHOLOGY, Issue 4 2006José Luís Albasanz PhD Adenosine A2A receptors (A2AR) are highly expressed in striatum. However, they are also present in extrastriatal structures. A2AR were studied in post-mortem human frontal cortex from Pick's disease (PiD) and age-matched non-demented controls by radioligand binding assays, Western-blotting, real-time PCR and adenylyl cyclase activity determination. Saturation binding assay using [3H]ZM 241385, a selective A2A antagonist, as radioligand revealed a significant increase in total adenosine A2AR numbers (Bmax) in frontal cortex from PiD samples (191% of control Bmax), suggesting up-regulation of this receptor. A significant increase in the level of A2AR was also detected by Western-blotting. Furthermore, expression of mRNA coding A2AR determined by quantitative real-time PCR was enhanced. In agreement, stimulation of adenylyl cyclase by CGS 21680, a selective A2A receptor agonist, was significantly strengthened. Up-regulation of A2B receptors and their corresponding mRNA was also observed. These results show that A2A adenosine receptor/adenylyl cyclase transduction pathway is up-regulated and sensitized in frontal cortex brain from PiD. [source] Lineage-specific overexpression of the P2Y1 receptor induces platelet hyper-reactivity in transgenic miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2003B. Hechler Summary., In order to investigate the role of the platelet P2Y1 receptor in several aspects of platelet activation and thrombosis, transgenic (TG) mice overexpressing this receptor specifically in the megakaryocytic/platelet lineage were generated using the promoter of the tissue-specific platelet factor 4 gene. Studies of the saturation binding of [33P]2MeSADP in the presence or absence of the selective P2Y1 antagonist MRS2179 indicated that wild-type (WT) mouse platelets bore 150 ± 31 P2Y1 receptors and TG platelets 276 ± 34, representing an 84% increase in P2Y1 receptor density. This led to a well defined phenotype of platelet hyper-reactivity in vitro, as shown by increased aggregations in response to adenosine 5,-diphosphate (ADP) and low concentration of collagen in TG as compared with WT platelets. Moreover, overexpression of the P2Y1 receptor enabled ADP to induce granule secretion, unlike in WT platelets, which suggests that the level of P2Y1 expression is critical for this event. Our results further suggest that the weak responses of normal platelets to ADP are due to a limited number of P2Y1 receptors rather than to activation of a specific transduction pathway. TG mice displayed a shortened bleeding time and an increased sensitivity to in vivo platelet aggregation induced by infusion of a mixture of collagen and epinephrine. Overall, these findings emphasize the importance of the P2Y1 receptor in hemostasis and thrombosis and suggest that variable expression levels of this receptor on platelets might play a role in thrombotic states in human, which remains to be assessed. [source] Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti,tumor necrosis factor , but not methotrexateARTHRITIS & RHEUMATISM, Issue 10 2009Katia Varani Objective To investigate A1, A2A, A2B, and A3 adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti,tumor necrosis factor , (anti-TNF,), as compared with those in age-matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNF, release and NF-,B activation. Methods Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A2A and A3 agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti-TNF,. In a separate cohort of RA patients, TNF, release and NF-,B activation were evaluated in plasma and nuclear extracts, respectively. Results In ERA patients, we found a high density and altered functionality of A2A and A3 receptors. The binding and functional parameters of A2A and A3 receptors normalized after anti-TNF,, but not MTX, treatment. TNF, release was increased in ERA patients and in MTX-treated RA patients, whereas in anti-TNF,,treated RA patients, release was comparable to that in the controls. NF-,B activation was elevated in ERA patients and in MTX-treated RA patients. Anti-TNF, treatment mediated decreased levels of NF-,B activation. Conclusion A2A and A3 receptor up-regulation in ERA patients and in MTX-treated RA patients was associated with high levels of TNF, and NF-,B activation. Treatment with anti-TNF, normalized A2A and A3 receptor expression and functionality. This new evidence of A2A and A3 receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component. [source] Ro 60 functions as a receptor for ,2 -glycoprotein I on apoptotic cellsARTHRITIS & RHEUMATISM, Issue 3 2009Joanne H. Reed Objective The autoantigens 60-kd Ro/SSA (Ro 60) and ,2 -glycoprotein I (,2GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether ,2GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti,Ro 60 IgG. Methods The interaction between soluble recombinant Ro 60 fragments and ,2GPI was investigated in vitro by direct and saturation binding assays using native human ,2GPI and recombinant domain deletion mutants. Binding of ,2GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti,Ro 60 IgG. Results The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (Kd = ,15 nM) to domain V of ,2GPI in vitro. Beta2 -glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, ,2GPI blocked the binding of anti,Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti,Ro 60 IgG inhibited the binding of ,2GPI. Moreover, ,2GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. Conclusion These results demonstrate that Ro 60 functions as a novel receptor for ,2GPI on the surface of apoptotic cells. The formation of Ro 60,,2GPI complexes may protect against anti,Ro 60 autoantibody,mediated tissue injury. [source] In vivo saturation binding of GABA-A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometryBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2009Seth C. Hopkins Abstract Typically, the dose-occupancy curves for GABA-A receptor ligands are determined using in vivo binding of [3H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA-A ligands zolpidem, (RS)-zopiclone, L-838417 and flumazenil, to demonstrate affinity and saturation of the filter-retained, membrane-bound fraction. The in vivo binding of flumazenil and L-838417 both saturated around 200,nm, at a similar level to the specific binding of (S)-zopiclone after doses of the racemic zopiclone, using (R)-zopiclone to estimate non-specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA-A receptors in vivo (200,nm). Dose-occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)-isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo. Copyright © 2009 John Wiley & Sons, Ltd. [source] Influence of N-Terminal Hydrophobicity of Cationic Peptides on Thermodynamics of their Interaction with Plasmid DNACHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2009Geetha N. Goparaju There is a need to understand the thermodynamics of interaction of cationic peptides with DNA to design better peptide based non-viral gene delivery vectors. The main aim of this study was to understand the influence of N-terminal hydrophobicity of cationic amphiphilic peptides on thermodynamics of interaction with plasmid DNA. The model peptides used were TATPTD and TATPTDs modified at the N-terminal with hydrophobic amino acids. The thermodynamic binding data from isothermal titration calorimetry were compared with ethidium bromide analysis and ultrafiltration to correlate the binding parameters with the structural features of the various peptides used. It was observed that peptides having a smaller hydrophobic domain at the N-terminal have good DNA condensing ability compared with the ones with a longer hydrophobic domain. Calorimetry of peptides that reached saturation binding indicated that enthalpy and entropy are favorable for the interaction. Moreover, the interaction of these peptides with DNA appears to be predominantly electrostatic. [source] | |||||||||||||